therapeutic effects of intravenous immunoglobulin, …...therapeutic effects of intravenous...
TRANSCRIPT
Therapeutic Effects of Intravenous Immunoglobulin, Platelet Transfusion and B
Cell Depletion on Murine Immune Thrombocytopenia (ITP)
By
Li Guo
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy (Ph.D.)
Institute of Medical Science University of Toronto
© Copyright by Li Guo 2016
ii
Therapeutic Effects of Intravenous Immunoglobulin, Platelet
Transfusion and B Cell Depletion on Murine Immune
Thrombocytopenia (ITP)
Li Guo
Doctor of Philosophy
Institute of Medical Science
University of Toronto
2016
ABSTRACT
Immune thrombocytopenia (ITP) is an autoimmune disease characterized by an isolated
thrombocytopenia and increased tendency of bleeding. The pathogenesis of ITP includes both
decreased platelet production by megakaryocytes in the bone marrow and increased peripheral
platelet destruction in the spleen due to antibody- and T cell-mediated immune responses
against self platelet antigens. To better understand the disease and its therapies, I carried out
three projects focusing on antibody-mediated ITP, T cell-mediated ITP and the relationship
between the two immune pathways. In the first project, I examined the number and morphology
of bone marrow megakaryocytes after induction of acute thrombocytopenia by anti-platelet
antibodies with different specificities and isotypes. Most of the antibodies did not appear to alter
megakaryocyte morphology or maturation within 24 hours. Moreover, although IVIg therapy
successfully rescued the thrombocytopenia, it did not exhibit any significant effect on the
megakaryocytes. Second, using an active ITP mouse model, I investigated the effect of
allogeneic platelet transfusion therapy on T cell-mediated ITP. I found an immunosuppressive
effect of the allogeneic transfusions on the T cell-mediated ITP associated with a decrease in the
cytotoxic activity of CD8+ T cells in vitro and normalized megakaryocyte number/morphology
in the bone marrow. Third, to investigate the relationship between B and T cells in ITP, I
depleted B cells in vivo and examined the changes in T cell-mediated ITP. CD8+ T cell-
mediated ITP was found ameliorated after B cell depletion therapy and was associated with a
iii
decreased proliferation of CD8+ T cells that could be rescued with interleukin (IL)-2 in vitro.
This study provided a novel explanation for the therapeutic effect of B cell depletion in ITP and
suggests a synergistic role between B cells and T cells in initiating the disease. In summary, this
thesis revealed a lack of effect of anti-platelet antibodies on megakaryocytes suggesting that T
cells may be involved in mediating bone marrow effects in ITP. Related to the T cells, it further
elucidated the therapeutic effects of platelet transfusions and B cell depletion therapy on T cell-
mediated ITP. These results may be of importance in decisions regarding the management of
ITP.
iv
ACKNOWLEDGEMENTS
First and foremost, I am forever thankful to my supervisor, Dr. John W. Semple who
inspired me with his passion and wisdom, welcomed me to the wonderful world of science,
guided me through all the challenges, encouraged me upon every failure, and took care of me
like a family member and friend. I am extremely fortunate to have such a great mentor during
my early career.
I am also grateful to all the lab members for their support and friendship. In particular, I
thank Dr. Rukhsana Aslam and Edwin R. Speck, for their tremendous support, patient teaching,
extreme kindness and loving friendship. I would also like to thank lab members Michael Kim,
Dr. Anne Zufferey, Dr. Rick Kapur, Dr. Christopher G. J. McKenzie, Dr. Mark McVey and all
our summer students. I will always cherish our time full of laughter and joy.
Furthermore, I would like to thank my committee members Drs. Alan H. Lazarus and
Gerald Prud’homme who have provided invaluable advice and continuous great support towards
my development as a researcher.
I would like to also thank our collaborators, Dr. Heyu Ni for his generous supply of
animals, Christopher M. Spring for his technical support, the Ontario Trillium Foundation, the
Chinese Scholarship Council, and the Institute of Medical Science at the University of Toronto
for their financial support.
Finally I would like to thank my parents and grandmother for their unconditional love and
support that travelled half of the planet with twelve hours of time difference. I could never have
accomplished my PhD without their love.
I dedicate this thesis to my mom Haiyan, dad Qiang, and my grandma Yuying.
v
CONTRIBUTIONS
Li Guo (author) solely prepared this thesis. All aspects of this body of work, including the
planning, execution, analysis, and writing of all original research and publications was
performed in whole or in part by the author. The following contributions by other individuals
are formally acknowledged:
Dr. John W. Semple (Supervisor and Thesis Committee Member) – mentorship; laboratory
resources; guidance and assistance in planning, execution, and analysis of experiments as well
as manuscript/thesis preparation.
Dr. Alan H. Lazarus (Thesis Committee Member) – mentorship; laboratory resources; guidance
in planning of experiments and interpretation of results.
Dr. Gerald Prud’homme (Thesis Committee Member) – mentorship; guidance in planning of
experiments and interpretation of results.
Dr. Margaret L. Rand – guidance in thesis preparation.
Dr. Rukhsana Aslam – assistance in execution of experiments and manuscript editing for
Chapter 2.
Dr. Rick Kapur – assistance in manuscript editing for Chapter 2 and Chapter 4.
Mr. Edwin R. Speck– assistance in execution of experiments.
vi
Table of Contents
ABSTRACT ................................................................................................................................... ii
ACKNOWLEDGEMENTS .......................................................................................................... iv
CONTRIBUTIONS ....................................................................................................................... v
LIST OF TABLES ....................................................................................................................... ix
LIST OF FIGURES ...................................................................................................................... x
LIST OF APPENDICES ............................................................................................................. xii
LIST OF ABBREVIATIONS .................................................................................................... xiii
CHAPTER 1 Introduction .......................................................................................................... 1
1.1 Preamble ............................................................................................................................... 2
1.2 Megakaryocyte and platelet biology .................................................................................... 4
1.2.1 Megakaryocyte development, differentiation, and platelet production ...................................... 4
1.2.2 Platelets in hemostasis and thrombosis .................................................................................. 8
1.2.3 The immunomodulatory role of platelets .............................................................................. 11
1.2.4 Thrombocytopenia induced by immune responses against platelets ....................................... 13
1.3 Immune thrombocytopenia (ITP) ....................................................................................... 16
1.3.1 Brief history of ITP ............................................................................................................. 16
1.3.2 Current definition and diagnosis of ITP ............................................................................... 20
1.3.3 Pathogenesis of ITP ........................................................................................................... 27
1.3.4 Treatment of ITP ............................................................................................................... 62
1.3.5 Intravenous immunoglobulin (IVIg) in ITP ........................................................................... 66
1.3.6 Platelet transfusions in ITP ................................................................................................. 68
vii
1.3.7 Rituximab and B cell depletion therapy ............................................................................... 70
1.3.8 Animal models of ITP ........................................................................................................ 72
1.4 Rationale, hypothesis and specific aims ................................................................................. 76
1.4.1 Rationale ........................................................................................................................... 76
1.4.2 Hypothesis ........................................................................................................................ 77
1.4.3 Specific aims ..................................................................................................................... 77
CHAPTER 2 Thrombocytopenia and Megakaryocyte Counts Are Differentially
Controlled By The Specificity Of Anti-platelet Antibodies In A Murine Model Of Passive
Immune Thrombocytopenia (ITP) .......................................................................................... 78
2.1 Abstract .............................................................................................................................. 79
2.2 Introduction ........................................................................................................................ 80
2.3 Materials and Methods ........................................................................................................... 83
2.4 Results ................................................................................................................................ 85
2.5 Discussion .......................................................................................................................... 90
CHAPTER 3 Allogeneic Platelet Transfusions Prevent Murine T Cell-Mediated ITP ..... 94
3.1 Abstract .............................................................................................................................. 95
3.2 Introduction ........................................................................................................................ 96
3.3 Materials and Methods ........................................................................................................... 98
3.4 Results .............................................................................................................................. 105
3.5 Discussion ........................................................................................................................ 114
CHAPTER 4. CD20+ B cell Depletion Therapy Suppresses Murine CD8
+ T Cell-mediated
Immune Thrombocytopenia (ITP) ......................................................................................... 117
4.1 Abstract ............................................................................................................................ 118
4.2 Introduction ...................................................................................................................... 119
4.3 Material and Methods .......................................................................................................... 120
viii
4.4 Results .............................................................................................................................. 126
4.5 Discussion ........................................................................................................................ 136
CHAPTER 5. Discussion And Future Directions ................................................................. 138
5.1 Discussion ........................................................................................................................ 139
5.2 Conclusion ........................................................................................................................ 149
5.3 Future Directions ............................................................................................................... 150
REFERENCES .......................................................................................................................... 155
APPENDIX ................................................................................................................................ 215
ix
LIST OF TABLES
Table 1.1 Proposed definitions of ITP ........................................................................................ 18
Table 1.2 Summary of the antigen presenting cell (APC) defects in ITP.................................... 42
Table 1.3 Summary of Fc receptor expression patterns and functions. ..................................... 56
Table 1.4 A brief summary of therapies for the management of ITP. ....................................... 62
Table 2.1 Details of polyclonal (serum) and monoclonal antibodies used for ITP induction. ... 80
Table 3.1: Summary of platelet populations, immune splenocytes used and expected immune
responses induced ....................................................................................................................... 97
x
LIST OF FIGURES
Figure 1.1 Major stages of megakaryocyte development. ............................................................. 5
Figure 1.2 Petechiae in the skin. .................................................................................................. 22
Figure 1.3 A schematic view of the pathogenesis of ITP. .......................................................... 28
Figure 1.4 Figure 1.4: Megakaryocytes from healthy individuals and patients with ITP. . ........ 35
Figure 1.5 Development of anti-platelet adaptive immune response. ......................................... 46
Figure 2.1 Platelet counts induced by the indicated anti-platelet antibodies and IVIg effects. ... 85
Figure 2.2 MWReg30 significantly increased the megakaryocyte counts. ................................. 86
Figure 2.3 Megakaryocyte morphology induced by MReg30 and IVIg effects. . ....................... 87
Figure 3.1 Protocol for the development of ITP. ......................................................................... 99
Figure 3.2 Generation of anti-platelet IgG antibodies in CD61 KO mice. ............................... 102
Figure 3.3 Allogeneic platelet MHC I antigens inhibit T cell-mediated ITP. .......................... 104
Figure 3.4 Allogeneic platelet transfusions inhibit T cell-mediated ITP. .................................. 106
Figure 3.5 Allogeneic platelet MHC I antigens rescue megakaryocyte abnormalities in T cell-
mediated ITP. ............................................................................................................................ 108
Figure 3.6 Allogeneic platelet MHC I antigens normalize the number of megakaryocytes in T
cell-mediated ITP. ...................................................................................................................... 109
Figure 3.7 Effect of allogeneic platelet MHC I antigens on in vitro cytotoxicity in antibody- and
T cell-mediated ITP. .................................................................................................................. 110
Figure 4.1 Overview of the experimental design. ...................................................................... 120
xi
Figure 4.2 Anti-CD20 antibody efficiently depletes B cells in vivo. ....................................... 126
Figure 4.3 B cell depletion therapy increases both CD4+ and CD8
+ T cells in the spleen and
lymph nodes. . ............................................................................................................................ 128
Figure 4.4 The increases of FOXP3+ Tregs were proportional to the increase of CD4
+ and CD8
+
T cells in the spleen and lymph nodes. . .................................................................................... 129
Figure 4.5 B cell depletion inhibits CD8+ T cell proliferation in vitro. .................................... 130
Figure 4.6 B cell depletion therapy increases Granzyme B expression levels within CD3+CD8
+
T cells. ....................................................................................................................................... 132
Figure 4.7 In vivo B cell depletion results in a normalization of platelet counts in a murine
model of T cell-mediated ITP. .................................................................................................. 133
xii
LIST OF APPENDICES
Appendix I Definition and grading of bleeding manifestations ................................................ 211
xiii
LIST OF ABBREVIATIONS
ADP adenosine diphosphate
APC antigen presenting cell
ATP adenosine 5'-triphosphate
B10 cells CD5+CD1d
hi B cells
Bdep B cell depletion
BlyS B-lymphocyte stimulator
Breg regulatory B cell
CCL5, RANTES Chemokine (C-C motif) ligand 5, or released upon activation, normal T
cell expressed and secreted
CCR chemokine receptor
cDC conventional dendritic cell
CDR complementarity determining region
CFSE 5-(6)-carboxyflourescein diacetate succinimidyl ester
CLEC-2 C-type lectin domain family 2
CPDA citrate-phosphate-dextrose with adenine
CR complement receptor
CTL cytotoxic T lymphocyte
CVX convulxin
CXCL4 chemokine (C-X-C motif) ligand 4
DC dendritic cell
DCIR2, CLEC4A4 dendritic cell inhibitory receptor 2
DMS demarcation membrane system
EBV Epstein–Barr virus
xiv
ELISA enzyme-linked immunoabsorbent assay
EM Electron microscopy
ER endoplasmic reticulum
FNAIT, NAIT, NAITP
or NATP
fetal and neonatal alloimmune thrombocytopenia
GC germinal center
GI gastrointestinal
GITR glucocorticoid-induced tumor necrosis factor ligand
GM-CSF granulocyte-macrophage–colony-stimulating factor
GP glycoprotein
GPCR G protein-coupled receptors
H&E hematoxylin and eosin
HCV hepatitis C virus
HIV human immunodeficiency virus
HLA human leukocyte antigen
HPA human platelet antigen
HPV human herpes virus
IBLS ITP Bleeding Scale
ICH intracranial hemorrhage
IDO1 indoleamine 2,3-dioxygenase 1
IFN interferon
Ig immunoglobulin
IL interleukin
IPF immature platelet fraction
xv
ITP immune thrombocytopenia
ITP-BAT ITP specific Bleeding Assessment Tool
IVIg intravenous immunoglobulin
IWG international working group
JAK Janus kinase
KO knockout
LPS lipopolysaccharide
MCP monocyte chemoattractant protein
mDC migratory dendritic cell or myeloid dendritic cell
MHC major histocompatibility complex
MIP macrophage inflammatory protein
MMR macrophage mannose receptor
MZ marginal zone
NAP-2 neutrophil-activating peptide-2
ND non-depleted
NET neutrophil extracellular trap
OCS open canalicular system
PAR protease-activated receptor
PAT passive alloimmune thrombocytopenia
PBP platelet basic protein
PBS phosphate buffered saline
pDC plasmacytoid dendritic cell
PF-4 platelet factor-4
PSGL-1 P-selectin glycoprotein ligand-1
xvi
PTP post-transfusion purpura
RBC red blood cell
RIG-I retinoic acid-inducible gene-I
RLR retinoic acid-inducible gene-I (RIG-I)-like receptor
ROS reactive oxygen species
SCID severe combined immunodeficient
SIGN intercellular adhesion molecule-3-grabbing nonintegrin
SIRPα signal regulatory protein α
STAT signal transducers and activators of transcription
T2-MZP transitional 2 marginal-zone precursor
TCR T cell receptor
TFH T follicular helper cell
Th T helper
TLR toll-like receptor
TNF tumor necrosis factor
TPO thrombopoietin
TRAP thrombin receptor activator peptide
Treg regulatory T cells
TxA2 thromboxane A2
vWF von Willebrand factor
WHO the world health organization
WT wildtype
1
Chapter 1
Introduction
2
1.1 Preamble
This thesis focuses on the immune mechanisms mediating immune thrombocytopenia
(ITP) and how therapies affect these responses.
ITP is defined as an isolated thrombocytopenia without other disorders that may cause
thrombocytopenia. It is a consequence of increased platelet destruction in the periphery and/or
impaired platelet production by megakaryocytes in the bone marrow. In Chapter One, I will first
introduce a brief review on megakaryocyte and platelet biology and then provide an extensive
review on ITP, including various immunological reasons that contribute to the platelet or
megakaryocyte abnormalities in ITP. At the end of the introduction, I will introduce the aims of
my study and hypotheses.
The original research work in the thesis will be introduced in Chapters Two to Four in a
paper-based format. The work includes our studies on antibody- and T cell-mediated ITP and
the effect of different treatments on the pathogenesis of the disease in two mouse models of ITP,
i.e. the passive ITP and active ITP mouse models.
In Chapter Two, we examined antibody-mediated ITP in a passive mouse model with or
without IVIg therapy. In particular, we were interested in how the anti-platelet antibodies
affected the numbers and morphology of megakaryocytes in vivo without interference from T
cell-mediated megakaryocyte destruction as this has not been well characterized. We also
evaluated the effect of intravenous immunoglobulin (IVIg) therapy on platelet counts and the
megakaryocytes in these mice.
Chapter Three focuses on T cell-mediated ITP without B cells, and the effectiveness of
allogeneic platelet transfusions in alleviating the thrombocytopenia. While allogeneic platelet
transfusions have been widely used clinically for decades in many other thrombocytopenic
conditions, they are generally limited for use in patients with ITP. However, clinical evidence
began to emerge that contradicted the “limited use” dogma showing the effectiveness of platelet
3
transfusions for the management of ITP. Moreover, whether there is any immunomodulatory
effect of allogeneic platelet transfusions on the pathogenesis of ITP is unclear.
Next, we were interested in the interactions between B cells and T cells in ITP
pathogenesis. One way to study this is by depleting B cells and monitoring the changes in T
cell-mediated ITP. B cell depletion in vivo is a commonly used therapy for ITP and was initially
thought to inhibit antibody-mediated ITP, however, it was found to have antibody-independent
mechanisms as well. Therefore in Chapter Four, we examined the effect of B cell depletion
therapy on T cell-mediated ITP in our active ITP mouse model.
Chapter Five will briefly summarize the key findings of the thesis, the limitations of the
current studies and directions for future studies.
4
1.2 Megakaryocyte and Platelet Biology
1.2.1 Megakaryocyte development, differentiation, and platelet production
Mammalian megakaryocytes are a population of specialized bone marrow cells whose
primary physiological role is to produce and release platelets into the peripheral circulation
(Michelson, 2013). In both humans and mice, megakaryocytes initially develop from
hematopoietic stem cells in the fetal yolk sac and shortly after, in the fetal liver and then later,
mainly in the bone marrow after birth (Long et al., 1982; Machlus et al., 2014; Ogawa, 1993).
Early megakaryocyte progenitors can be identified by surface markers such as CD34 and CD41,
and transcription factors such as Flt3 (Battinelli et al., 2007). During their differentiation, they
become lineage restricted and become promegakaryoblasts after they begin to express CD61
(also known as integrin β3 chain, or glycoprotein (GP)IIIa) and elevated CD41 (integrin αIIb
chain, or GPIIb) molecules (Michelson, 2013).
Based on their morphological characteristics, megakaryocyte development can be
divided into 3 different stages: megakaryoblasts, promegakaryocytes, and mature
megakaryocytes (Dameshek and Miller, 1946; Long and Williams, 1981; Michelson, 2013). The
megakaryoblasts are about 10-30μm in diameter with an irregular agranular nucleus (sometimes
kidney-shaped) and a basophilic cytoplasm. The promegakaryocytes are approximately 15-80
μm in diameter with a more condensed nucleus, reduced nucleus/cytoplasm ratio and scant-to-
moderate amounts of granules. The mature megakaryocytes on the other hand, may have a
variable size from 18 to >100 μm in diameter with a condensed multilobed nucleus and a very
low nucleus/cytoplasm ratio with plenty amounts of granules in the cytoplasm. Figure 1.1
highlights the major stages of megakaryocyte development (Ebbe, 1976; Machlus et al., 2014;
Michelson, 2013).
There are several unique characteristics that occur during megakaryocyte development
which are necessary for platelet production and eventual hemostasis. These include endomitosis
and polyploidy, development of a demarcation membrane system (DMS), granule
5
Figure 1.1: Major stages of megakaryocyte development. Modified from Michelson, A.D.
(2013). Platelets (London ; Waltham, MA: Academic Press), Figure 2-1. (Michelson, 2013).
In the bone, a common myeloid progenitor cell commits to megakaryocytic lineage,
with the expression of lineage marker CD41 (integrin αIIb, GPIIb) on the cell surface.
Another important lineage marker is CD61 (integrin β3, GPIIIa). The
promegakaryoblast is the first morphologically recognizable megakaryocyte precursor
in bone marrow, which then become a megakaryoblast, and eventually mature as a
megakaryocyte. The megakaryocyte releases pro-platelets into circulation which then
become platelets. Thrombopoietin (TPO) is the principle regulator during the
megakaryocyte development.
6
formation/accumulation and proplatelet formation. The ploidy increase in megakaryocytes is
thought to be essential for the significant gene transcription events required to make proteins for
the approximately 1,000 platelets made from each megakaryocyte (Trowbridge et al., 1984).
The mechanism of this process is not well understood but there are a few clues that have
emerged and suggest both external growth factors and specific genetic processes are required.
For example, it was found that endomitosis was critically dependent on a megakaryocyte growth
factor, thrombopoietin (TPO), because defective signaling through its receptor, Mpl, leads to
decreased megakaryocyte proliferation and increased circulating platelet volumes (Gurney et al.,
1994). Actually, TPO is the most important growth factor during the whole process of
megakaryocyte maturation, proplatelet formation and platelet production. Through binding to its
receptor Mpl, TPO activates the tyrosine kinase Janus kinase 2 (JAK2) and triggers the
activation of the JAK/STAT signaling pathway (STAT for ‘signal transducers and activators of
transcription’) which further promotes megakaryocyte proliferation (Pallard et al., 1995; Sattler
et al., 1995). In addition to TPO, IL-11 also stimulates the proliferation of megakaryocytes and
ploidy maturation, synergistic to IL-3 (Kobayashi et al., 1993; Teramura et al., 1992). More
recently, Kahr et al reported that mutations in both the human and mouse NBEAL2 gene induce
decreased ploidy of megakaryocytes and developmental/structural defects such as a lack of
alpha granules (Kahr et al., 2011; Kahr et al., 2013). Deppermann et al found similar alpha
granule defects in Nbeal2-/- mice, but they did not observe megakaryocyte differentiation
defects (Deppermann et al., 2013). In other studies, Mazharian et al showed that mutations in
the SH2 domain-containing protein-tyrosine phosphatases, Shp1 and Shp2 were associated with
arrested ploidy and decreased numbers of megakaryocytes together with a
macrothrombocytopenia in vivo (Mazharian et al., 2013). These studies have contributed to our
understanding of how megakaryocytes increase their ploidy.
The DMS system is an elaborate invaginated membrane system derived from the
megakaryocyte plasma membrane and contacts both the extracellular environment and the
megakaryocyte cytoplasm. It serves as a reservoir for extra membrane surface to generate
proplatelets (Battinelli et al., 2007; Behnke, 1968, 1969; Nakao and Angrist, 1968; Radley and
7
Haller, 1982; Schulze et al., 2006). Schulze et al observed that megakaryocyte DMS
invagination relies on actin polymerization (Schulze et al., 2006). A recent study by Eckly et al
identified a pre-DMS structure and proposed DMS biogenesis in four steps: ‘(1) focal
membrane assembly at the cell periphery; (2) plasma membrane invagination and formation of
a perinuclear pre-DMS; (3) expansion through membrane delivery from Golgi complexes; and
(4) ER-mediated lipid transfer.’ (Eckly et al., 2014)
During megakaryocyte maturation, another characteristic is the progressive formation
and appearance of secretory granules, which are ultimately delivered to and stored in platelets.
These granules are essential for platelet activation and function and there are several different
types of granules in megakaryocyte and platelets, including α granules, dense granules, and
lysosomes (Machlus et al., 2014). α -granules are the most abundant, usually 200-500nm in
diameter containing hundreds of different proteins that are acquired through both protein
synthesis and endocytosis/pinocytosis from the extracellular environment (Blair and
Flaumenhaft, 2009; Handagama et al., 1987). Endogenously synthesized proteins include, for
example, platelet factor 4, β-thromboglobulin, and von Willebrand factor (vWF). Exogenous
proteins captured by the megakaryocytes and platelets include fibrinogen, albumin and factor V
etc (Blair and Flaumenhaft, 2009; de Larouziere et al., 1998). Another type of secretory
granules called dense granules are approximately 250 nm in diameter and contain, for example,
serotonin, a non-metabolic pool of adenine nucleotides [adenosine 5'-diphosphate (ADP) and
adenosine 5'-triphosphate (ATP)], high concentrations of calcium, and other bioactive
substances that are released upon platelet activation (Youssefian and Cramer, 2000).
During the final stages of megakaryocyte maturation, the cytoplasm begins to form
proplatelets, which are released into the bloodstream and eventually become circulating
platelets. The development of proplatelets from megakaryocytes has been visualized in vitro and
in vivo (Behnke, 1969; Italiano et al., 1999; Junt et al., 2007; Patel et al., 2005). Proplatelet
production begins with the extension of pseudopodia from the plasma membrane with
subsequent elongation to yield thick proplatelet branches and eventually bifurcating the
proplatelet ends to form thinner proplatelet shafts (Italiano et al., 1999). This process occurs
8
repetitively until the entire cytoplasm of megakaryocyte is consumed (Italiano et al., 1999; Patel
et al., 2005). The proplatelet elongation is driven by microtubules at a speed of approximately
1μm/min and bifurcation is driven by actin polymerization (Hartwig and Italiano, 2006; Italiano
et al., 1999; Tablin et al., 1990). Microtubule arrays extend throughout the entire membrane
system and use a dynein-powered motor to slide bidirectionally to transport protein cargo from
the megakaryocyte cytoplasm into the developing proplatelets (Hartwig and Italiano, 2006;
Patel et al., 2005). The microtubules also function as scaffolds to move mitochondria and
granules from the megakaryocytes to the developing proplatelets (Richardson et al., 2005).
Furthermore, the extracellular matrix has also been shown to be involved in megakaryocyte
maturation and proplatelet production. Matrix proteins such as vitronectin, collagen, and
fibrinogen have all been shown to regulate proplatelet formation, possibly through interaction
with integrins and adhesion receptors on the megakaryocytes surface which provide stimulatory
signals for protein synthesis and skeletal remodeling (Balduini et al., 2008; Kunert et al., 2009;
Larson and Watson, 2006; Sabri et al., 2004; Thon and Italiano, 2010). During proplatelet
formation, megakaryocytes migrate from the bone marrow niches to the extravascular area and
release proplatelets directly into the circulation (Junt et al., 2007). Ultimately, the released
proplatelets divide and become platelets that are then available to maintain hemostasis (Figure
1.1) (Michelson, 2013).
1.2.2 Platelets in hemostasis and thrombosis
Platelets were first described independently by Gulliver and Addison and Donné in the
1840s. Subsequently, detailed drawings of platelets by Osler in the 1870s distinguished them
from leukocytes and red blood cells (RBCs) (Robb-Smith, 1967). In 1882, Bizzozero observed
platelets microscopically in the circulating blood of living animals and showed that they were
the first component of the blood to adhere to damaged blood vessel walls (Bizzozero, 1881;
Blanchette and Freedman, 1998). In 1910, James Wright coined the term ‘platelets’ which has
now been universally accepted (Brown, 1913).
Platelets are anucleated cells with a biconvex discoid structure and are the smallest
circulating cell type in the blood, with a size of approximately 2-4μm in diameter in humans and
9
1-3μm in mouse (Michelson, 2013; Thon and Italiano, 2010; Trowbridge et al., 1984). The
normal platelet count in human peripheral blood is between 150 - 450×109/L and their life span
is approximately 10 days (Harker and Finch, 1969; Michelson, 2013; Nugent et al., 2009)
whereas in mice, the normal platelet count (depending on the strain) is between 600 -
1600×109/L with a life span of approximately 3-5 days (Michelson, 2013).
The primary physiological function of platelets is the maintenance of hemostasis. Upon
vascular damage, platelets are rapidly activated and recruited to the site of injury and eventually
form a platelet plug to stop bleeding and infectious microbe dissemination. Our understanding
of the biological and molecular mechanisms has been largely improved in the last few decades.
Platelets have unique structures that are closely related to their hemostatic functions. For
example, their membranes (like the DMS in megakaryocytes) are invaginated, providing
additional membrane area during activation. These invaginations form the open canalicular
system (OCS, or ‘surface-connected canalicular system’) which communicates with the
extracellular environment and can allow extracellular proteins to be readily taken up by platelets
(Michelson, 2013; White and Escolar, 1993). Platelets also have a dense tubular system which is
the site of calcium storage and contain α and dense granules which are derived from
megakaryocytes and can be secreted upon platelet activation. (Rodan and Feinstein, 1976).
Probably the most important feature of platelets and their ability to mediate hemostasis is the
expression of a variety of surface receptors including (1) integrin receptors αIIbβ3 (also known
as GPIIbIIIa, or the CD41/CD61 complex) and α2β1 (GPIaIIa), (2) the leucine-rich repeat
family receptor GPIb-IX-V, (3) the G protein-coupled receptors (GPCRs) including protease
activation receptor 1 (PAR1) and PAR4 and the ADP receptors, P2Y1 and P2Y12, (4)
immunoglobulin receptor GPVI, and (5) the tyrosine kinase receptor Mpl, etc. Upon activation,
platelets also express additional receptors which further promote platelet function. For example,
the C-type lectin receptor P-selectin is expressed on activated platelets and facilitates platelet
adhesion.
10
Of these receptors, αIIbβ3 is unique to platelets and critical for platelet activation and
aggregation. It is the most abundant glycoprotein on the platelet plasma membrane, with
approximately 80,000 copies per platelet. αIIbβ3 can bind multiple ligands, such as fibrinogen,
vWF, fibronectin, vitronectin etc. When platelets are activated, the αIIbβ3 transforms from a
low affinity resting state into a high affinity state and this conformational change promotes its
binding to its ligand, such as vWF and fibrinogen; a process known as ‘inside-out signaling’
(Plow and Ma, 2007).
Upon vascular injury, circulating platelets become exposed to the vascular subendothelial
matrix, and undergo adhesion, activation, aggregation and eventually form a plug that physically
stops the flow of blood out of the vessel. Initially, vascular injury exposes collagen which can
bind to platelet receptors GPIaIIa and GPVI as well as vWF and the immobilized vWF is
recognized by platelet GPIb-IX-V, initiating platelet adhesion. Platelet adhesion is followed by
platelet activation which is characterized by a series of changes in platelets including
thromboxane A2 (TxA2) synthesis, activation of αIIbβ3 (inside-out signaling), platelet cytosol
calcium elevation, increased surface expression of multiple receptors as well as platelet shape
change (platelet spreading) and granule secretion. The activated αIIbβ3 molecules can bind to
vWF and fibrinogen with high affinity and this can then bridge activated αIIbβ3 receptors on
different platelets resulting in platelet aggregation. In addition, elevated calcium elevation also
induces platelet shape change and phosphatidylserine (PS) exposure on platelet surface which
provides a procoagulant surface and promotes thrombin generation. Concurrently, granule
secretion releases bioactive substances including vWF, fibrinogen, ADP and these released
soluble platelet agonists further amplify platelet activation and aggregation through interaction
with their receptors e.g. between ADP and P2Y1 and P2Y12 receptors, thrombin and PAR1 and
PAR4, TxA2 and GPCR (Furie and Furie, 2008; Li et al., 2010).
Platelet activation and aggregation is also amplified by the activation of the coagulation
cascade. This cascade is activated when tissue factor molecules are exposed on injured
endothelial or subendothelial cells and when exposed to the circulation, it leads to the
production of thrombin and the formation of fibrin. Thrombin could further promote αIIbβ3
11
activation and platelet aggregation. Fibrin polymerizes into long threads and forms a network
that further traps platelets. Eventually with the accumulation of platelet aggregation and fibrin
deposition, the formed platelet plug is stabilized which arrests blood loss from the injured site
and this can subsequently recruit leukocytes for healing (Furie and Furie, 2008; Li et al., 2010).
1.2.3 The immunomodulatory role of platelets
Although hemostasis is the primary role for platelets, it has become clear that they are also
immune-like cells with innate immune functions and with the capability to interact with
adaptive immune cells. Platelets are involved in many infectious diseases and have the ability to
recognize and bind to viruses, bacteria, and parasites (Assinger, 2014; Kapur et al., 2015b;
Yeaman, 2010). To list just a few of the organisms that are able to directly interact with platelets
are the human immunodeficiency virus (HIV), hepatitis C virus (HCV), Epstein–Barr virus
(EBV), Staphylococcus aureus, Streptococcus sanguis and malarial parasites (Assinger, 2014;
Yeaman, 2010).
Platelets can directly recognize pathogens through their extensive repertoire of surface
receptors. For example, Hantavirus and adenoviruses as well as bacteria S. aureus have been
shown interact with platelets via GPIIbIIIa (Cheung et al., 1991; Mackow and Gavrilovskaya,
2001; Niemann et al., 2004; Yeaman, 2010). Platelets can also interact with some viruses and
bacteria through their GPIaIIa and GPVI receptors (Assinger, 2014; Coulson et al., 1997;
Flaujac et al., 2010; Zahn et al., 2006). Besides their various receptors that are important in
hemostasis, platelets also express toll-like receptors (TLRs) and lectin receptors that are
specialized for infectious antigen detection. TLRs are a family of proteins that have protective
properties against microbial infections by recognizing structurally conserved molecules of
microbes and activating the host immune system (McKenzie et al., 2013). It has been shown
that human platelets express all TLRs 1-9 (Kapur et al., 2015b) but whether they express TLR10
has not been reported. Among those receptors, TLR4 is the most studied and has been shown to
be functional on platelets (Andonegui et al., 2005; Aslam et al., 2006). The activation of
platelets through TLR4 is triggered by the binding of TLR4 to lipopolysaccharides (LPS), a core
12
antigen in the outer membrane of Gram-negative bacteria (Andonegui et al., 2005). This process
can further promote platelet-neutrophil interaction and the subsequent formation of neutrophil
extracellular traps (NETs) (Andonegui et al., 2005; Clark et al., 2007). Lectin receptors such as
the C-type lectin domain family 2 (CLEC-2) on platelets have also been shown to be able to
bind HIV and dengue virus (Assinger, 2014; Chaipan et al., 2006). Moreover, platelets can
detect infectious agents through their complement (C1q) receptor and chemokine receptors
(CCR) CCR1, CCR3 and CCR4 etc (Assinger, 2014; Clemetson et al., 2000; Nguyen et al.,
2000; Othman et al., 2007; Scott et al., 1987; Yeaman, 2010). In addition to direct antigen
binding, human platelets also express FcγRII and FcεRI receptors through which they can detect
antigen-immunoglobulin complexes (Joseph et al., 1983; Rosenfeld et al., 1985; Yeaman, 2010).
All the receptors mentioned above could induce platelet activation and promote a series
of responses as will be introduced below. Perhaps the most common pathway of platelet
activation upon exposure of exogenous infections is through thrombin production and this is
because a broad range of microbial agents can trigger the release of tissue factor from injured
endothelial cells and subendothelial stroma. Nonetheless, the mechanisms of how platelet
activation in vivo is induced after exposure to infectious agents is probably multifactorial
(Smyth et al., 2009).
Upon activation, platelets can kill or help to kill pathogens in many ways. Firstly,
through degranulation, platelets can release a variety of microbialcidal proteins such as
kinocidins, platelet factor-4 (PF4/ CXCL4), β-defensins, all of which have anti-microbial
characteristics (Assinger, 2014; Auerbach et al., 2012; Mohan et al., 2010; Solomon Tsegaye et
al., 2013; Wilson et al., 2013; Yeaman, 2010). Secondly, through the upregulation of CD40L
(CD154) expression on platelets, the interaction between platelets and CD40 expressing
endothelial cells promotes several inflammatory reactions (Henn et al., 1998). Thirdly, activated
platelets express P-selectin which interacts with its counter-receptor P-selectin glycoprotein
ligand-1 (PSGL-1) on the surface of leukocytes (Polgar et al., 2005). Fourthly, activated
platelets, via chemokine secretion, can recruit dendritic cells (DCs) and monocytes and promote
their maturation (Hagihara et al., 2004; Langer et al., 2007; Metcalf Pate et al., 2013; Singh et
13
al., 2012). Fifthly, platelets can interact with neutrophils which not only promotes neutrophil
extracellular trap (NET) formation for trapping pathogens but also stimulates reactive oxygen
species (ROS) production and boosts phagocytosis by neutrophils (Elzey et al., 2005; Henn et
al., 1998; Yeaman, 2010).
Platelets also have other active immunomodulatory roles on the immune system. For
example, we previously showed improved skin graft survival after allogeneic platelet
transfusions (Aslam et al., 2008). In addition, part of my PhD work has investigated the
immunomodulatory role of allogeneic platelet transfusions in a murine model of immune
thrombocytopenia (ITP) (Guo et al., 2014).
1.2.4 Thrombocytopenia induced by immune responses against platelets
Platelets can be the target of pathological immune responses which lead to increased
platelet destruction and/or decreased platelet production. This eventually causes the
development of thrombocytopenia, which is usually defined as the peripheral platelet count
below 150 × 109/L in humans (Greer, 2014). Thrombocytopenia mediated by immunological
factors can be generally divided into two categories: autoimmune and alloimmune
thrombocytopenia. Thrombocytopenia caused by autoimmune mechanisms is induced by
abnormal immune responses against self-platelet antigens, including primary immune
thrombocytopenia (ITP) or drug/infection-induced secondary ITP. In contrast, alloimmune
thrombocytopenia refers to thrombocytopenia induced after allogeneic platelet transfusions
where antibodies develop against the transfused platelet alloantigens (Greer, 2014). Platelet-
specific alloimmune responses can also induce thrombocytopenia and are exclusively triggered
by allelic human platelet antigens (HPA) (Hayashi and Hirayama, 2015). The differences in
HPA antigens can induce alloimmune responses that cause fetal and neonatal alloimmune
thrombocytopenia (FNAIT), post-transfusion thrombocytopenia (PTP), and passive transfer of
platelet alloantibodies.
1.2.4.1 Fetal and neonatal alloimmune thrombocytopenia (FNAIT)
14
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is also known as neonatal
alloimmune thrombocytopenia (NAIT or NAITP or NATP). It is characterized by
thrombocytopenia in the fetus or newborn and develops when the mother is negative for a
certain HPA antigen but carries a fetus who inherits the HPA antigen from the father.
Subsequently, the HPA on fetal platelets can be recognized as foreign by the mother’s immune
system and the mother develops IgG anti-HPA antibodies that migrate across the placenta, bind
to the fetal platelets and/or megakaryocytes and induce fetal thrombocytopenia (Kamphuis et al.,
2014; Liu et al., 2015). The most common HPA epitopes on platelets are HPA-1a located on
GPIIIa, and HPA-5b on GPIa (Mueller-Eckhardt et al., 1989; Thienelt and Calverley, 2009; von
dem Borne et al., 1981). In Caucasians, 85% of FNAIT is induced by anti-HPA-1a
alloantibodies (Kamphuis et al., 2010) but although the mismatch of HPA between the mother
and the fetus is as high as 1/50, the incidence of severe FNAIT (platelet count <50×109/L) is
below 1/500 (Kamphuis et al., 2014; Thienelt and Calverley, 2009). FNAIT may develop during
the first pregnancy with a recurrence rate of 80% during subsequent pregnancies (Radder et al.,
2003). It is usually diagnosed after the presence of bleeding symptoms such as petechiae or after
detection of intracranial hemorrhage (ICH) by ultrasound examination in the newborns. ICH
may be life-threatening and happens in 10-20% of severe FNAIT cases (Kamphuis et al., 2014).
Intravenous immunoglobulin (IVIg) and platelet transfusions are the recommended treatment
for newborns with severe thrombocytopenia (Murphy and Bussel, 2007). Due to the high
recurrence rate, prophylactic antenatal treatment is provided for pregnant women with a history
of FNAIT, especially for mothers with previous pregnancies associated with ICH. High dose
(0.5-2g/Kg) IVIg combined with steroids is the most common treatment.
1.2.4.2 Post-transfusion purpura (PTP)
Post-transfusion purpura (PTP) is a rare adverse complication after red blood cell or platelet
transfusions which is mediated by alloantibodies against HPA antigens. It usually abruptly
develops 7-10 days after the transfusion and symptoms are very similar to those seen in patients
with ITP (Chapman et al., 1987; Shulman et al., 1961; Thienelt and Calverley, 2009; Vogelsang
et al., 1986). Patients may have widespread purpura and in some severe cases, life-threatening
15
bleeding symptoms such as ICH (Li et al., 2012a). PTP occurs most commonly in women who
have had history of multiple pregnancies or transfusions. It appears that patients who have
developed alloimmune responses against HPAs and re-exposed to the same antigens after
transfusion produce high titers anti-HPA antibodies. The most common antigens involved in
PTP are HPA-1a, HPA-1b, HPA-3a, HPA-3b, and HPA-5a (Hayashi and Hirayama, 2015;
Kickler et al., 1988; Simon et al., 1988). The pathophysiology of PTP is unclear but several
hypotheses have been proposed including (a) autologous platelets being destroyed because of
binding of immune complexes to their surface; (b) recipient platelets acquiring the phenotype of
the donor's platelets because they bind soluble antigens from the transfused blood product or (c)
exposure to foreign transfused platelets induces the formation of cross-reactive autoantibodies
against the recipient's platelets (Thienelt and Calverley, 2009). Although most cases of PTP are
self-limiting, therapeutic interventions may shorten the period of thrombocytopenia and include
IVIg, plasmapheresis, or steroids (Thienelt and Calverley, 2009).
1.2.4.3 Passive alloimmune thrombocytopenia (PAT)
Passive alloimmune thrombocytopenia (PAT) usually occurs within a few hours after a
blood transfusion and is mediated by alloantibodies contained in the plasma of blood donors
including alloantibodies against HPA-1a and HPA-5b (Santoso and Kiefel, 2001; Warkentin and
Smith, 1997; Warkentin et al., 1992; Webert et al., 2014). Patients usually recover after the
alloantibodies are cleared.
16
1.3 Immune thrombocytopenia (ITP)
Immune thrombocytopenia (ITP) is an autoimmune disorder and can be classified as either
primary or secondary ITP. As primary ITP is the main focus of this thesis, from now on the
abbreviation ITP will stand for primary chronic immune thrombocytopenia unless specified.
1.3.1 Brief history of ITP
The history of ITP has been investigated and comprehensively reviewed (Blanchette and
Freedman, 1998; Freedman and Blanchette, 1998). The acronym “ITP” was historically defined
as “idiopathic thrombocytopenic purpura” but the disorder has had several terms associated with
it including “autoimmune thrombocytopenic purpura”, “immune thrombocytopenic purpura”
and “autoimmune thrombocytopenia”. Recently, because of the improved understanding of the
immunological pathogenesis and diagnosis of ITP, the disease’s nomenclature has been
standardized and is now termed “immune thrombocytopenia” (Rodeghiero et al., 2009).
The word “purpura” was derived from the Greek word porphyra (Πρϕυρα) which is a
name of a purple fish from which a purple dye was obtained (Freedman and Blanchette, 1998).
Purpura has been used in medicine to describe a clinical symptom characterized with “red
spots” or “red eminences” during, for example, pestilential fevers in the Greco-Roman period
and was coined by the ancient physicians Hippocrates and Galen. In the 10th
century, purpura in
its chronic form was described by an Arab physician Avicenna (Blanchette and Freedman,
1998; Jones and Tocantins, 1933). In 1580, purpura without fever was described by Amatus
Lusitanus in his “Curatonum Medicinalium centuriae septem” (Blanchette and Freedman, 1998;
Lusitanus, 1965) and in 1658, Lazarus de la Rivière (Riverius), physician to the King of France,
described purpura as “purple spots like Flea-bitings, called....Peliculae or Pelechiae ... ''
(Blanchette and Freedman, 1998; Riverius, 1668).
Paul Gottleib Werlhof is credited with probably the first description of ITP in 1735
(Morbus Maculosus Haemorrhagicus). Subsequently, ITP was also known as Werlhof’s
Disease. “In his Opera Omnia (printed posthumously in 1775) he described `an adult girl,
17
robust, without manifest cause,' who bled from her nose and mouth and vomited `very thick,
extremely black blood. Immediately there appeared about the neck & on the arms, spots partly
black, partly violaceous or purple...'. ” (Blanchette and Freedman, 1998; Werlhof, 1965). In
1802, Robert Willan, a London physician distinguished four types of purpura in his book ``On
Cutaneous Diseases'' (Blanchette and Freedman, 1998; Willan, 1808).
Although platelets were discovered in the 1840s by Gulliver, Addison and Donné, the
relationship between platelets and purpura was not discovered until 1883 by Krauss in Germany
and 1887 by Denys in Louvain (Blanchette and Freedman, 1998; Denys, 1887; Krauss, 1883;
Robb-Smith, 1967). In 1889, Georges Hayem in Paris documented the first actual platelet count
in a patient with purpura (Blanchette and Freedman, 1998; Denys, 1887; Krauss, 1883). In 1915,
Frank in Poland described normal numbers of megakaryocytes in ITP (Blanchette and
Freedman, 1998; Frank, 1915) and a year later, George Minot in the USA studied a case of ITP
in detail (Blanchette and Freedman, 1998; Minot, 1916). In the same year, Kaznelson in Prague
described the first case of splenectomy as a successful therapy in a patient with ITP and since
then, splenectomy has become an important treatment (Blanchette and Freedman, 1998;
Kaznelson, 1916). The success of splenectomy in ITP treatment also provided solid evidence to
support the theory that the development of ITP is due to platelet destruction in the spleen.
During that time, however, an alternative theory supported by Frank et al was that ITP was
related to decreased platelet production (Blanchette and Freedman, 1998). Now it has been
recognized that both theories are correct in patients with ITP.
In the second half of the twentieth century, the understanding of the pathogenesis of ITP
as well as the therapeutic management of the disease was greatly improved. For example, 1951
was an important year in the history of ITP. Harrington et al showed for the first time that a
factor in the plasma of patients with ITP was responsible for inducing thrombocytopenia;
transferring plasma from patients with ITP into healthy volunteers including himself induced
significant thrombocytopenia in most recipients (Harrington et al., 1951). This classic
experiment provided substantial evidence that there is a factor in the plasma that could directly
induce thrombocytopenia. In the same year, Hirsch and Dameshek differentiated the acute and
18
Table 1.1 Proposed definitions of ITP. Modified from Rodeghiero, F., Stasi, R., Gernsheimer,
T., Michel, M., Provan, D., Arnold, D.M., Bussel, J.B., Cines, D.B., Chong, B.H., Cooper, N., et
al. (2009). Standardization of terminology, definitions and outcome criteria in immune
thrombocytopenic purpura of adults and children: report from an international working group.
Blood 113, 2386-2393. (Rodeghiero et al., 2009).
Primary ITP Primary ITP is an autoimmune disorder characterized by
isolated thrombocytopenia (peripheral blood platelet count
<100 × 109/L) in the absence of other causes or disorders that
may be associated with thrombocytopenia.
The diagnosis of primary ITP remains one of exclusion; no
robust clinical or laboratory parameters are currently
available to establish its diagnosis with accuracy.
The main clinical problem of primary ITP is an increased
risk of bleeding, although bleeding symptoms may not
always be present.
Secondary ITP
All forms of immune-mediated
Thrombocytopenia except primary ITP
Phases of the disease Newly diagnosed ITP: within 3 months from diagnosis
Persistent ITP: between 3 to 12 months from diagnosis.
Includes patients not reaching spontaneous remission or
not maintaining complete response off therapy.
19
Chronic ITP: lasting for more than 12 months
Severe ITP: Presence of bleeding symptoms at
presentation sufficient to mandate treatment, or
occurrence of new bleeding symptoms requiring
additional therapeutic intervention with a different
platelet-enhancing agent or an increased dose.
20
chronic forms of ITP (Hirsch and Dameshek, 1951). Moreover, the first application of
corticosteroids for the treatment of ITP was also performed by Wintrobe et al and
corticosteroids have now become the major first-line therapy for ITP (Blanchette and Freedman,
1998; Wintrobe et al., 1951). In 1965, Shulman et al identified the plasma factor belonging to
the IgG fraction of plasma and in the mid 1970’s, Dixon and Cines demonstrated that platelet-
associated IgG could be quantified and verified that the plasma factor was actually anti-platelet
antibodies (Blanchette and Freedman, 1998; Cines and Schreiber, 1979; Dixon et al., 1975;
Shulman et al., 1965). In 1981, Imbach reported the first application of IVIg for the treatment of
ITP (Imbach et al., 1981) and in 1991 Semple et al found increased platelet-reactive IL-2
secreting CD4+ T helper (Th) cells in patients with ITP which provided probably the first
evidence of abnormal T cell reactivity in the development of ITP (Semple and Freedman,
1991). It has now been widely accepted that the etiology of ITP includes both humoral and
cellular immune responses and in the last two decades, a considerable amount of immune
investigations have been published on ITP.
1.3.2 Current definition and diagnosis of ITP
It is now well understood that ITP is an autoimmune disease of children and adults,
characterized with an insufficient number of platelets in the circulation due to decreased platelet
production in the bone marrow and/or increased peripheral platelet destruction in the spleen.
According to the International Working Group (IWG) report, primary ITP is now defined as “an
autoimmune disorder characterized by an isolated thrombocytopenia (peripheral blood platelet
count <100 × 109/L) in the absence of other causes or disorders that may be associated with
thrombocytopenia. The diagnosis of primary ITP remains one of exclusion; no robust clinical or
laboratory parameters are currently available to establish its diagnosis with accuracy. The
main clinical problem of primary ITP is an increased risk of bleeding, although bleeding
symptoms may not always be present” (Rodeghiero et al., 2009). Secondary ITP refers to
immune-related thrombocytopenia secondary to other etiologies, such as those caused by drugs,
21
infections or other diseases e.g. Lupus. The proposed definition of ITP as well as phases of ITP
are summarized in Table 1.1.
In the IWG report, the phases of ITP have also been significantly altered and are no longer
simply divided into acute or chronic, but instead are now divided into 3 categories: newly
diagnosed (from diagnosis to 3 months), persistent (3-12 months after diagnosis) and chronic
(more than 12 months after diagnosis)(Rodeghiero et al., 2009). An advantage of the new
diagnostic criteria is avoiding the vagueness of ITP at diagnosis, at which time point patients
may have developed the disease recently or alternatively may have had the disease
asymptomatically for a long time. Persistent ITP includes patients who did not achieve
spontaneous remission or no response after initial treatment but within a year from initial
diagnosis, they were able to eventually maintain a response after treatment. Patients who remain
thrombocytopenic after 12 months from diagnosis are categorized as chronic ITP. Patients with
newly diagnosed and persistent ITP have significantly higher remission rates than patients with
the chronic form of ITP (Imbach et al., 2006; Rodeghiero et al., 2009; Sailer et al., 2006; Stasi et
al., 1995). Therefore more aggressive treatment was recommended for patients with the chronic
form of the disorder (Rodeghiero et al., 2009).
Incidence and prevalence of ITP
The incidence of ITP varies according to age, gender and geographical areas. The
incidence of childhood ITP is approximately 4 -5/100,000 person-year in Europe and 8/100,000
in North America, with a peak incidence at the age of 1-5 years (median 4 years) with a male
dominated pattern (Hedman et al., 1997; Kuhne et al., 2001; Segal and Powe, 2006; Terrell et
al., 2012; Yong et al., 2010; Zeller et al., 2005). The prevalence of ITP in children is about
4.6/100,000 children in Europe and 7.2/100,000 in North America (Hedman et al., 1997; Segal
and Powe, 2006). The incidence of childhood ITP is also influenced by the season and
prevalence of infections, showing a peak of new diagnoses in winter, especially in February, and
platelet count nadirs in autumn (Kuhne et al., 2001; Moulis et al., 2014; Zeller et al., 2005). In
adults, the overall incidence of ITP is about 2.9/100,000 in European countries and about
22
Figure 1.2: Petechiae in the skin of patients with ITP. Adopted from Greer, J.P. (2014),
Wintrobe's clinical hematology (Philadelphia: Wolters Kluwer, Lippincott Williams & Wilkins
Health), Figures 45.1 and 47.4. (Greer, 2014).
23
10-12/100,000 person-year in North America (Koylu et al., 2015; Moulis et al., 2014; Segal and
Powe, 2006; Terrell et al., 2012). The prevalence of adult ITP is approximately 35-50/100,000
in Europe and about 9-10/100,000 in North America but the latter prevalence may be
underestimated due to the limitation of local registries (Koylu et al., 2015; Moulis et al., 2014;
Segal and Powe, 2006; Terrell et al., 2012). In adult ITP, the incidence is higher in woman than
men in patients below age 64, and vice versa in patients of age 65 and older (Bennett et al.,
2011; Terrell et al., 2012). The overall incidence and prevalence of ITP is also increased after
65, at about 90/100,000 in the UK and 20/100,000 in North America (Bennett et al., 2011;
Terrell et al., 2012). A limitation to these statistics is the lack of consistency of clinical criteria
among the reported investigations. Although the standard for the definition and diagnostic
criteria has been recommended by the IWG in 2009, the published data that adhere to the IWG
are still limited (Rodeghiero et al., 2009). In children, the onset of ITP is usually preceded by an
infectious history within 3 weeks and infections such as Varicella zoster virus or Epstein-Barr
virus infection tend to be the most common (Kuhne et al., 2003; Liel et al., 2014; Lusher and
Iyer, 1977; Lusher and Zuelzer, 1966; Provan et al., 2010). In addition, Measles-Mumps-
Rubella (MMR) vaccination may also lead to an increased risk of ITP (Miller et al., 2001;
Provan et al., 2010). In adults, the onset of ITP is usually insidious and an infectious history is
less common (den Ottolander et al., 1984; Jacobs et al., 1986; Liel et al., 2014; Pizzuto and
Ambriz, 1984; Stasi et al., 1995).
Clinical manifestations of ITP
Clinical manifestations of ITP are mainly hemorrhagic symptoms such as purpura,
bruising in the skin, oral cavity bleeding, gastrointestinal (GI) bleeding and/or rarely,
intracranial hemorrhage (ICH). The definition and category of bleeding manifestations was also
provided by the IWG recently and is listed in Appendix I (Rodeghiero et al., 2013). As
mentioned in Appendix I, petechiae are a typical symptom of ITP and refer to small dots in the
skin caused by intradermal hemorrhages. They are red when fresh and become purplish within a
few days as shown in Figure 1.2, adopted from Greer, J.P. (2014), Wintrobe's clinical
hematology (Greer, 2014). Ecchymoses (bruises) are another type of typical bleeding symptom
24
in the skin, larger than petechiae and are usually observed on the back and thighs. Other
bleeding symptoms include hematoma (localized swelling filled with blood), epistaxis (nasal
bleeding), menorrhagia (excessive amount of menstrual bleeding) hematochezia (stools with
blood) and ICH, etc.
In general the severity of bleeding correlates with the platelet counts (Bolton-Maggs and
Moon, 1997; Cines et al., 2009; Kuhne et al., 2001; Neunert et al., 2008; Page et al., 2007) but
in patients with platelet counts less than 20×109/L, there is no significant correlation. It has been
controversial whether the management of ITP should be based on absolute platelet count or
bleeding symptoms. Therefore, investigation and validation of bleeding assessment in ITP
would provide a useful guidance for the management of ITP.
With these considerations, efforts have been made since the 1990s to produce different
bleeding assessment tools (Bolton-Maggs et al., 2004; Bolton-Maggs and Moon, 1997;
Freedman, 2003). For example, in 1997, Bolton-Maggs categorized pediatric ITP patients
registered in the UK into four groups: “no symptoms; mild symptoms (bruising and petechiae,
occasional minor epistaxis, very little or no interference with daily living); moderate symptoms
(more severe skin manifestations with some mucosal lesions, and more troublesome epistaxis
and menorrhagia); and severe symptoms (bleeding episodes [epistaxis, melaena, and/or
menorrhagia] requiring hospital admission and/or blood transfusion— symptoms interfering
seriously with quality of life).” (Bolton-Maggs and Moon, 1997). Although these four categories
seem to correlate with platelet counts ranging from <10, 10-19, 20-50, 51-100 to >100 (×109/L),
most patients only had mild symptoms in all groups. Therefore this assessment tool was not
specific enough to indicate the severity of the disease or provide any prognostic clues and this
was confirmed by similar findings in the following decade (Kuhne et al., 2001; Neunert et al.,
2008). In 2002, Buchanan and Adix proposed another bleeding assessment criteria that includes
overall bleeding assessment with 6 grades from none to life-threatening as well as 5 grades of
epistaxis, oral bleeding and skin bleeding (Buchanan and Adix, 2002). However there was a
lack of assessment of the prognostic value in their studies. The world health organization (WHO)
also provided an evaluation tool for hemorrhage/bleeding in 2006, which was designed for
25
adverse event evaluation after chemotherapy in cancer patients (NCI [2006] Common
Terminology Criteria for Adverse Events [CTCAE] version 3.0. National Cancer Institute, U.S.
National Institutes of Health. Available at http://ctep.info.nih.gov.). This tool provided grads of
1 to 5 for hemorrhage, based not only on the manifestation but also on the medical intervention
and outcome; for example 5 refer to death. In 2007, Page et al proposed an advanced assessment
tool, the “ITP Bleeding Scale (IBLS)” (Page et al., 2007). In the IBLS, bleeding symptoms were
assessed at nine anatomical sites including the skin, oral, nasal, GI, urinary, gynecological,
pulmonary, intracranial and subconjuntival sites. Bleeding in each individual site could be
further graded from 0 to 2 based on the severity. Similar to Bolton-Maggs Assessment tool,
although the bleeding grade correlated with platelet counts in all patients, the IBLS score did not
correlate when platelet counts in patients were below 30×109/L (Bolton-Maggs and Moon,
1997; Page et al., 2007). Despite this, the IBLS provided a useful tool to capture more details in
bleeding manifestations as compared to previous assessment tools. More recently, the IWG
provided an ITP specific Bleeding Assessment Tool (ITP-BAT), which provided a systemic
evaluation including “skin (S), visible mucosae (M), and organs (O), with gradation of severity
(SMOG)” with clear definitions of the manifestations and descriptions of grading standards
(listed in Appendix I) (Rodeghiero et al., 2013). Under each of the “S” “M” and “G” categories,
the bleeding severity could be further graded from 0 to 4 and the highest grade would be taken if
there are more than one symptom within in the category (Rodeghiero et al., 2013). In this way,
the ITP-BAT not only allows physicians to distinguish bleeding in different sites to allow more
consistent evaluations but also aids in quicker decision making for ITP management. Although
ITP-BAT provides the most refined bleeding assessment standard to date, its application needs
to be validated and the relationship between the severity of bleeding symptoms and the outcome
of the patients with/without treatment need to be further investigated (Rodeghiero et al., 2013).
Despite the lack of standardization in bleeding assessments, it has been shown that ITP patients
with a bleeding history have a significantly higher risk of bleeding than those without
(Rodeghiero et al., 2013). In pediatric ITP, more than half of the patients would have
spontaneous remission without any intervention, although most patients only have mild skin
26
manifestations, whereas ICH is rare but has a mortality of 25% (Kuhne et al., 2001; Psaila et al.,
2009; Zeller et al., 2005).
Efforts have been made to look for prognostic markers to predict the bleeding risk in ITP.
Recently, there are a few studies on the immature platelet fraction (IPF) and function of platelets
in ITP. IPF is a technical description of a circulating platelet population characterized by a
larger flow cytometric forward light scatter which, in general, indicates a larger diameter of the
platelets (Briggs et al., 2004). In 2011, two groups independently reported increased IPF in the
peripheral blood of patients with ITP (Barsam et al., 2011; Psaila et al., 2009). The increase in
IPF percentage in ITP, as compared to control healthy volunteers, indicates more platelet
production and more immature platelets in the circulation and was found associated with less
clinically severe bleeding events (Barsam et al., 2011). Moreover, IPF was found significantly
increased in pediatric patients with chronic ITP compared with patients with acute ITP, which
may thus be an useful prognostic tool (Adly et al., 2015). In 2014, van Bladel et al found an
inverse correlation between bleeding events and platelet functions in pediatric chronic ITP (van
Bladel et al., 2014). They first used a microaggregation test to test the aggregation potential of
isolated platelets by flow cytometry (van Bladel et al., 2014). In addition, platelets were
stimulated with adenosine diphosphate (ADP), convulxin (CVX), thrombin receptor activator
peptide (TRAP), GPVI and proteinase-activated receptor 1 receptors respectively and their
activation level was measured using fluorescent-conjugated antibodies against P-selectin and
fibrinogen (van Bladel et al., 2014). Frelinger et al have recently confirmed that platelet function,
as indicated by TRAP-stimulated P-selectin and activated GPIIbIIIa and CD42b expression
levels, is correlated with the severity of bleeding in pediatric ITP (Frelinger et al., 2015).
Therefore, it appears that in the near future, prognostic markers/assays may be available for the
prediction of bleeding events and for the improvement of ITP management.
Laboratory tests for ITP
Current laboratory findings are still non-specific in ITP. Blood tests most often reveal an
isolated thrombocytopenia and sometimes show normocytic anemia proportional to blood loss.
27
White blood cell counts and differential counts are usually normal (Liel et al., 2014) and
although more than half of the patients with ITP have detectable antiplatelet antibodies in their
circulation, antiplatelet antibody tests are not commonly used clinically (Liel et al., 2014). This
is generally because there are several limitations to the antibody testing. First, the reliability of
the tests needs to be improved, as antiplatelet antibodies are found in some healthy individuals
(Chong and Ho, 2005; Liel et al., 2014). The presence of antiplatelet antibodies in healthy
individuals has also evoked further questions relating to the specificity of anti-platelet
antibodies and the value of the test itself (Arnold et al., 2015). In addition, there is lack of
evidence for the prognostic value of antiplatelet antibody tests and the tests are not readily
available in most hospitals and therefore their application for clinical diagnosis is limited. Bone
marrow examinations in ITP often reveal normal or an increased number of megakaryocytes,
with an increased proportion of immature megakaryocytes (Dameshek and Miller, 1946;
Houwerzijl et al., 2004).
ITP is a diagnosis of exclusion
As none of the symptoms or laboratory tests described above are specific enough and these
characteristics are often observed in secondary ITP and other diseases, the diagnosis of ITP still
remains one of exclusion (Provan et al., 2010). However, the history, physical examination,
complete blood count and peripheral blood film need to be considered for diagnosis.
1.3.3 Pathogenesis of ITP
ITP is an autoimmune disease initiated by a breakdown of immunological self-
tolerance (Cines et al., 2009). The consequence of the failure of self-tolerance results in
decreased platelet production from megakaryocytes in the bone marrow and increased
peripheral platelet destruction in the spleen (Cines et al., 2014; McKenzie et al., 2013).
Although the development of anti-platelet specific immune responses in ITP is a complicated
process that has not been completely understood, it has been shown to be related to but not
limited to: potential platelet and megakaryocyte defects, contributions from antigen presenting
cells (APCs), increases in autoreactive B and T cells, inefficient clearance of those autoreactive
28
Figure 1.3: A schematic view of the pathogenesis of ITP.
29
The intrinsic defects in megakaryocytes and platelets may contribute to the initial activation of the
immune system. Other unknown environmental factors or microbial infections may also activate the
megakaryocytes and/or platelets which then activate antigen presenting cells (APCs) to present platelet
antigens and to stimulate autoreactive B and T cell responses. The activated B cells mature into plasma B
cells and produce anti-platelet autoantibodies that could mediate platelet and megakaryocyte destruction
with the help of Th1, th17 and Th22 cells. These CD4+ T cells also promote the rise of platelet
autoreactive CD8+ T cells (CTLs) which can induce direct platelet and megakaryocyte lysis. Defects in
Bregs and Tregs as well as imbalances in cytokine production all contribute to the development of ITP.
30
immune cells, and dysregulation of cytokine production. A schematic view of the pathogenesis
of ITP is shown in Figure 1.3.
1.3.3.1 Overview of self-tolerance
Under physiological situations, the immune system protects our body from exogenous
infectious invasions, however does not attack self-antigens. This self-tolerance is maintained
both in the thymus and bone marrow during T and B cell development (usually referred to as
‘central tolerance’), as well as in the periphery (peripheral tolerance) (Paul, 2013).
Thymic tolerance of T cells
T cells are characterized by the expression of T cell receptors (TCRs) on their cell surface
which enables them to recognize virtually any peptide antigen presented by MHC molecules
(Chaplin, 2010). TCRs are in association with CD3 (altogether form the TCR complex) and
need support from their co-receptors: CD4 or CD8 (Huppa and Davis, 2013). The TCR
complexes together with CD4 recognize peptide: MHC class II complexes whereas TCR
together with CD8 recognize peptide: MHC class I complexes (Chaplin, 2010; Matsui et al.,
1986).
T cells originate from hematopoietic stem cells in the bone marrow and migrate into the
thymus for maturation (hence called “T” cells) (Boehm, 2008; Rothenberg and Champhekar,
2013). A key feature of T cells is the diversity of TCRs. TCRs genes are composed of multiple
V, D and J gene segments. In the thymus, these early pre-T cells undergo VDJ rearrangement in
their TCR beta genes leading to the functional expression of a rearranged β chain of the TCR on
their cell surface. Cells that fail to generate functional β chains cannot proliferate (β-selection)
and are eliminated whereas those T cells that successfully rearrange their β chains undergo
extensive proliferation, express both CD4 and CD8 molecules (called double positive T cells)
and eventually express a rearranged TCR- chain (Paul, 2013). After this, T cells express either
CD4 or CD8 exclusively (the CD4/CD8 lineage commitment) and eventually a small population
of the cells mature from the thymus as either CD4+ or CD8
+ naïve T cells (Shah and Zuniga-
Pflucker, 2014).
31
During the process of T cell development, the most important two mechanisms of self-
tolerance for T cells called ‘positive selection’ and ‘negative selection’ take place (Goodnow
and Ohashi, 2013; Shah and Zuniga-Pflucker, 2014). Positive selection happens in double
positive T cells that survived β-selection. It is a process in which only T cells that recognize
self-peptide: MHC complexes expressed on thymic epithelial cells (TECs) receive survival
signals from the TECs and proceed to further maturation. Those T cells that fail to recognize
self MHC undergo apoptosis (self MHC restriction). Positive selection ensures the TCR can
recognize either self MHC class I or class II molecules and enables the T cells distinguish self
from non-self. Another mechanism termed negative selection happens after CD4/CD8
commitment. It is a process of deleting autoreactive T cells with high affinities of TCR for self-
MHC (Laky and Fowlkes, 2007; Shah and Zuniga-Pflucker, 2014). Most of the highly
autoreactive T cells are eliminated and a small fraction become suppressive regulatory T cells
(Tregs) (Baldwin et al., 2005). Therefore negative selection is crucial to avoid autoimmune
diseases.
Regulatory T cells (Tregs) derived from the thymus are also important for self-tolerance, as
genetic Treg deficiencies lead to spontaneous development of autoimmune diseases (Brunkow
et al., 2001; Ochs et al., 2007; Sakaguchi et al., 2006). The majority of Tregs are CD4+FOXP3
+
T cells (FOXP3 = forkhead box P3) and usually express high levels of CD25 (Ramsdell and
Ziegler, 2014; Sakaguchi et al., 2010; Sakaguchi et al., 1995). A mutation in the Foxp3 gene in
the scurfy mouse was associated with a severe autoimmune phenotype (Brunkow et al., 2001;
Godfrey et al., 1991; Ramsdell and Ziegler, 2014). Consistently, in 2001, the Foxp3 mutation
was found responsible for the immune dysregulation in X‑linked syndrome (IPEX) in humans
(Bennett et al., 2001; Wildin et al., 2001).
Medullary TECs (mTECs) also regulate T cell tolerance by the presentation of a repertoire
of self antigens by their MHC molecules for T cells to recognize. mTECs have been shown
capable of expressing a wide array of antigens including organ-specific genes like insulin
(Jolicoeur et al., 1994). An important gene related to their antigen presentation is the
autoimmune regulator (AIRE) (Heino et al., 1999).
Peripheral T cell tolerance
32
Despite of the mechanisms inducing central tolerance within the thymus, a small fraction
of autoreactive T cells are still released into the periphery, however, self-tolerance is usually
well maintained in the periphery and autoimmune diseases rarely occur. Despite our limited
knowledge, peripheral T cell tolerance is maintained through clonal deletion, anergy and
ignorance of autoreactive T cells as well as regulation by Tregs (Ohashi and DeFranco, 2002).
After leaving the thymus, naïve T cells circulate in the blood and the secondary lymphoid
organs such as lymph nodes and spleen and are constantly in contact with dendritic cells (DCs)
and other antigen presenting cells (APCs) (Chow et al., 2002). Under quiescent situations, APCs
constitutively present self-antigens through their MHC class I and class II complexes at a
relatively low concentration and with rapid turnover (Eisenlohr et al., 2007; Schlosser et al.,
2007). Their interactions with mature naïve T cells that recognize self antigen peptides: MHC
complexes at low affinity promote naïve T cell survival and self-tolerance (Eisenlohr et al.,
2007; Jenkins, 2013). Upon infection, naïve T cells are able to be activated when they receive
three signals from APCs (Hansen and Roche, 2013). Signal one is the activation of TCR through
interaction either between TCR/CD4 and peptide: MHC class II, or between TCR/CD8 and
peptide: MHC class I on DCs (Fooksman, 2014; Rosette et al., 2001). Signal two is a co-
stimulation signal such as the interaction between CD28 on T cells and CD80/86 on DCs
(Gimmi et al., 1991). Moreover, DCs can further promote the activation, expansion and
differentiation of T cells through cytokine production (signal 3) (Blomgren and Larsson, 1978;
Schorle et al., 1991). Of these three signals, signal one and two are necessary for T cell
activation (Jenkins, 2013). After activation, T cells can then differentiate into effector T cells
which no longer need co-stimulatory signals for their function when encountering their target
cells (Jenkins, 2013).
Autoreactive T cells in the periphery could bind to their cognate MHC on APCs at high
affinity (signal 1), but without signal 2 and 3, those T cells undergo clonal deletion and anergy
(Lamb et al., 1983). For example, T cell clones cultured with cognate virus peptides in vitro
without APCs have been found with blocked proliferation when restimulated with antigens
together with APCs (Lamb et al., 1983). Accumulated evidence has shown impaired T cell
proliferation when T cells are provided with their cognate antigens on APCs but without
signaling through CD28 (Boise et al., 1995; Paul, 2013). This decreased proliferation of
33
autoreactive T cells is called clonal deletion and the unresponsiveness of T cells upon antigen
stimulation is termed anergy. Besides clonal deletion and anergy, autoreactive T cells can also
literally ignore self-antigens if they circulate but do not bind to self antigens (Goodnow and
Ohashi, 2013; Schietinger and Greenberg, 2014).
In the periphery, APCs also play an important role in self-tolerance. First, antigen
presentation by APCs without providing co-stimulatory signals (signal 2) and proinflammatory
cytokines (signal 3) induces T cell clonal deletion and anergy (Lin et al., 2010). In addition,
DCs also take up and present antigens from extracellular matrix and other cells and induce
cross-tolerance of autoreactive T cells (Lin et al., 2010).
Despite of the different mechanisms of T cell anergy induction, certain self-reactive T cells
are still able to survive. At this stage, FOXP3+ Tregs play an important role in the maintenance
of self-tolerance in the periphery, as Foxp3 defects in both human and mouse are associated
with spontaneous development of autoimmune diseases (Bennett et al., 2001; Brunkow et al.,
2001; Godfrey et al., 1991; Ramsdell and Ziegler, 2014; Wildin et al., 2001). In addition to
thymic FOXP3+ Tregs discussed above, FOXP3
+ Tregs can also derive from naïve T cells in the
periphery upon stimulation (Wing and Sakaguchi, 2010). Tregs are able to suppress
autoimmune responses through contact induced apoptosis of targeted responder T cells or APCs,
as well as through the production of anti-inflammatory cytokines such as IL-10 and TGF-β
(Wing and Sakaguchi, 2010). Recently it has been shown that autophagy is important for Treg
lineage stability and function by integrating environmental cues to Tregs (Wei et al., 2016).
B cell tolerance
B cells originate from common lymphoid progenitor cells in the bone marrow. The
common lymphoid progenitor cells first develop into CD19+ pro-B cells, then become pre-B
cells that express a pre-B receptor and eventually transform into naïve B cells that express
immunoglobulin M (IgM) (Hardy, 2003; Hardy and Hayakawa, 2001). When mature naïve B
cells encounter antigens that express complementary epitopes, their BCRs bind, internalize and
digest the antigens and present the antigen peptide on the cell surface through peptide: MHC
class II complexes (signal 1). If the antigen peptide:MHC class II complexes on B cells is
recognized by activated Th cells from whom they receive Th cell-derived co-stimulatory signals
34
(signal 2) together with cytokines (signal3), the B cells become activated and differentiate into
plasmablasts (Paul, 2013). These cells migrate to primary lymphoid follicles together with Th
cells and T follicular helper (TFH) cells and form germinal centers (GCs) (Shlomchik and Weisel,
2012; Victora and Mesin, 2014). In the GCs, activated B cells undergo extensive proliferation,
somatic hypermutation and class switching that allows them to eventually become either
memory B cells or plasma B cells that produce high affinity antibodies of different isotypes
(Victora and Mesin, 2014).
Similar to T cells, B cell tolerance is regulated both centrally within the bone marrow
during development and in the periphery after maturation. B cell tolerance is also regulated by
APCs as well. In contrast to T cells, however, autoreactive B cells may not undergo apoptosis in
the bone marrow but instead further rearrange their immunoglobulin genes (receptor editing)
and if their B cell receptor (BCR) become low/negative self-reactive, the B cells can proceed to
maturation (Ohashi and DeFranco, 2002). In addition, it has been shown that the deletion of
autoreactive B cells in the bone marrow is not as restrictive as T cells in the thymus.
Autoreactive B cells could be found frequently in the spleen and lymph nodes, however, without
activation signals from APCs and T cells, most of these autoreactive B cells are anergic and
short lived (Goodnow et al., 1989).
Regulatory B cells (Bregs) also play an important role in the maintenance of self-tolerance
as reviewed by Rosser et al and Kim et al recently (Kim et al., 2015; Rosser and Mauri, 2015).
In 1996, Wolf et al showed that B cell deficient mice were unable to recover from experimental
autoimmune encephalomyelitis (EAE) (Wolf et al., 1996). Later, the production of IL-10 by B
cells was found critical for the anti-inflammatory role of B cells (Fillatreau et al., 2002; Mauri et
al., 2003). Thus Bregs are generally defined as IL-10+ B cells with anti-inflammatory functions.
Different IL-10+ Breg populations involved in self-tolerance and control of inflammation have
been reported, such as CD19+CD21
hiCD23
hiCD24
hi transitional 2 marginal-zone precursor (T2-
MZP) cells, CD5+CD1d
hi B (B10) cells, CD19
+CD21
hiCD23
- marginal zone (MZ) B cells
(Rosser and Mauri, 2015). In addition to IL-10+ Bregs, other Breg populations with anti-
autoimmunity characters have been reported in the last few years. For example, Ray et al
showed an IL-10 independent role of B cells in the recovery of mice from EAE through the
engagement of T cells and maintenance of Tregs via the glucocorticoid-induced tumor necrosis
35
factor ligand (GITR) expressed on B cells (Ray et al., 2012). B cell specific deletion of IL-35 in
mice also showed impaired recovery from EAE similar as B cell specific IL-10 deficient mice,
suggesting an important role of IL-35+ Bregs in self-tolerance maintenance (Shen et al., 2014).
Unlike Tregs with the signature FOXP3+ expression, no lineage specific transcription factor
could be found within Bregs (Kim et al., 2015; Rosser and Mauri, 2015). Instead, Bregs have
been reported at different stages from naïve B cells to plasmablasts and most of the Bregs are
mainly found upon stimulation/inflammation (Bodogai et al., 2013; Matsumoto et al., 2014;
Rosser and Mauri, 2015). In particular, the discovery of CD20dim
or CD20- Bregs and their
regulation on T cell-mediated immune responses may help to explain the therapeutic effects of
B cell depletion therapy in CD8+ T cell-mediated ITP as will be discussed in Chapter 4
(Bodogai et al., 2013; Matsumoto et al., 2014).
In summary, T cells and B cells are normally non-responsive to self antigens under
physiological situations which is maintained by mechanisms such as apoptosis or anergy of
autoreactive cells. Despite a great increase of knowledge in the last two decades, our
understanding of the mechanisms of self-tolerance still remains incomplete. In ITP and other
autoimmune diseases, it is thought that self-tolerance mechanisms are breached and autoreactive
T cells and B cells are allowed to induce organ or tissue damages.
1.3.3.2 Evidence of decreased platelet production and increased destruction in ITP
ITP is a heterogeneous autoimmune disease and although a failure of maintaining self-
tolerance leading to anti-platelet specific immune responses is a complicated process, it has
been shown to be related to a number of different immune mechanisms. These include but are
not limited to: potential platelet and megakaryocyte defects, contributions from antigen
presenting cells (APCs), increases in autoreactive B and T cells, inefficient clearance of those
autoreactive immune cells and dysregulation of cytokine production. A schematic view of the
pathogenesis of ITP is shown in Figure 1.3.
There are two main reasons for the inadequate number of platelets in the circulation of
patients with ITP: decreased platelet production from megakaryocytes and increased peripheral
platelet destruction (Cines et al., 2014; McKenzie et al., 2013).
36
In ITP, impaired platelet production by megakaryocytes was suspected a century ago
(Nugent et al., 2009). Indeed, as early as 1915, Frank hypothesized that the thrombocytopenia of
ITP may be due to a platelet production defect (Blanchette and Freedman, 1998; Frank, 1915).
In the 1940s, light microscopy of the bone marrow from patients with ITP revealed normal or
only slightly increased numbers of megakaryocytes, which were disproportional to the degree of
thrombocytopenia (Dameshek and Miller, 1946; De La Fuente, 1949; Diggs and Hewlett, 1948;
Nugent et al., 2009). In addition, morphological abnormalities of megakaryocytes were
observed under light microscopy, including an increased proportion of premature
megakaryocytes and decreased platelet producing megakaryocytes, as indicated by scant
evidence of platelet formation, lack of granularity, vacuolization of the cytoplasm, and pyknotic
nuclei (Figure 1.4) (Dameshek and Miller, 1946; De La Fuente, 1949; Diggs and Hewlett, 1948;
Nugent et al., 2009). Electron microscopy (EM) studies confirmed these findings showing
swollen mitochondria, distended DMS, condensed chromatin, all of which were consistent with
features of apoptosis and this was confirmed by the observation of increased caspase-3 staining
in megakaryocytes (Figure 1.4) (Houwerzijl et al., 2004; Nugent et al., 2009; Stahl et al., 1986).
Platelet turnover studies also provided consistent findings by estimating the platelet
production rate based on the measurements of radiolabeled platelet survival in vivo with time
(with the assumption that the platelet destruction equals production when the platelet count is
stable). Under thrombocytopenic conditions such as secondary to blood loss, the
megakaryocytes in healthy individuals had the potential of approximately a 10-fold increase in
platelet production capacity (Cines et al., 2014). However, this was not observed in patients
with ITP, where only decreased or normal platelet production was found (Ballem et al., 1987;
Branehog et al., 1975; Harker, 1970; Heyns Adu et al., 1986; Nugent et al., 2009; Stoll et al.,
1985).
Increased platelet destruction in ITP was initially suggested by the studies of Harrington et
al (Harrington et al., 1951) and this was confirmed by platelet kinetic studies. Using 51
chromium
labeled allogeneic platelets, these studies showed that the life span of platelets was about 10
days in healthy humans (Harker and Finch, 1969) and only a few hours to days in patients with
ITP (Harker, 1970; Harker and Finch, 1969). The shorter life span of platelets in ITP was also
37
Figure 1.4: Megakaryocytes from healthy individuals and patients with ITP. Images adapted from Dameshek and Miller, 1946
and Houwerzijl et al., 2004 (Dameshek and Miller, 1946; Houwerzijl et al., 2004).
38
(A, B) Normal megakaryocytes, magnification ×1000. (C, D) ITP megakaryocytes with rare or no platelet formation, sharp
cytoplasmic edges and pyknotic nuclei, magnification ×1000. (E) Normal megakaryoblast showing a lobulated nucleus (N). In the
cytoplasm, characteristic demarcation membrane system (asterisks) and normal mitochondria (arrowheads) can be found,
magnification × 3000. (F) Megakaryoblast from a patient with ITP in the process of para-apoptosis; N indicates nucleus. The cell
has an intact enlarged peripheral margin (large arrow), magnification × 4500. (G) The inset of panel F shows a higher
magnification with mitochondria, some of which are slightly swollen (open arrowheads) and others which have completely
collapsed cristae which appear as empty vacuoles (asterisks). The enlarged peripheral margin in the cytoplasm is free of organelles
(open arrow), magnification × 15 000. (A-D) Light microscopy images of bone marrow megakaryocytes. (E-F) Electron
microscopy images of bone marrow megakaryocytes.
39
confirmed by other groups using 51
chromium labeled allogeneic platelets or 111
indium-labeled
autologous platelets (Branehog et al., 1975; Branehog et al., 1974; Kernoff et al., 1980). The
increased platelet destruction theory was also supported by the effectiveness of splenectomy in
raising platelet count as a treatment for ITP (Blanchette and Freedman, 1998; Branehog et al.,
1975; Louwes et al., 2001; Nugent et al., 2009).
Despite the clear evidence of decreased platelet production as well as increased
destruction in ITP, the factors that trigger the immune responses against self-platelets and
megakaryocytes are still elusive.
1.3.3.3 Defects in platelets in ITP
Certain dysfunctions of platelets and megakaryocytes may contribute to the development
of ITP. For example, as mentioned in section 1.1.3, healthy human platelets express all the
TLRs, which have a protective function against infections (Kapur et al., 2015b). However, the
expression of TLR2 and TLR4 on platelets was found to be significantly decreased in both acute
and chronic ITP (Wang et al., 2009). The platelets with decreased expression of TLRs might be
recognized as ‘abnormal cells’ that leads to their destruction and development of ITP.
Alternatively, it may also be possible that the development of ITP is due to increased platelet
destruction mediated by activation of TLRs after infections as suggested by animal studies
(Aslam et al., 2006; Semple et al., 2007). Perhaps this may help to explain the observation that
in children with ITP, febrile illnesses and infections are quite common preceding the
development of thrombocytopenia (Cines et al., 2009).
HLA class II complexes are one of the most important proteins upregulated upon infection.
Platelets do not normally express HLA class II antigens, however, Boshkov et at reported the
expression of HLA-DR on the platelets from a child with active ITP which became negative
after remission (Boshkov et al., 1992). Enhanced expression of HLA-DR in ITP was also
confirmed by other groups (Semple et al., 1996) and although it was suggested that aberrant
HLA-DR expression on platelets may give rise to autoreactive T cells in ITP, further studies are
required to confirm this.
40
Another platelet defect that may potentially trigger ITP is the dysregulation of CD47
expression. CD47 is expressed on virtually all cells (Seiffert et al., 1999) and is a ligand for
signal regulatory protein α (SIRPα) on neutrophils, monocytes, and dendritic cells (Seiffert et al.,
1999). The interaction between CD47 and SIRPα down-regulates phagocytosis by SIRPα+ cells
(Oldenborg et al., 2000; Ravetch and Lanier, 2000; Seiffert et al., 1999). It has been shown that
CD47 expression on aged platelets is significantly lower in patients with ITP as compared to
healthy controls (Catani et al., 2011), however, whether this enhances the platelet destruction in
ITP is still not known.
Senescent platelets are cleared from the circulation continuously and primarily via
macrophage-mediated phagocytosis in the spleen (Cines et al., 2014; Clarkson et al., 1986;
Harker, 1970). In the last few years, however, a new theory has been proposed that implicates
the Ashwell-Morell receptor on liver hepatocytes and macrophages being capable of
recognizing asialoglycoproteins (glycoproteins lacking sialic acid) on aged platelets (Grewal et
al., 2008; Rumjantseva et al., 2009) and promoting platelet clearance. This Ashwell-Morell
receptor mediated platelet clearance might be significantly increased under pathological
situations and lead to the development of ITP. In a recent case study reported by Shao et al, a
patient with refractory ITP had significantly increased platelet desialylation (Shao et al., 2015)
and with oseltamivir phosphate (tamiflu) treatment, a sialidase inhibitor, the desialylation of the
platelets gradually decreased and the platelet count of the patient recovered (Shao et al., 2015).
Of note, this patient was positive for GPIb-IX antibody, although the antibody titer was
unchanged before or after the remission (Shao et al., 2015). Further investigations are still
needed to further confirm the relationship among anti-GPIb antibodies, platelet desialylation
and ITP development.
Furthermore, increased apoptosis of platelets in ITP may contribute to the development of
the disease but this remains controversial. In a study by Catani et al, platelet apoptosis in adult
ITP was increased and this was associated with an upregulated T cell stimulation potential of
DCs (Catani et al., 2006). However, a study of platelet apoptotic markers in children with ITP
by Wang et al showed inconsistent findings (Wang et al., 2010). Different than the findings in
adults, the authors examined apoptotic markers on platelets including annexin V, caspase 3, and
mitochondrial inner transmembrane potential depolarization, and could not find any significant
41
changes. In addition, platelets from these paediatric patients with ITP were found to be resistant
to apoptosis as suggested by increased CXCR4 expression in the plasma (Wang et al., 2010).
These discrepancies between the studies might be due to different pathogenic mechanisms in
paediatric versus adult ITP, the stage of ITP or the various therapies the patients may have
received.
Overall, defects in platelets could directly increase their phagocytosis by macrophages or
indirectly increase platelet destruction through stimulation of DCs and activation of antibody- or
T cell-mediated anti-platelet immune responses. This may eventually lead to the development of
ITP but more research is required to confirm whether platelet defects are responsible for the
autoimmune initiation of the attack against self platelets.
1.3.3.4 Dysregulation of APCs and initiation of adaptive immune responses in ITP
Apart from platelet abnormalities, dysregulation of antigen presenting cells (APC) also
seems to be involved in the pathogenesis of ITP. APC is a general term for several different cell
types all of which have the capability of taking up and digesting protein antigens into peptides
(known as antigen processing) and loading their peptides onto the major histocompatibility
complex (MHC) molecules which are the ligands for activating naïve T cells (antigen
presentation) (Banchereau et al., 2000; Watts, 1997). There are two types of MHC molecules
termed MHC class I and class II. While MHC class I molecules are universally expressed on all
cells except the RBCs, MHC class II molecules are almost exclusively expressed on APCs and
facilitate their function in priming CD4+ T cells (Banchereau et al., 2000). Another important
feature of APCs is the expression of various co-stimulation receptors which are important for T
cell activation and proliferation such as CD80/86 (Banchereau et al., 2000). APCs include DCs,
monocytes (also known as macrophages when activated), B cells and activated epithelial cells
etc (Chaplin, 2010). Under normal situations, APCs endocytose exogenous antigens and digest
the proteins into peptides with the help of the proteasome in the cytosol or lysosomes. The
peptides are then transported into the endoplasmic reticulum (ER) or endosomes for loading
onto to MHC class I or class II molecules respectively (Banchereau et al., 2000; Chaplin, 2010).
The peptide: MHC complexes are then transported to the cell surface where they can potentially
interact with antigen-specific TCRs expressed on T cells (Banchereau et al., 2000; Chaplin,
2010).
42
Genetic studies of the human leukocyte antigen (HLA) polymorphisms showed an
association of certain HLA class II alleles and a predisposition of ITP suggesting a contribution
of APCs in initiating ITP. For example, in 1979, Karpatkin et al found an association of HLA-
DRw2 on B cells from patients with ITP (Karpatkin et al., 1979) and subsequently,
polymorphisms in several HLA class II alleles were found associated with anti-GPIIbIIIa or
anti-GPIb-IX antibody production in Japanese patients with ITP (Kuwana et al., 2000; Nomura
et al., 1998). However, no association could be found in other Asian populations including
patients from China and India (Leung et al., 2001; Negi et al., 2012). In addition polymorphisms
in HLA class II alleles were also reported in Italian patients with ITP (Veneri et al., 2002). The
differences among these studies could be perhaps explained by the differences in the distribution
of HLA alleles in a particular population or may also be due to the heterogeneity of the disease.
DCs are the most important APCs for the initial activation of T cells and have specialized
features for antigen processing and presentation. DCs do not only actively macropinocytose
extracellular fluid and phagocytose extracellular pathogens through Fc-receptors, but also
express a broad variety of anti-microbial pattern recognition receptors (PRRs) to recognize
microbial invasions efficiently (Liu and Nussenzweig, 2013). For example, they express nearly
all known TLRs for the detection of, for example, bacterial lipopolysaccharides (LPS),
lipoproteins, lipopeptides, flagellin, as well as double-stranded RNA of viruses. They also
express the intercellular adhesion molecule-3-grabbing nonintegrin (SIGN) family receptors,
and multiple C-type lectins including the macrophage mannose receptor (MMR), DEC205
(CD205), and dendritic cell inhibitory receptor 2 (DCIR2, i.e. Clec4a4) and also retinoic acid-
inducible gene-I (RIG-I)-like receptors (RLR) etc (Liu and Nussenzweig, 2013). When activated
by one or more of these receptors, DCs not only process and present antigens but also increase
the expression of co-stimulatory signals and the production of cytokines (Banchereau et al.,
2000). This enables DCs to activate naïve T cells and promote T cell proliferation and
differentiation. For example, activated DCs regulate the differentiation of naïve CD4+ T cells
into Th1, Th2, Th17 cells or suppressive regulatory T cells (Tregs) (Liu and Nussenzweig,
2013). DCs can be divided into at least three groups: plasmacytoid DCs (pDCs), conventional
DCs (cDCs), and migratory DCs (mDCs). cDCs can be further divided into CD8α+ and CD8α
-
cDCs whereas mDCs are also known as myeloid DCs due to their non-lymphatic distribution
43
(Gilliet et al., 2008; Liu and Nussenzweig, 2013; Shortman and Liu, 2002). Under normal
situations, platelets can be taken up by DCs but the platelet peptide:MHC complexes generated
by the DCs do not trigger T cell activation because those platelet peptides would be recognized
by T cells as “self” through weak peptide:MHC/TCR interactions and lack of co-stimulatory
signals (Banchereau et al., 2000). However, abnormal DC activation, under inflammatory
situations may provide simultaneous co-stimulation signal (signal 2) together with signal 1 and
lead to the activation of T cells against self-platelet antigens and the development of ITP. In
2006, Catani et al found significantly increased CD86 expression on cultured DCs from the
blood and spleens of patients with ITP and this could act as a co-stimulatory ligand through the
interaction with CD28 and activates the T and B cells to attack platelets (Catani et al., 2006;
McKenzie et al., 2013). When the DCs were pulsed with platelets, those derived from patients
with ITP induced higher T cell proliferation responses in vitro (Catani et al., 2006). This
suggested an association between increased antigen presenting capability of DCs and an
expansion of autoreactive T and B cells in ITP. The increased expression of CD86, together
with increased expression of CD80 and HLA-DR on DCs in ITP were also recently confirmed
by Zhang et al (Zhang et al., 2015). In addition, these authors found decreased CD205
expression on splenic DCs in ITP which is important for DC antigen presentation and immune
tolerance induction (Bozzacco et al., 2007; Fukaya et al., 2012; Zhang et al., 2015; Zhou et al.,
2012). The decreased CD205 on DCs in ITP may help to explain the impaired self-tolerance and
the Treg deficiency and dysfunction observed in the disease (Liu et al., 2007; Sakakura et al.,
2007). Moreover, Saito et al found that ITP was associated with a pDC but not a mDCs
deficiency in the peripheral blood, and the pDC deficiency was normalized in responders to H.
pylori eradication therapy (Saito et al., 2012).
In addition to DC dysfunction, monocytes have also been shown to be involved in the
initiation and promotion of anti-platelet adaptive immune responses in ITP. Monocytes (or
macrophages when activated) are another important member of APCs and like DCs, they also
contribute to the recognition of foreign antigens and the initiation of adaptive immune responses
(Chaplin, 2010). Moreover, activated macrophages are highly phagocytic, engulfing most of the
senescent and abnormal cells, and also antibody-opsonized immune complexes, including anti-
platelet antibody opsonized platelets in ITP. In ITP, CD16+ (FcγRIIIA) monocytes were found
44
Table 1.2 Summary of the antigen presenting cell (APC) defects in ITP
Parameter Major biological
function
Response
compared to
normal
Reference
Polymorphisms in
HLA class II
antigen
presentation,
activation of CD4+
T cells
Association with
HLADRB1*0410,
HLADRB1*0405,
HLADRB1*0401
Kuwana et al., 2000
Nomura et al., 1998
Veneri et al., 2002
nonchanged Leung et al., 2001
Negi et al., 2012
plasmacytoid DC type I interferon
production
decreased Saito et al., 2012
CD80/86 on DCs co-stimulation increased Catani et al., 2006
Zhang et al., 2015
CD205 on DCs immune tolerance
induction
decreased Zhang et al., 2015
indoleamine 2,3-
dioxygenase 1 in
DCs
immune tolerance
induction
decreased Zhang et al., 2015
TLR7 in DCs antiviral, DC
activation
increased Yu et al., 2011
CD16+ monocytes activation of CD4
+
T cells
increased Zhong et al., 2012
DC= dendritic cell
45
increased and could directly interact with T cells to promote the expansion of IFN-CD4
+ T
cells while inhibiting the expansion of Tregs (Zhong et al., 2012).
It thus appears that the activation of APCs is a prerequisite for the initiation of anti-platelet
specific adaptive immune responses in ITP. Abnormalities in the number, distribution, and
function of the APCs as discussed above could all contribute to the development of ITP. A brief
summary of the APC defects in ITP is listed in Table 1.2.
1.3.3.5 The role of T cells in ITP
Antigen specific immune responses elicited by T cells are called “cell-mediated” immune
responses and together with the “humoral” immune responses elicited by B cells comprise the
adaptive immune system (Chaplin, 2010; Paul, 2013). There are two types of TCRs: composed
of either a TCR heterodimer of α and β polypeptides or of γ and δ polypeptides (Haas et al.,
1993; Huppa and Davis, 2013). The αβ TCR is the major T cell group and the receptor is
composed of a variable (V) region (the region responsible for antigen specificity), a constant (C)
region, and a transmembrane domain on both chains (Novotny et al., 1986; Paul, 2013). When
activated, T cells become effector cells. Effector CD4+ T cells are important for promoting the
activation of other T and B cells and regulating immune responses. They can be further divided
into different subtypes including Th1, Th2, Th17, Th22, TFH and Tregs (Paul, 2013). Activated
effector CD8+ T cells are usually referred to as CD8
+ cytotoxic T lymphocytes (CTLs) (Johnson
et al., 1985) and can recognize target cells through TCRs or Fas ligand and kill the target cells
through the release of perforin and granzymes (Hamann et al., 1997).
The involvement of T cells in the pathogenesis of ITP has been suspected since the early
1970s even before the discovery of the TCR. In fact, T cell involvement in ITP might date back
to the early 1950s when Harrington showed that only 16 of 26 plasma transfusions induced
thrombocytopenia suggesting alternative mechanisms (Harrington et al., 1951). In 1970,
Piessens et al found increased lymphocyte proliferation in response to autologous platelets in
vitro from a patient with ITP as measured by the uptake of tritiated thymidine (Piessens et al.,
1970). Consistent findings were reported shortly afterward by Clancy and Wybran et al
independently (Clancy, 1972; Wybran and Fudenberg, 1972). These studies provided early
evidence of activated lymphocytes in ITP. Clancy also noted inhibited leukocyte migration by
46
autologous platelets in vitro (Clancy, 1972). Subsequently, accumulated evidence suggested that
ITP may be associated with T cell abnormalities. For example, in 1978, Stuart et al enumerated
T cells in the blood of both the patients with ITP and their families and found decreased
numbers of T cells in some of the families (Stuart et al., 1978). In 1984, Borkowsky and
Karpatkin found elevated leukocyte migration inhibition factor from patients with ITP, which is
known to be produced by T lymphocytes (Borkowsky and Karpatkin, 1984). In 1987,
thymectomy in a 13- year-old girl rescued her myasthenia gravis and ITP suggesting that T cells
played a significant role in the disease (Jansen et al., 1987). However the T cell theory of ITP
was not defined until 1991 when Semple and Freedman found increased anti-platelet
proliferation of peripheral blood mononuclear cells (PBMC) from patients with ITP which was
associated with increased IL-2 production (Semple and Freedman, 1991). This study was
considered the first clear evidence of the involvement of T cells in the pathogenesis of ITP.
Subsequently, the sequencing of complementarity determining region 3 (CDR3) in the β chain
of TCRs revealed antigen-induced oligoclonality in T cells from patients with ITP, in particular,
non-responders to splenectomy (Fogarty et al., 2003; Shimomura et al., 1996). In 2001,
autoreactive T cells were found with specificities against GPIIbIIIa epitopes in ITP (Kuwana et
al., 2001) and recently, Zhang et al reported an increased lipid raft aggregation on T cells from
patients with ITP, suggesting abnormal T cell signaling in ITP (Zhang et al., 2016). Figure 1.5
shows the potential process of the activation of T cells and development of autoreactive anti-
platelet immune responses in ITP.
CD4+
T cells in ITP
In addition to the study by Semple et al, increased proliferation of CD4+ T cells in ITP was
also confirmed by other groups (Ma et al., 2012; Ware and Howard, 1993). Ma et al also found
upregulated Bmi-1 expression associated with increased proliferation and survival of CD4+ T
cells (Ma et al., 2012; Yamashita et al., 2008). Altogether, these findings suggest that there is a
breach in self-tolerance and an activated anti-platelet CD4+ T cell response in ITP. Further
studies on CD4+ T cells and their subsets revealed that in ITP, CD4
+ T cells are commonly Th1
polarized. Under physiological situations, Th1 and Th2 constitute the major population of
activated CD4+ T cells(Liao et al., 2011; Paul, 2013). Th1 cells are distinguished by their
production of IL-2 and IFN-and are important for targeting macrophages for clearance of
47
Figure 1.5: Development of the anti-platelet adaptive immune response. Modified from Coopamah et al (2003). Transfusion
Medicine Reviews, 17 (1):69-80, Fig 2. (Coopamah et al., 2003) .
48
.
(A) Senescent platelets are consistently taken up by macrophages (APCs). Platelet autoantigen peptides are digested and
presented by MHC class II on the APC surface. The peptide: MHC class II complex could be recognized by cognate TCRs
on CD4+ T cells (signal 1). Signal 1 alone would not activate the CD4
+ T cells. When activated, however, APCs increase
their expression of co-stimulatory molecules e.g. CD80/86 and CD40 and produce inflammatory cytokines such as type I
interferons (IFN) and IL-12. (B) Activation of autoreactive CD4+ T cells. The costimulatory proteins on activated APCs
interact with their ligands expressed on CD4+ T cells (signal 2) and together with signal 1, activate autoreactive CD4
+ T
cells. Both APC and T cell derived cytokines (signal 3) further promotes the activation. (C) Activated autoreactive CD4+ T
cells differentiate, proliferate, and produce cytokines in favor of a Th1 responses including increased production of IL-2,
IFN-γ, IL-17, IL-22, and decreased IL-10 and TGF-β. The activation of autoreactive CD4+ T cells then drives the activation
of autoreactive B cells (D) and autoreactive CD8+ T cells (E). The activated B cells differentiate into plasma B cells which
produce anti-platelet antibodies and the CD8+ T cells differentiate into CTLs which can directly kill platelets and
megakaryocytes.
49
intracellular bacteria and amplification of cell-mediated immune responses (Liao et al., 2011).
Th2 cells on the other hand, are characterized by the production of IL-4, IL-13 and IL-5 and are
important for immunity against extracellular helminthes and initiating the production of IgE
(Prussin et al., 2010). Both Th1 and Th2 cells are often called helper T cells but they suppress
the proliferation and differentiation of each other (Paul, 2013). It is now well established that an
imbalance of Th1/Th2 cells is associated with autoimmune diseases. Work by Semple et al
showed increased serum IL-2, IFN- and/or IL-10, not IL-4 nor IL-6 in patients with ITP
suggesting an early activation of CD4+ T cells and a Th1 polarized response (Semple et al.,
1996). Th1 polarized T cell responses in ITP were further confirmed by increased IFN-
+CD4+/IL-4
+CD4
+ (Th1/Th2) ratios in patients (Stasi et al., 2007; Wang et al., 2006) and these
studies suggest that CD4+ T cell activation and Th1 responses may be responsible for the
activation of CD8+ T cell- and antibody-mediated platelet destruction in ITP. Alternatively, it is
possible that the Th1/Th2 imbalance is a consequence of thrombocytopenia as it has been shown
that platelets are a source of multiple cytokines and thrombocytopenia is associated with their
decreased levels which may be important for the Th1/Th2 balance (Feng et al., 2012; Khan et al.,
2005). In addition, there is also a possibility that thrombocytopenia secondary to infection leads
to a decreased level of soluble CD40L in vivo which induces a Th1 polarized immune response
(Kuwana et al., 2003). Taken together, it appears that the most consistent T cell abnormality in
ITP is a Th1 cytokine skewing (Figure 1.5). However, other T cell subsets have also been shown
involved.
Th17 cells in ITP
Th17 cells are a relatively minor CD4+ T cell population characterized by their production
of their signature cytokines including IL‐17 (IL-17A and IL-17F), IL‐21, and IL‐22. Th17 cells
are induced when both IL-6 and transforming growth factor (TGF-β) are present and when IL-4
and IL-12 are absent (Paul, 2013). The major function of Th17 cells is to enhance neutrophil
generation against extracellular bacteria and fungi infection under physiological situations
(Isailovic et al., 2015; Schwarzenberger et al., 1998). However, Th17 cells have been found to
play a proinflammatory role in the pathogenesis of several autoimmune diseases including ITP
(Bettelli et al., 2006; McKenzie et al., 2013; O'Shea, 2013; Wilson et al., 2007). In 2009, Zhang
50
et al first reported increased Th17 cells in the peripheral blood of patients with ITP and this
correlated with an increase in Th1 populations (Zhang et al., 2009). However, quantification of
Th17 cytokines including IL-17A, TGF- and IL-6 in patient plasma showed comparable levels
to controls. Two years later, Cao et al reported no significant difference of Th17 populations in
peripheral blood mononuclear cells (PBMC) between patients with ITP and healthy individuals
(Cao et al., 2011). In the same year, however, a larger prospective study by Rocha et al, showed
a 5 fold increase of IL-17A in the plasma of patients with ITP and increased expression of IL-23,
IL-6, IL-1, IL-2, IL-12 but not TGF- (Rocha et al., 2011)The increased Th17 and Th17
cytokines in active ITP were then confirmed by several other studies (Hu et al., 2012; Hu et al.,
2011; Ji et al., 2012; Ye et al., 2015). Furthermore, the levels of IL-17, IL-22 and IL-23 were
found decreased in patients who responded to treatment (Cao et al., 2012; Li et al., 2015c; Ye et
al., 2015). In general, IL-17 is increased in active ITP and appears to be positively correlated
with Th1 immune responses and negatively correlated with the remission of the disease.
However, it is worth noting that there are some discrepancies in the literature. While this may
be related to limited sample sizes and the sensitivity of quantitative studies, further
investigations are needed to better understand the role of Th17 cells in ITP.
Th22 cells in ITP
Th22 cells are another type of CD4+ T cell subset that may be involved in the development
of ITP. Th22 cells have both pro- and anti-inflammatory effects under different circumstances
but in combination with Th17 cells, they are overtly pro-inflammatory. In the study by Cao et al,
although the authors did not observe any significant Th17 levels, they did find increased IL-22
in the plasma from patients with active ITP and of interest, they found that the IL-22 was
decreased after treatment (Cao et al., 2012). In another study by Hu et al, both Th17 and Th22
cells were found increased in ITP together with an increase of IL-22 in patients’ plasma (Hu et
al., 2012). In both studies, the increased IL-22 correlated with increased Th1 responses
suggesting the IL-22 produced by Th22 cells promoted T cell differentiation towards Th1 which
further promotes the development of ITP (Cao et al., 2011; Hu et al., 2012).
Regulatory T cells (Tregs) in ITP
51
Tregs are a subset of T cells which are important for the maintenance of self-tolerance and
suppression of both T cell and B cell responses (Ramsdell and Ziegler, 2014). Tregs have been
shown to suppress immune responses through contact-dependent mechanisms and the
production of soluble factors, such as the cytokines TGF-β and IL-10 (Buckner, 2010;
Sakaguchi et al., 2010; Vignali et al., 2008). Treg deficiency is associated with many
autoimmune diseases (Bennett et al., 2001; Brunkow et al., 2001; Godfrey et al., 1991;
Ramsdell and Ziegler, 2014; Wildin et al., 2001).
Many studies have now confirmed that in patients with active ITP, there is a significant
deficiency/dysfunction of Tregs (Reviewed in McKenzie et al, 2013). Of interest, the peripheral
deficiency of Tregs could be restored after several different types of treatments such as
dexamethasone, rituximab, TPO receptor agonists (TPO-RAs) or IVIg (Bao et al., 2010; Ling et
al., 2007; Stasi et al., 2008; Yu et al., 2008). These results suggest that perhaps the rise in
platelet counts by the treatments may be responsible for affecting Treg numbers in vivo. The
decreased number of Tregs has also been shown to be associated with impaired cross-talk
between Tregs and DCs, for example, due to decreased expression of the immunomodulatory
enzyme indoleamine 2,3-dioxygenase 1 (IDO1) on DCs (Catani et al., 2013).
The Treg abnormalities in ITP and other autoimmune diseases may be due to several
reasons (Buckner, 2010; Vignali et al., 2008). For example, Treg deficiency may occur when
target cells express inadequate cell surface markers that are involved in contact-dependent
suppression such as cytotoxic T lymphocyte antigen 4 (CTLA-4). Although genetic studies did
not find any associations between CTLA-4 polymorphism and a predisposition to ITP, plasma
CTLA-4 levels have been found decreased in acute ITP and elevated after treatment (Li et al.,
2011; Pavkovic et al., 2003; Radwan and Goda, 2015; Zhu et al., 2015). In addition, it was
found that CTLA-4-Ig stimulated DCs from patients with ITP to promote formation of Tregs in
vitro (Xu et al., 2012). Alternatively, the Treg deficiency may be due to a dysfunction of Treg
populations, for instance, a deficiency in the transcription of FOXP3 or in the production of
suppressive cytokines, such as TGF-β or IL-10 (Catani et al., 2013; Chen et al., 2014; Li et al.,
2015a). In addition, the local milieu may play a role in the dysfunction of Tregs, as they may be
significantly influenced by different cytokines such as IL-2, IL-12, and IL-21 (Andersson et al.,
2002; Clough et al., 2008; King and Segal, 2005; Tesse et al., 2012; Thornton et al., 2004).
52
The imbalance of Th17/Treg in ITP
In addition to the independent role of either Th17 or Tregs, the Th17/Treg ratio is also
considered as an indicator of the immune balance, because of the dichotomy in the generation of
Th17 cells and Tregs. On the one hand, the presence of TGF-β with IL-6 and IL-23 favors Th17
differentiation from naïve CD4+ T cells and inhibits Treg development (Afzali et al., 2007;
Bettelli et al., 2006; Mangan et al., 2006). On the other hand, TGF-β without IL-6 favors Treg
differentiation and FOXP3 transcription which in turn inhibits Th17 development (Afzali et al.,
2007; Bettelli et al., 2006; Mangan et al., 2006). The Th17/Treg balance is an important
regulator of Th1/Th2 responses, and a Th17/Treg shift towards Th17 predominance is
associated with inflammation and autoimmune diseases (Afzali et al., 2007).
The Th17/Treg ratio may be a more sensitive indicator to distinguish ITP severity
compared with changes in either Th17 or Tregs. For example, in the study by Ji et al, patients
with ITP were divided into two groups: an observational group whose platelet counts were
above 30×109/L without active bleeding, and those in a treatment required group, whose platelet
counts were lower or with active bleeding symptoms (Ji et al., 2012). Significant skewed
Th17/Treg ratios towards Th17 were found in the latter group (Ji et al., 2012). In addition, a
recent study by Yu et al, using an inhibitor of the notch signaling pathway DAPT, showed that
the Th17/Treg ratios were decreased in a dose-dependent manner (Yu et al., 2015). Their study
not only confirmed the role of the notch signaling pathway in Th17/Treg regulation, but also
provided a potential immunotherapy strategy for ITP.
As is shown in Figures 1.3 and 1.5, CD4+ T cells play a central role for the activation and
regulation of both antibody- and CTL- mediated immune responses. Abnormalities in the
distribution of different subsets of CD4+ T cells as well as biased cytokine production leads to a
breach of self-tolerance and development of autoreactive plasma B cells and CTLs that
eventually can mediate the destruction of platelets and/or megakaryocytes.
CD8+ T cells in ITP
Beside CD4+ T cells, another important type of T cell subset participating in the
pathogenesis of ITP is CD8+ T cells. When naïve CD8
+ T cells are activated after their TCR
53
recognizing specific peptide:MHC class I complexes on target cells, they begin to express
granule effector molecules and Fas Ligand (FasL) and differentiate into effector cells called
cytotoxic T lymphocytes (CTLs) (Cui and Kaech, 2010; Hanabuchi et al., 1994). In contrast to
CD4+ T cells whose primary function is to enhance or inhibit immune cells, CTLs can directly
induce apoptosis of target cells through their granule-mediated pathway or death receptor
pathway (Cui and Kaech, 2010; Kelso et al., 2002). In granule-mediated cell death, CTLs
release cytotoxic granules that contain apoptosis-inducing proteins such as perforin and
granzymes A and B into target cells (Lieberman, 2010; Lieberman, 2013; Masson et al., 1990;
Metkar et al., 2002). In the death receptor pathway, CTLs express Fas ligand (FasL) that
recognizes Fas expressed on abnormal cells and triggers programmed cell death of the target
cells (Fisher et al., 1995; Lieberman, 2010). The target cells subsequently break up into small
apoptotic bodies that are phagocytosed by macrophages (Kerr et al., 1972). The classical in
vitro assay to measure antigen-specific CTL function is called a cytotoxicity assay. Purified
CD8+ T cells (effectors) are co-cultured in vitro with radioactive or fluorescence labeled target
cells that express cognate peptide:MHC class I complexes for various times and then the
apoptosis/death of the target cells is measured (Guo et al., 2014; Kay et al., 1977; Olsson et al.,
2003). This assay has been instrumental in defining the role of CTLs and has shown that they
are important in anti-virus and anti-tumor immunity and also in the pathogenesis of a number of
autoimmune diseases, such as diabetes, multiple sclerosis and ITP (Faustman and Davis, 2009;
Li et al., 2007; Olsson et al., 2003; Saxena et al., 2011; Spits et al., 1986; Zhang et al., 2006). In
addition, a genetic deficiency in the granule-mediated T cell killing pathway can lead to
increased incidence of spontaneous lymphomas and a deficiency in the Fas-FasL pathway is
associated with a predisposition to autoimmune diseases (Lieberman, 2013). Thus, the
activation and function of CTLs are important parameters of adaptive immune responses.
In 2003, Olsson et al showed increased cytotoxic activity of T cells against autologous
platelets in patients with active ITP, suggesting for the first time the involvement of CD8+ T cell
mediated platelet lysis in the pathogenesis of the disease (Olsson et al., 2003). In addition to
platelet destruction, Li et al demonstrated that CTLs could also induce megakaryocyte para-
apoptosis in ITP (Li et al., 2007). A gene expression/pathway analysis by Sood et al revealed the
activation of several death receptor pathways e.g. JAK/ STAT, all of which suggest CD8+ T cell
54
activation and cell-mediated cytotoxicity in ITP (Sood et al., 2008). Although the total number
of CD8+ T cells seemed to be comparable between patients with ITP and healthy individuals, the
IFN-+CD8
+/ IL-4
+CD8
+ ratio was found significantly higher in active ITP, and decreased after
treatment, indicating an imbalance of subpopulations of CD8+ T cells in the pathogenesis of the
disease (Stasi et al., 2007). Furthermore, Audia et al investigated the spleens from patients with
ITP who were rituximab nonresponders and found increased IFN-+CD8
+ T cells together with
increased CD27−CD28
−CD8
+ effector memory T cells (Audia et al., 2013). In addition, the
authors found increased expression of HLA-DR and granzyme B on CD8+ T cells, indicating
increased CD8+ T cell activation in ITP (Audia et al., 2013). Taken together, it appears that the
activation and clonal expansion of autoreactive CD8+ T cells also play a significant role in the
development of ITP, possibly due to the activation of APCs and upregulation of MHC class
I/co-stimulation molecules or the activation of CD4+ T cells favoring Th1 and Th17 cells. The
autoreactive CD8+ T cells could induce direct lysis of platelets and megakaryocytes, which
process is known as CD8+ T cell-mediated ITP.
In summary, dysregulation of T cells plays an important role in ITP (Figure 1.5). T cell
abnormalities in ITP include increased proinflammatory Th1, Th17 and Th22 cells, decreased
Th2 and CD4+ Treg cells, as well as increased IFN-
+CD8
+ and decreased IL-4
+CD8
+ T cells.
These abnormalities are associated with the activation of APCs and moreover, the activation of
CD4+ T cells promotes the activation of autoreactive B cells in ITP.
1.3.3.6 Activation of B cells and anti-platelet antibody production in ITP
Antibody-mediated ITP was the earliest mechanism discovered in the pathogenesis of ITP
(Harrington et al., 1951) and a failure of B cell tolerance maintenance may also give rise to
autoreactive B cells which produce anti-platelet antibodies.
In 1951, Harrington showed that the transfusion of plasma from patients with ITP into
healthy volunteers induced transient thrombocytopenia in most recipients, clearly indicating an
ITP inducing factor in the plasma (Harrington et al., 1951). In 1965, Shulman et al showed that
the thrombocytopenia-inducing factor in the plasma of patients with ITP was quantitatively and
qualitatively found in the gammaglobulin fraction of plasma, suggesting that the ITP inducing
factor was IgG (Shulman et al., 1965). In 1972, using rabbit anti-human IgG to absorb anti-
55
platelet antibodies in the sera, Karpatkin et al confirmed that there were anti-platelet IgG
antibodies in the plasma of approximately 65% of patients with ITP (Karpatkin et al., 1972).
Subsequently, Hymes et al examined the isotype of IgG in antibody positive ITP patients, and
found IgG1 the most abundant (Hymes et al., 1980). It was estimated that about two thirds of
patients with ITP have detectable anti-platelet antibodies (Chan et al., 2003; Karpatkin et al.,
1972). The specificity of anti-platelet antibodies in ITP was not clear until 1982 when studies
using platelets from healthy donors and GPIIbIIIa deficient patients with Glanzmann
thrombasthenia showed that the majority of autoantibodies in ITP were against platelet
GPIIbIIIa (van Leeuwen et al., 1982). Later, anti-platelet IgM and IgA antibodies were also
reported in ITP (Liel et al., 2014) and several studies have now confirmed that the majority of
anti-platelet antibodies in ITP target GPIIbIIIa and GPIb-V-IX, while some antibodies can also
react with GPIV, α2β1, P-selectin (CD62P), αvβ3, glycosphingolipids and cardiolipin (Liel et al.,
2014).
The production of autoreactive anti-platelet antibodies by plasma B cells and its regulation
may involve multiple factors such as APC activation, enhanced Th1 and Th17 responses, Treg
dysfunction/deficiency as well as defects within B cells and related cytokines (Paul, 2013). For
example, B-lymphocyte stimulator (BlyS) produced by APC was found significantly increased
in the circulation of ITP patients, which is important for promoting B cell survival, proliferation
and differentiation (Yu et al., 2011). In addition, CD19+CD24
hiCD38
hi human regulatory B cells
(Bregs) that have suppressive function for CD4+ T cells were found to be decreased in ITP (Li et
al., 2012b). This was found to be associated with decreased expression of IL-10 upon
stimulation in vitro, which was rescued in patients who responded to treatment, suggesting that
like the Treg deficiency observed in ITP, a Breg deficiency is also involved in the development
of the disease (Li et al., 2012b).
1.3.3.7 Anti-platelet antibody-mediated ITP
Antibodies, also known as soluble immunoglobulins, are roughly Y-shaped molecules
consisting of two identical heavy chains and light chains which are produced by mature B cells
and plasma B cells. The two arms of the Y are two identical variable regions (V regions), each
of which is composed of V domains from a heavy and a light chain. The V region determines
the antigen specificity and affinity of the Ig while the trunk of the Y is the constant region (C
56
region) and determines the isotype of the Ig (Murphy et al., 2012; Paul, 2013; Williams and
Barclay, 1988). The protease papain cleaves the antibody into three pieces: two antigen-binding
fragments (Fab) that contain the antigen binding V regions, and a crystallized fragment (Fc)
which is part of the C region capable of interacting with Fc receptors (FcRs) (Kalmanson and
Bronfenbrenner, 1942; Petermann, 1946; Schirrmacher et al., 1975). Another protease pepsin,
on the other hand, cuts the antibody into an F(ab’)2 fragment in which the two arms of the
antibody remain linked and several fragments of the remaining constant region (Deutsch et al.,
1946; Kirkham and Schroeder, 1994; Petermann and Pappenheimer, 1941). The antigen binding
site of each V region is dependent on the three CDRs, CDR1, CDR2, and CDR3 (Kirkham and
Schroeder, 1994). Similar to TCRs, it is estimated that up to1012
different antibodies can be
made in an individual through VDJ recombinations, heavy chain and light chain combinations,
class switching and B cell hypermutation (Schroeder et al., 1998; Schroeder et al., 1995). In
contrast, whereas TCRs only recognize antigen peptides presented through MHC class I or class
II complexes, the antibody molecule can directly bind to antigens that bear either peptides
(linear determinant) or conformational determinants in the native antigen (Murphy et al., 2012;
Paul, 2013).
The immunoglobulins produced by plasma B cells contribute to antigen specific immunity
in mainly three different ways: neutralization, opsonization, and complement activation
(Bottazzi et al., 2010; Michaelsen et al., 1991). Neutralization refers to the binding of antibodies
to antigens and preventing the penetration of antigen into cells (Bottazzi et al., 2010).
Opsonization refers to the phagocytosis enhancement of antigen-antibody complexes by FcR
expressing cells such as macrophages (Bottazzi et al., 2010). The activation of the complement
system is triggered by the binding of the complement C1q fragment to IgG which ultimately
leads to cell death by osmotic lysis (Bottazzi et al., 2010). Under physiological situations, there
might be a few self-reactive B cells but these B cells and the antibodies they produce are not
pathogenic, due to their low-affinity against self-antigens or lack of support from helper T cells
(Paul, 2013).
Autoreactive IgG anti-platelet antibodies could induce the development of ITP in several
different ways, including increasing platelet destruction, decreasing platelet production, or
interfering with platelet function. Of these mechanisms, antibody-mediated platelet destruction
57
Table 1.3 Summary of Fc receptor expression patterns and functions. Modified from Paul
W. E.. Fundamental Immunology (Philadelphia : Wolters Kluwer Health/Lippincott
Williams & Wilkins, c2013) Figure 24.1. (Paul, 2013).
Nomenclature Expression Function
FcγRI Macrophages, Neutrophils,
Eosinophils, DCs
Activation, enhance effector
response
FcγRIIA Macrophages, Neutrophils, Mast cells,
Eosinophils, Platelets, DCs
Activation, effector activation
FcγRIIB Macrophages, Neutrophils, Mast cells,
Eosinophils, DCs, B cells
Inhibition, set threshold for effector
cell activation, maintain tolerance
FcγRIIIA Macrophages, Basophils, Mast cells,
NKs, DCs, B cells
Activation, dominant pathway for
effector activation by IgG
FcγRIIIB Neutrophils Decoy, synergize with FcγRIIIA
FcεRI Basophils, Mast cells, Eosinophils,
platelets, DCs
Activation, degranulation, allergy
FcαRI Macrophages, Neutrophils,
Eosinophils
Activation, effector cell activation
58
and enhanced clearance in vivo is considered the most important mechanism of
thrombocytopenia in ITP and has been well studied (Provan et al., 2010). It can be further
divided into Fc receptor (FcR) mediated phagocytosis, antibody-dependent cell-mediated
cytotoxicity (ADCC) by NK cells, complement activation-induced platelet lysis, and direct
platelet fragmentation.
Clearance of antibody-bound platelets by Fc receptors
Fc receptor (FcR) mediated phagocytosis seems to be the dominant mechanism of platelet
destruction in ITP. FcRs refer to a family of receptors that recognize the Fc fragments of
immunoglobulins and the receptors for IgG are termed Fcγ receptors (FcγR) (Fridman, 1991).
Both DCs and monocytes express all four types of FcγRs, including the activating receptors
FcγRI, FcγRIIA, FcγRIIIA (FcγRIV in mouse) that promote phagocytosis, and the inhibitory
receptor FcγRIIB (Ravetch and Nimmerjahn, 2013) that suppresses phagocytosis. As the Fc
portion of IgG antibodies could be recognized by multiple FcγRs with different affinities, the
overall response of DCs or monocytes is dependent on the activation/inhibition ratio (Ravetch
and Nimmerjahn, 2013). A summary of the FcRs is listed in Table 1.3.
The importance of FcγR dependent phagocytosis in ITP is supported by several
observations. Early evidence came indirectly from the observation that splenectomy could
significantly increase the platelet counts in most of the patients with ITP (still considered one of
the “gold standard” therapies) (Blanchette and Freedman, 1998; Hirsch and Dameshek, 1951). It
was found that platelet-associated IgG was increased after splenectomy in some patients but
patients could still maintain remission, suggesting that remission is not necessarily due to
decreased antibody production but rather the absence of platelet destruction in the spleen
(Luiken et al., 1977; McMillan, 1981). In 1974, Handin et al showed that antibody-coated
platelets could be phagocytosed by autologous peripheral leukocytes in vitro, in particular by
neutrophils, suggesting the phagocytosis of antibody-platelet complexes in vivo in ITP (Handin
and Stossel, 1974). Later, the effectiveness of IVIg in ITP management and related studies
further improved our understanding of the FcγR mediated phagocytosis of platelets in the
pathogenesis of the disease. In 1981, Imbach et al reported that IVIg was effective in pediatric
chronic ITP, and in the following two years Fehr et al and Newland et al showed similar
findings in adult ITP (Fehr et al., 1982; Imbach et al., 1981; Newland et al., 1983). It was
59
suspected that the high dose IVIg raised platelet counts in ITP through competitive binding to
FcRs leading to decreased Fc dependent phagocytosis of anti-platelet IgG-coated platelets. This
was supported by the study of Clarkson and colleagues in 1986 showing that infusion of purified
monoclonal antibody against both FcγRII and FcγRIIIA into patients could induce an immediate
increase of platelet counts and ameliorate ITP (Clarkson et al., 1986). In an analogous set of
observations, intravenous anti-D immunoglobulin (IV anti-D) infusions were also found to
increase platelet counts in Rh+ ITP patients, but not in Rh
− patients, suggesting anti-D opsonized
RBC may competitively block FcR-mediated platelet phagocytosis in ITP (Lazarus and Crow,
2003; Salama et al., 1984; Salama et al., 1986). It is now well accepted that FcR-mediated
phagocytosis of antibody-bound platelets is a major mechanism responsible for the increased
platelet destruction in ITP and spleen is the major site for the attack. This process in the spleen
appears to be blocked by therapies such as splenectomy, IVIg, IV anti-D and purified anti-FcγR
antibodies.
It is worth noting that although antibody-mediated platelet destruction is considered the
major mechanism and the spleen is considered the major site for platelet destruction in patients
with ITP, more than one third of patients fail splenectomy and this suggests that the immune
response against platelets can linger outside of the spleen (Provan et al., 2010).
In the last decade, there have been further studies on different subtypes of FcRs in ITP. In
2008, Asahi et al found decreased expression of the inhibitory FcRIIB on monocytes from H.
pylori positive Japanese patients with ITP compared with H. pylori negative patients suggesting
a decreased inhibition of macrophage activation in some patients (Asahi et al., 2008). Later,
Satoh and colleagues found a genetic polymorphism of the FcRIIB gene in patients with ITP
(Satoh et al., 2013). However, this could not be confirmed in another study (Xu et al., 2010).
Overall, the increased platelet destruction in ITP may be a consequence of the increased
activation/inhibition ratio of the differing activating and inhibitory FcR, e.g. increased FcRIIIA
activation with reduced FcRIIB suppression. Further studies are still needed to confirm the role
of FcRIIB receptors in ITP.
The role of natural killer (NK) cells in the development of ITP is controversial. In the study
by Engelhard et al, NK cell activity was higher than normal in a patient with ITP, and was
60
downregulated by IVIg therapy (Engelhard et al., 1986). In 1991, Semple et al showed that
PBMC from patients with ITP have decreased NK cell cytotoxic activity against the NK-
sensitive cell line K562 suggesting defective NK cell activity in ITP (Semple et al., 1991).
Related to this, Soubrane et al found that the decreased NK function in vitro could be rescued by
the injection of soluble anti-CD16 (FcRIII) antibody in a patient with secondary ITP (HIV
associated) (Soubrane et al., 1993). In apparent contrast, however, Garcia-Suarez et al found
increased numbers of CD56+CD3
− NK cells in patients with chronic ITP but they did not
address cytotoxicity (Garcia-Suarez et al., 1993). Later it was shown that the polymorphism of a
pseudogene FcRIIc in ITP was associated with a significantly increased expression of FcRII
on NK cells and NK activation (Breunis et al., 2008). NK cells also express FcRIII and
therefore are capable of recognizing IgG-coated targets to induce lysis of the target cells, a
process known as antibody dependent cellular cytotoxicity (ADCC) but few studies have
examined this in ITP. More studies are required to sort out these observations but it is possible
that the FcRs on CD16+ NK cells are occupied by IgG-platelet complexes, leading to destruction
of platelets and/or megakaryocytes by ADCC.
1.3.3.8 Other pathways of antibody-mediated ITP
Activation of the complement system may also be involved in the platelet destruction in
ITP. In addition to recognition by FcR, the Fc portion of antibodies can also be recognized by
the C1q component of complement leading to activation of the complement cascade and
eventual lysis of the antibody bound targets. In 1979, Cines et al reported increased C3 on the
surface of platelets in a subpopulation of patients with ITP suggesting activation of the
complement system in ITP. The elevated C3 in ITP was confirmed by other groups with
additional findings of increased C4 on the platelet surface (Winiarski and Holm, 1983). More
recently, Peerschke found increased complement activation capacity (CAC) in the plasma of
patients with ITP and this was associated with a decreased circulating absolute IPF and
thrombocytopenia (Peerschke et al., 2010). These studies suggested that complement mediated
platelet destruction may have a role to play in ITP.
It has also been proposed that in certain forms of secondary ITP such as the ITP associated
with HIV infections, autoreactive anti-platelet antibodies may induce platelet destruction by
61
directly binding the platelet and inducing reactive oxygen species (Cines et al., 2014; Nardi et
al., 2001; Nardi et al., 2007). Perhaps related to this, the oxidative stress level was found
significantly increased in pediatric chronic ITP (Zhang et al., 2011). Through gene expression
analysis of whole blood, they found that the oxidative stress-related pathways were among the
most significantly activated pathways in chronic ITP, which was different from acute ITP in
children. Moreover, the authors found that the overexpression of vanin-1, an oxidative stress
sensor in epithelial cells was positively associated with disease progression suggesting potential
prognostic value of vanin-1 in the clinical management of ITP (Zhang et al., 2011).
Increased platelet activation induced by autoreactive antibodies in ITP has also been
proposed but remains disputable (Cines et al., 2014). In 1987, Sugiyama et al identified an
antibody in a patient with ITP that induced significant platelet aggregation (Sugiyama et al.,
1987) and Yanabu et al subsequently showed the presence of anti-platelet CD9 IgG
autoantibodies in ITP plasma capable of activating platelets in an FcγRII-dependent manner
(Yanabu et al., 1993). More recently, Urbanus et al demonstrated that anti-GPIbα antibodies
significantly increased platelet activation via the translocation of GPIbα into lipid rafts and
activation of FcγRIIa in a single case of ITP (Urbanus et al., 2013).
In addition to their binding to platelets, autoreactive antibodies also impair platelet
production through their interactions with megakaryocytes in the bone marrow of patients with
ITP (Cines et al., 2014). As is mentioned above, it has been noted that in ITP there is an
increased apoptosis of megakaryocytes and a decreased platelet production from
megakaryocytes (Dameshek and Miller, 1946; Houwerzijl et al., 2004). In 2003, Chang et al
showed that plasma from ITP patients as well as purified anti-platelet monoclonal antibodies
could inhibit the yield of megakaryocytes from umbilical cord blood mononuclear cells in vitro
(Chang et al., 2003). McMillan et al further showed that anti-platelet antibodies were not only
able to inhibit megakaryocytopoiesis but also decreased ploidy of megakaryocytes, suggesting
impaired megakaryocyte maturation in patients with ITP (McMillan et al., 2004). In addition,
Balduini showed that anti-GPIbα but not anti-GPIIbIIIa antibodies inhibited pro-platelet
formation from umbilical cord blood derived megakaryocytes in vitro, suggesting an additional
mechanism that contributes to the immune thrombocytopenia in ITP (Balduini et al., 2008). Of
interest, however, work in this thesis suggests that in vivo, anti-platelet antibodies may not have
62
a significant role to play in megakaryocyte destruction in passive ITP (Chapter 2). Reasons for
the differences between the in vitro and in vivo megakaryocytes results are not clear.
In conclusion, ITP is a heterogeneous disease and multiple compartments of the immune
system all contribute to the development of platelet and megakaryocyte destruction. The major
immune mechanisms involved are shown in Figure 1.3. Initially, there could be intrinsic defects
in platelets and megakaryocytes or environmental triggers that promote increased clearance of
platelets by activated APCs and enhanced antigen-presentation of platelet epitopes under
inflammatory situations. This could trigger the activation of adaptive autoimmunity against
platelets. Activated autoreactive Th1 and Th17 CD4+ T cells and impaired Tregs may promote
both antibody-mediated and CD8+ T cell-mediated ITP. While CD8
+ T cells mediate ITP
through direct platelet lysis and/or megakaryocyte apoptosis induction, the antibody-mediated
ITP involves several mechanisms including Fc receptor mediated phagocytosis of platelets,
impaired production of platelets, as well as increased activation and consumption of platelets.
1.3.4 Treatment of ITP
A few years ago, the IWG published a guideline for the diagnosis and management of
ITP based on current evidence from the literature and expert opinions (Provan et al., 2010). The
review here is based on that guideline with updated evidence from clinical trials in the last few
years. A therapeutic summary is shown in Table 1.4. A principle recommended in the guideline
was that treatment options should be considered based on the symptoms and needs of the
patients, not on their platelet counts. Two of the treatments are related to my PhD study, i.e. the
allogeneic platelet transfusions and B cell depletion therapy, will be further discussed in future
sections.
Treatment of adult ITP
Patients with platelet counts above 50×109/L should be under observation without any
treatment unless there is active bleeding or under special circumstances that demand a safe level
of platelet counts such as surgery or anticoagulation therapy (Provan et al., 2010). Patients with
platelet counts below 50×109/L but above 20×10
9/L are not necessarily treated without
significant bleeding symptoms. Patients with platelet counts below 20×109/L, however, have a
63
Table 1.4 A brief summary of therapies for the management of ITP. Modified from Provan et al. International consensus report
on the investigation and management of primary immune thrombocytopenia. Blood. 2010;115:168-186.(Provan et al., 2010).
Clinical
situation
Therapy option Recommended dose Approximate
response rate
Toxicities
First-line Prednis(ol)one 0.5-2 mg/kg/d 70%-90% of patients respond
initially. 20-50% of patients
have sustained platelet count.
Vary with length of administration: mood
swings, weight gain, anger, anxiety, insomnia,
Cushingoid faces, dorsal fat, diabetes, fluid
retention, osteoporosis, hypertension, GI
distress and ulcers, etc.
Dexamethasone
40 mg daily for 4 d,
every 2-4 wk for 1-
4 cycles
Methylprednisolone 30 mg/kg/d for 7 d
IV anti-D 50-75 μg/kg Initial response rate similar to
IVIg, typically last 3-4 wk but
may persist for months in
some patients.
Hemolytic anemia (dose-limiting toxicity),
fever/chills.
IVIg 0.4 g/kg/d for 5 d or
infusions of 1
g/kg/d for 1-2 d
Initial response rate up to 80%,
usually transient; occasionally
persists for months.
Headaches, transient neutropenia, renal
insufficiency, aseptic meningitis, thrombosis,
flushing, fever, chills, fatigue, nausea,
diarrhea, blood pressure changes and
tachycardia.
Second-
line
Rituximab 375 mg/m2/wk×4 Initial response rate 60-80%, 2-
year response rate above 40%,
5-year response rate about 20%.
Fever/chills, rash, or scratchiness in throat,
serum sickness and (very rarely)
bronchospasm, anaphylaxis, pulmonary
embolism, retinal artery thrombosis, infection.
Splenectomy Initial response rate above 80%, Hemorrhage, peripancreatic hematoma,
64
approximately two-thirds
achieve a lasting response.
subphrenic abscess, wound infection, death,
pneumococcal infection, fever, overwhelming
sepsis syndrome, thrombosis.
TPO-RAs Romiplostim doses
1-10 μg/kg
Initial response rate above 80%,
sustained response with
continual administration of the
drug.
Headaches, fatigue, epistaxis, arthralgia and
contusion, increased bone
marrow reticulin, worsening thrombocytopenia
upon discontinuation, thrombosis.
65
higher risk of bleeding and therefore most should be treated despite of bleeding symptoms
(Provan et al., 2010; Thienelt and Calverley, 2009).
Fist-line treatments for ITP include steroids, IV anti-D and IVIg (Provan et al., 2010).
Steroids therapies are a conventional and affordable first-line treatment option but have
systemic and sometimes severe side effects that greatly reduce the quality of life of patients
while IV anti-D and IVIg are more expensive but better tolerated (Provan et al., 2010).
Commonly prescribed steroids include prednisone, dexamethasone, and methylprednisolone.
Prednisone is recommended to be administrated at 0.5-2 mg/kg/d until the platelet count
increase is above 30-50×109/L and then administered at tapered doses or tapered if there is no
response after 4 weeks. Dexamethasone was recommended at 40 mg/day for 4 days for once or
for 4 cycles at an interval of 2 weeks. In the literature, high dose methylprednisolone is usually
prescribed at 30 mg/kg for 7 days to patients who had failed other first-line therapies. IV anti-D
is recommended for RhD+ non-splenectomized patients with ITP without autoimmune
hemolytic anemia at a dose between 50-75 μg/kg for 4-5 days. IVIg is recommended at 0.4 g/kg
for 5 days or 1 g/kg for 1-2 days.
Second-line treatments are generally recommended for patients who fail one or more of the
first-line therapies with the goal of achieving relatively safe platelet counts (Provan et al., 2010).
Second-line treatment options include azathioprine, cyclosporin A, cyclophosphamide, danazol,
dapsone, mycophenolate mofetil, rituximab, splenectomy, TPO-RAs and vinca alkaloids
(Provan et al., 2010). Of these, splenectomy has shown the highest long-term response rate,
however about 20% fail to respond and another 10% patients may relapse after an initial
response. Therefore, splenectomy is not recommended to patients within 6 months from the
initial diagnosis of ITP. Other commonly prescribed second-line treatments include rituximab
and the TPO-RAs, eltrombopag and romiplostim, with clear advantages including low toxicity
and improved quality of life (Provan et al., 2010).
Emergency treatments are recommended to patients ‘needing surgical procedures, at high
risk of bleeding, or with active central nervous system (CNS), GI, or genitourinary bleeding’,
66
including switching among first-line treatments or a combination of them or together with
platelet transfusions (Provan et al., 2010).
Treatment of pediatric ITP
A difference between adult and pediatric ITP is that most children undergo spontaneous
remission whereas adults usually do not. Moreover, only 3% of the children with ITP have
clinically significant bleeding symptoms. Therefore, for newly diagnosed patients whose
platelet counts are above 20×109/L without serious bleedings, observation without intervention
is recommended (Provan et al., 2010; Thienelt and Calverley, 2009). For patients with platelet
counts below 20×109/L or with serious bleedings, IV anti-D, IVIg and prednisone are
recommended as first-line treatments. For the management of life-threatening bleedings, platelet
transfusions together with IVIg, IV anti-D, or prednisone were recommended (Provan et al.,
2010; Thienelt and Calverley, 2009).
Management of ITP in pregnancy
Both newly diagnosed ITP during pregnancy and preexisting ITP present during pregnancy
are usually investigated and managed differently than regular adult ITP. During the first two
trimesters of pregnancy, interventions are not recommended unless necessary when maternal
platelet counts are below 20×109/L or there is significant bleeding. In the last a few weeks of
pregnancy, a platelet count of 50-75×109/L or higher was recommended for anesthesia. First-
line treatments are similar as with adult ITP, however, low dose steroids are recommended to
minimize side effects (Provan et al., 2010; Thienelt and Calverley, 2009). Evidence for second-
line treatments are limited but combinations of first-line treatments and rituximab have been
shown effective. Splenectomy was recommended only during the second trimester when
necessary.
1.3.5 Intravenous immunoglobulin (IVIg) in ITP
Intravenous immunoglobulin (IVIg) is prepared from pooled plasma of thousands of blood
donors and mainly contains polyvalent IgG antibodies (Lazarus and Crow, 2003). It was initially
used to treat hypogammaglobulinemic diseases (Etzioni and Pollack, 1989) but now, it has
67
become a first-line treatment for the management of ITP as well as other autoimmune diseases
(Etzioni and Pollack, 1989; Lazarus and Crow, 2003).
In 1981 Imbach et al first reported increased platelet counts in pediatric patients with ITP
after IVIg infusion in a pilot study (Imbach et al., 1981). After the initial success, Imbach and
collaborators recruited more patients for IVIg therapy and showed that more than eighty percent
of patients responded to IVIg, with a mean response time of 9 days (Imbach et al., 1985).
Subsequently, IVIg was found effective in upregulating the platelet counts of adults with ITP
with comparable initial response rates (Bussel et al., 1983; Fehr et al., 1982; Newland et al.,
1983; Provan et al., 2010). However the treatment free response rate 4-6 weeks after the IVIg
treatment was below 50% (Bussel et al., 1983; Fehr et al., 1982; Newland et al., 1983; Provan et
al., 2010). Adverse events of IVIg therapy mostly are benign (Provan et al., 2010; Ryan et al.,
1996). Chills and headaches are the most common, while other symptoms may occur including
‘transient neutropenia, renal insufficiency, aseptic meningitis, thrombosis, flushing, fever’,
‘fatigue, nausea, diarrhea, blood pressure changes and tachycardia’ (Provan et al., 2010; Ryan
et al., 1996).
Possible mechanisms of action of IVIg have been investigated since its early application
in ITP. In the study by Fehr et al in 1982, administration of autologous 99m
Tc-labeled anti-Rh(D)
IgG antibody opsonized RBCs in addition to IVIg showed prolonged RBC clearance time,
suggesting that IVIg inhibited platelet clearance by macrophages (Fehr et al., 1982). Increased
hemolysis determined by haptoglobin level after IVIg therapy supported this theory (Salama et
al., 1983). Studies comparing intact immunoglobulins and Fc-depleted F(ab’)2 fragments
showed that the Fc portion of immunoglobulins is needed for the function of IVIg (Burdach et
al., 1986; Crow et al., 2001; Tovo et al., 1984). These studies clearly indicate that at least one
mechanism for the action of IVIg is through an inhibition of FcR-mediated platelet clearance by
macrophages (Burdach et al., 1986; Crow et al., 2001; Tovo et al., 1984).
As mentioned in session 1.3.2.8, Clarkson et al showed that the blockade of FcγRII and
FcγRIIIA receptors ameliorated ITP, indicating a major role of these two receptors in the
clearance of platelets in ITP (Clarkson et al., 1986). Using an anti-platelet monoclonal antibody
induced transient thrombocytopenia (passive ITP) mouse model in FcRI, FcRIIB and FcRIII
68
deficient mice, Samuelsson et al showed that IVIg downregulated the platelet clearance through
inhibition of FcRIII in association with upregulation of the inhibitory receptor FcRIIB
(Samuelsson et al., 2001). This was concurrently confirmed in the studies by Crow and Teeling
(Crow et al., 2003a; Teeling et al., 2001). Furthermore, Siragam et al found that IVIg
specifically downregulated FcRIIIA on CD11c+ DCs independent of FcRIIB (Siragam et al.,
2006).
In addition to the modulation of FcR mediated platelet destruction, IVIg also ameliorates
ITP through other mechanisms. For example, in 1985, Delfraissy et al proposed a mechanism of
IVIg through the improvement of suppressor cells, based on their findings of normalized
proliferation of the PBMCs from IVIg responders upon Concanavalin A stimulation in vitro
(Delfraissy et al., 1985). Kaneko et al in Ravetch group associated the anti-inflammatory role of
IVIg with the sialylation of its Fc fragments (Kaneko et al., 2006).In the same year, Leytin et al
examined the effect of IVIg on platelets and found significantly decreased platelet apoptosis
after IVIg treatment (Leytin et al., 2006). Our previous work a few years ago showed increased
suppressive Tregs in IVIg treated ITP mice (Aslam et al., 2012). More recently, Leontyev et al
examined the impact of IVIg therapy on cytokine production in antibody-mediated passive ITP
mice (Leontyev et al., 2014). They found that in passively-induced ITP mice of either the
BALB/c or C57BL/6J genetic backgrounds, IVIg stimulated the production of IL-4, IL-10, IL-
11, IL-23, granulocyte-macrophage–colony-stimulating factor (GM-CSF), monocyte
chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β) and tumor
necrosis factor α (TNF-α). Increased MCP-3 and chemokine (C-C motif) ligand 5 (CCL5, also
known as RANTES, an abbreviation of ‘released upon activation, normal T cell expressed and
secreted’) were also increased by IVIg treatment, but only BALB/c strain mice (Leontyev et al.,
2014). Taken together, it is clear that the mechanism of action of IVIg is complicated and still
not fully elucidated.
1.3.6 Platelet transfusions in ITP
Since the 1950s, allogeneic platelet transfusions have been used clinically to rescue
platelet counts in thrombocytopenic disorders such as the severe thrombocytopenia secondary to
leukemia (Han et al., 1966; Hirsch and Gardner, 1952; Slichter, 1980). However, in the case of
69
ITP, historically, allogeneic platelet transfusions were not recommended. In the report by
Sprague et al in 1952, entitled: “Platelet transfusions and the pathogenesis of idiopathic
thrombocytopenic purpura”, it was suspected that platelets transfused into patients with ITP
‘disappear within several hours from the circulation’ as suggested by the quick drop of platelet
counts after platelet transfusions, whereas in patients with aplastic anemia, transfused platelets
could survive for 4-6 days (Sprague et al., 1952). Therefore, although substantial evidence was
lacking, platelet transfusions were subsequently considered ‘unnecessary and undesirable,
considering the risks of hepatitis and the lack of effectiveness’ (Ahn and Harrington, 1977).
There were also assumptions that patients with ITP would not require platelet transfusions
‘because the compensatory marrow response is associated with a reduction in megakaryocyte
maturation time and release of young hyperfunctional platelets’ (Slichter, 1980). Although
platelet production by megakaryocytes has clearly been shown to be impaired in ITP (Chang et
al., 2003; Li et al., 2007) and to date, no supporting evidence exists that the platelets from
patients with ITP are actually ‘hyperfunctional’, the concept that platelet transfusions should be
avoided for the management of ITP is still prevailing (Provan et al., 2010; Thienelt and
Calverley, 2009).
Contrary to the dogma that platelet transfusions should be avoided in ITP, there is evidence
that supports the effectiveness of platelet transfusions in the immediate control of active
bleeding. In 1986, a retrospective evaluation of 11 patients with ITP showed efficient control of
active bleeding after platelet transfusions with approximately half of the transfusions increasing
immediate platelet counts and 16% of the transfusions inducing next-day platelet count
increments (Carr et al., 1986). This study not only challenged the dogma that platelet
transfusions were not effective in ITP, but suggested a therapeutic benefit of platelet
transfusions in ITP. Furthermore, Salama et al showed that massive platelet transfusions given
as the only treatment for 10 cases of refractory ITP increased the platelet counts and stopped
active bleeding in all patients without serious side effects (Salama et al., 2008). In another study,
Spahr et al demonstrated that platelet transfusions administered concurrently with IVIg could
significantly raise the platelet counts to 50×109/L (averaged below 10×10
9/L before treatment)
in more than half of patients with ITP (Spahr and Rodgers, 2008). Similarly, in the IWG
guideline, platelet transfusions in combination with other first-line treatments, including IVIg,
70
were recommended for the management of life-threatening bleeding or preparation for surgery
(Provan et al., 2010). Concurrently, Thachil et al also recommended that in ITP and other
immune mediated thrombocytopenic disorders, platelet transfusions ‘should not be withheld if a
safe platelet count is preferable’ (Thachil, 2010). Overall, to date, the clinical evidence suggests
that platelet transfusions may in fact be effective in ITP and even more importantly, could
control active bleeding within short time periods. However, since there is limited evidence in
the literature, future evaluations of the effectiveness and safety of allogeneic platelet
transfusions in the management of ITP are still needed.
1.3.7 Rituximab and B cell depletion therapy
Rituximab is a purified monoclonal chimeric antibody derived from fusing the V region of
a mouse anti-human CD20 antibody to a human IgG1 constant segment (Reff et al., 1994).
Rituximab targets CD20 on B cells and induces B cell depletion in vivo. It was initially licensed
as an immunotherapy for B cell lymphoma and now is also used as a treatment for several
autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, autoimmune
haemolytic anemia, multiple sclerosis, diabetes and ITP (Brodie et al., 2008; Cohen et al., 2006;
Cross et al., 2006; Gurcan et al., 2009; Hu et al., 2007; Leandro et al., 2002; Lund and Randall,
2010; Pescovitz et al., 2009; Stasi et al., 2001). Several mechanisms of how rituximab causes B
cell depletion in vivo have been proposed including Fc-receptor mediated phagocytosis of
antibody coated B cells, killing of B cells through antibody-dependent cellular cytotoxicity
(ADCC), complement-dependent lysis of B cells or sensitization of B cells to apoptosis
(Cardarelli et al., 2002; Golay et al., 2000; Gurcan et al., 2009; Lefebvre et al., 2006; Pedersen
et al., 2002). The application of rituximab in ITP management was first reported by Stasi et al in
2001 (Stasi et al., 2001). In their study, rituximab was given at a dose of 375 mg/m2 once
weekly for 4 weeks to 25 patients with chronic ITP who had failed at least 2 different therapies.
More than 50% of the patients responded to rituximab, and 20% of them had a complete
response and 28% still had treatment-free remission after 6 months. The therapy was well
tolerated in all patients with only minor side effects that were mostly fevers and chills. With
larger scale patient recruitment and longer term follow up, the overall response of rituximab in
adult ITP has been reported to be approximately 60%, with a 2-year response rate of 40% and a
5-year remission rate of 20% (Khellaf et al., 2014; Patel et al., 2012).
71
Rituximab in combination with other therapies, such as corticosteroids or
thrombopoietin receptor agonists, have also shown synergistic effects (Gudbrandsdottir et al.,
2013; Zhou et al., 2015). For example, in a randomized clinical trial, rituximab and
dexamethasone combined therapy showed significantly higher response rates than
dexamethasone monotherapy after 6 months and longer time to relapse (Gudbrandsdottir et al.,
2013). Moreover, compared with classical corticosteroid therapy or splenectomy, rituximab is
well-tolerated with fewer side effects. Of the 248 rituximab treated ITP patients in the study by
Khellaf et al, only 19% of patients had adverse events, most of which were minor except 3
events (approximately 1%) that required interruption of the treatment (Khellaf et al., 2014).
Therefore with the consideration of the balance between efficacy and side effects, rituximab is a
preferred treatment for the management of ITP.
Efforts have been made to understand the therapeutic mechanisms of rituximab since its
initial success in ITP management (Stasi et al., 2001). It was suspected that rituximab could
rescue the antibody-mediated ITP through removal of B cells (Stasi et al., 2001). Although it
was confirmed in subsequent studies that the antibody-platelet antibody titers decreased after
remission in some patients, it was also found that the antibody titer may be unchanged in
patients under remission as well (Cooper et al., 2012). This suggests that there must be some
other antibody-independent mechanism(s) of rituximab therapy. The effectiveness of rituximab
in some antibody negative patients also indicates alternative explanations for the in vivo effect
of rituximab (Cooper et al., 2012). In 2007, Stasi et al reported normalized Th1/Th2 (IFN-γ+/IL-
4+ of CD4
+ T cells) and Tc1/Tc2 (IFN-γ
+/IL-4
+ of CD8
+ T cells) ratios in the peripheral blood of
rituximab responders at 3 and 6 months after treatment but not in patients with active ITP nor in
rituximab non-responders, suggesting an immunomodulatory effect of rituximab on both CD4+
and CD8+ T cell responses (Stasi et al., 2007). Nazi et al also showed that the number of IFN-γ
+-
producing T cells upon exogenous antigen stimulation was decreased in patients with ITP who
received rituximab administration compared with patients without rituximab treatment (Nazi et
al., 2013). Subsequently, further examinations of the CD4+ T cells revealed restored Treg
number and function in the peripheral blood in rituximab responders (Stasi et al., 2008). Of
interest, the number of Tregs remained low in the spleen of rituximab non-responders (Audia et
72
al., 2011). However, it remains unclear how rituximab regulates T cell-mediated immune
responses in ITP.
1.3.8 Animal models of ITP
Animal models of human diseases provide a valuable tool to facilitate investigations into
the pathogenesis of disease and to evaluate therapeutic reagents and procedures prior to clinical
trials. Several animal models of ITP have contributed to our understanding of ITP and are still
of relevance today in human medicine as reviewed by Semple (Semple, 2010). Animal models
of ITP can be divided into three categories: passive ITP (anti-platelet serum/antibody induced),
secondary ITP (drug/infections/other diseases induced) and active ITP (anti-platelet immune
cell-induced).
Passive ITP
Passive ITP models were the earliest developed and are still the most commonly used
animal models of ITP. Since the 1950s, passive ITP models induced by anti-platelet sera in
guinea pigs, swine, rodents, and rabbits etc have been used for the understanding of the
pathology and treatments of thrombocytopenia (Semple, 2010). For example, in 1960, using a
guinea pig passive ITP model induced by anti-platelet serum, Rossolini et al showed that
cortisone was effective in ameliorating thrombocytopenia. A few years later, cortisone was
further found effective in a neonatal thrombocytopenia model as well (Chieffi et al., 1964;
Semple, 2010). In the 1970s and 1980s, rodent passive ITP models were used for the study of
TPO and megakaryopoiesis during thrombocytopenia (Dassin et al., 1983; Kalmaz and
McDonald, 1981; Nakeff and Roozendaal, 1975; Rolovic et al., 1970; Semple, 2010). These
early models using immune animal sera laid the groundwork for understanding different
mechanisms related to ITP. With the advent of monoclonal antibody technology, anti-platelet
monoclonal antibodies are commonly used to induce thrombocytopenia today.
In 1981, the earliest anti-platelet monoclonal antibodies (mAb) were developed by
McMichael and Newman respectively (McMichael et al., 1981; Newman et al., 1981). Since
then, passive ITP models induced by monoclonal antibodies have been widely used. For
example, the passive ITP model was used to improve our understanding of the therapeutic
73
effects of IVIg in ITP. In 1987, using the monoclonal antibody induced passive ITP rat model,
Tsubakio et al showed the IVIg as an effective therapy for ITP (Tsubakio et al., 1987). Later, the
anti-platelet antibody-mediated passive ITP mouse model was commonly used in further studies
of different FcRs in ITP and IVIg therapies by several groups, including the studies by
Samuelsson et al and Siragam et al mentioned in session 1.3.4 (Crow et al., 2003a; Samuelsson
et al., 2001; Siragam et al., 2006; Teeling et al., 2001). More recently, the passive ITP mouse
model also helped to evaluate the role of FcRIIB in ITP development. In FcRIIB deficient
mice, passively infused anti-platelet antibodies could still mediate thrombocytopenia and IVIg
as well as anti-CD44 antibodies were found effective in certain strains of FcRIIB deficient
mice suggesting a dispensable role of the FcRIIB in ITP (Crow et al., 2015; Leontyev et al.,
2012). In addition, there are studies using splenectomized mice to help understand the role of
IVIg in splenectomized patients (Leontyev et al., 2012; Schwab et al., 2012). Taken together,
these studies using the passive ITP mouse model in genetically altered animals not only
improved our understanding of both ITP and the in vivo effects of IVIg on the immune system.
In the last decade, the passive ITP model has also been utilized for the study of the immune
functions of platelets and their interaction with other immune cells as well. In 2004, our group
showed that LPS could bind to TLR4 on platelets and induce increased platelet destruction and
development of thrombocytopenia in vivo (Aslam et al., 2006). The LPS also significantly
enhanced FcR-mediated phagocytosis of anti-platelet IgG opsonized platelets in vitro,
suggesting that signaling through TLRs on platelets may exacerbate ITP (Semple et al., 2007).
Passive ITP models also allowed for comparisons of different monoclonal antibodies in the
pathogenesis of ITP. For instance, it has been shown that anti-GPIb antibodies but not anti-
GPIIbIIIa antibodies are capable of inducing Fc-independent desialylation of sialic acid on
platelets and this leads to increased platelet destruction through the Ashwell-Morell receptors
and development of thrombocytopenia in vivo (Li et al., 2014; Li et al., 2015b). However, there
are still controversies and future investigations are still needed to confirm the role of anti-GPIb
antibodies in platelet desialylation and ITP development. We also compared different anti-GPIb
and anti-GPIIbIIIa antibodies and their effects on megakaryocytes in vivo and this will be
discussed in Chapter 2.
74
Studies using CD47 deficient mouse models also revealed a potential novel mechanism
in ITP. It has been shown that CD47 deficient platelets from CD47+/−
or CD47−/−
mice induced
increased phagocytosis by macrophages in vitro as well as rapid platelet clearance in vivo
(Olsson et al., 2005). The CD47 deficient platelets also exacerbated the antibody-mediated
passive ITP in mice (Olsson et al., 2005). Moreover, Guo et al have shown that a bacteria strain
Escherichia coli O157:H7 could down regulate CD47 expression on platelets and upregulate the
phagocytosis of platelets by macrophages to induce thrombocytopenia in mice (Guo et al., 2009).
Secondary ITP Models
In the literature, there are a broad variety of different secondary ITP animal models which
are relevant to the study of secondary ITP in humans. For example, quinine induced ITP was
probably the earliest secondary ITP animal model developed. In 1964, Wakisaka et al studied
quinidine induced ITP in dogs (Semple, 2010; Wakisaka et al., 1964) and in 1977, Kekomaki et
al studied aspirin induced ITP in rabbits. In both cases, it was the binding of the drug to the
animal’s platelets that triggered antibody production against platelet-drug complexes and the
ultimate destruction of platelets (Kekomaki et al., 1977). Other drugs that may cause secondary
ITP in mice include sulphonamide, phenobarbitol, pantethine, gold, and heparin.
Infection-induced secondary ITP in animals was first reported by Musaji et al (Musaji et
al., 2004). The authors showed exacerbated thrombocytopenia when mice with passive ITP were
infected with mouse viruses (Musaji et al., 2004). Subsequently, Tremblay et al confirmed
Semple et al’s in vitro results by showing that LPS could exacerbate passive ITP in mice
(Tremblay et al., 2007). These latter models may be a useful tool to improve our understanding
of the immunological functions of platelets and their role in regulating the immune system.
Active ITP
Active murine ITP was developed as an adoptive transfer model to induce both antibody-
and T cell-mediated ITP by our group (Chow et al., 2010). In this model, GPIIIa (CD61, β3
integrin) knockout (KO) mice were immunized by multiple wildtype (WT, CD61+) platelet
transfusions and when immune, they are sacrificed and their splenocytes adoptively transferred
into mice with severe combined immunodeficiency (SCID) to induce ITP (Chow 2010). By
75
removal of either CD8+ T cells or B cells from the splenocytes before adoptive transfer,
antibody-mediated or CD8+ T cell-mediated ITP respectively, could be induced. It was found
that while IVIg treatment was effective in raising platelet counts in antibody-mediated ITP, it
did not affect the thrombocytopenia mediated by CD8+ T cells (Chow et al., 2010). Moreover,
subsequent examination of the thymus from ITP SCID mice revealed increased retention of
Tregs in the thymus which may help explain the Treg deficiency in human ITP (Aslam et al.,
2012). In this thesis, I examined two ITP therapies in our active murine model: allogeneic
platelet transfusion therapy and B cell depletion therapy (Chapters 3 and 4). My work not only
showed consistent findings as in human patients but also revealed novel mechanisms that may
help to explain certain phenomena in human ITP.
In summary, animal models of ITP have improved our understanding of the anti-platelet
immune responses in ITP and the actions of therapeutic reagents. Furthermore, it appears that
the active ITP model could be helpful for the preclinical evaluation of novel therapies for
patients with ITP.
76
1.4 Rationale, Hypothesis and Specific Aims
1.4.1 Rationale
Immune thrombocytopenia (ITP) is a heterogeneous autoimmune disease where both B
cell and CD8+ T cell abnormalities are involved. In the pathogenesis of the disease, the relative
contributions of B cells and CD8+ T cells are difficult to distinguish from each other in patients.
In addition, the evaluation of how different therapies rescue the disease under these different B
or T cell circumstances is even more complicated. Moreover, megakaryocytes are not routinely
examined in ITP despite their critical role for platelet production and therefore our knowledge
of megakaryocytes before and after treatments is still limited. Animal models of ITP thus are
valuable tools that will allow us to differentiate the antibody-mediated and CD8+ T cell-
mediated ITP and examine the mechanisms of treatments as well as their effect on the
megakaryocytes and platelet production.
Although anti-platelet antibodies with different antigen specificities and isotypes have been
widely reported in human ITP, the antibody effects on megakaryocytes and thrombopoiesis have
not been well characterized. In addition, IVIg is a first-line treatment for ITP and has been
found effective in reducing anti-platelet antibody-mediated peripheral platelet destruction but its
effect on megakaryocytes in vivo is not clear. Thus, characterization of the effects of different
antibodies and IVIg on megakaryocytes may improve our understanding of the disease and the
IVIg therapy.
Allogeneic platelet transfusion therapy is another treatment option under emergent
situations for the management of ITP but its application in ITP is still controversial.
Historically, its use was frowned upon for the management of ITP, however, limited clinical
evidence has emerged that suggests that transfusions may be effective in raising platelet counts
and controlling bleeding symptoms in ITP. Investigations on allogeneic platelet transfusions in
either antibody-mediated or T cell-mediated ITP using our active ITP mouse model may provide
helpful information for clinical practice.
B cell depletion in vivo using rituximab is also a common treatment for the management of
ITP. The unchanged antibody titer in rituximab responders and the effectiveness of rituximab in
77
certain anti-platelet antibody negative patients suggest that antibody-independent therapeutic
effects of rituximab exist but their mechanism(s) is unknown. Examination of the effect of B
cell depletion on CD8+ T cell-mediated ITP in our mouse model may uncover novel
mechanisms of the treatment and improve our understanding of the interaction between B cells
and T cells in ITP.
1.4.2 Hypothesis
1) Antibodies of different specificities and isotypes induce thrombocytopenia in vivo but do
they interfere with megakaryocytes in the bone marrow?
2) IVIg can rescues antibody-mediated thrombocytopenia but does it have effects on
megakaryocytes?
3) Allogeneic platelet transfusions have immunomodulatory effects and may suppress CD8+ T
cell-mediated ITP.
4) Does B cell depletion therapy affect suppress CD8+ T cell-mediated ITP?
1.4.3 Specific aims
1) Compare the effects of different anti-platelet antibodies/sera on megakaryocytes and
thrombopoiesis and evaluate the efficacy of IVIg on megakaryocytes in antibody-mediated
passive ITP.
2) Examine the effect of allogeneic platelet transfusions on CTL-mediated active ITP, including
their effects on megakaryocytes and analyze the CTL responses against platelet CD61 versus
MHC antigens in active ITP.
3) Evaluate the efficacy of anti-mouse CD20 IgG2a antibody on B cell depletion in vivo and
investigate the effects of the anti-CD20 antibody on CTL-mediated ITP.
78
Chapter 2
Thrombocytopenia And Megakaryocyte Counts Are
Differentially Controlled By The Specificity Of Anti-
Platelet Antibodies In A Murine Model Of Passive
Immune Thrombocytopenia (ITP)
Li Guo*,1-3
Rukshana Aslam*,1,2
Kristin Hunt,1,2
Yu Hou,1,2
Anne Zufferey, 1,2
Edwin R.
Speck,1,2
Rick Kapur,1,2,4
Alan H. Lazarus,1-6
Heyu Ni,1-6
and John W. Semple.1-7
1The Toronto Platelet Immunobiology Group,
2Keenan Research Centre for Biomedical Science
of St. Michael's Hospital, 3Institute of Medical Science, University of Toronto,
4Canadian
Blood Services, Departments of 5 Medicine,
6 Laboratory Medicine and Pathobiology, and
7
Pharmacology, University of Toronto, Toronto, Ontario, Canada.
*L.G and *R.A. contributed equally to the paper.
79
2.1 Abstract
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by
increased peripheral immune platelet destruction and megakaryocyte defects in the bone
marrow. Although ITP was originally thought to be primarily due to antibody-mediated
autoimmunity, it is now clear that T cells can also play a significant role in the disease.
However, the exact interplay between platelet destruction, megakaryocyte dysfunction, and the
elements of both humoral and cell-mediated immunity in ITP remain incompletely understood.
While most studies have focused on immune platelet destruction in the spleen, an additional
possibility is that the anti-platelet antibodies also have effects on bone marrow megakaryocytes.
To address this, we utilized an in vivo passive ITP mouse model, where BALB/c mice were
administered various anti-GPIIb, GPIIIa or GPIb monoclonal antibodies or anti-platelet sera,
followed by platelet counts and bone marrow megakaryocytes enumeration. Our results show
that after 24 hours, all the different anti-platelet antibodies/sera induced significant
thrombocytopenia in recipient mice. IVIg raised the platelet counts in all the thrombocytopenic
mice except mice in NIT-E antibody (an anti-GPIb antibody) induced group. Compared with
naïve control mice, however, histological examination of the bone marrow revealed that only
two antibody preparations (mouse-anti-mouse β3 sera and an anti-GPIIb monoclonal antibody,
MWReg30) increased bone marrow megakaryocyte counts, and IVIg only partially normalized
the number of megakaryocytes in MWReg30 group. Our study suggests that most of the anti-
platelet antibodies may not have direct effect on megakaryocytes in vivo, and the bone marrow
effects observed in ITP may involve T cell responses. Moreover, IVIg may not directly affect
thrombopoiesis.
80
2.2 Introduction
Immune thrombocytopenia (ITP) is an autoimmune bleeding disease characterized by an
isolated thrombocytopenia without any other hematological abnormalities except a possible
anemia due to blood loss (Rodeghiero et al., 2009). The pathogenesis of ITP is complex both at
the level of thrombohaemostasis and immune system regulations. For example, the
thrombocytopenia of ITP is due to at least two major mechanisms: either increased peripheral
platelet destruction in the spleen or reduced platelet production from bone marrow (Ballem et
al., 1987; Cines et al., 2014; Shulman et al., 1965). The mechanism of peripheral platelet
destruction in ITP has been studied extensively and is thought to be primarily due to increased
FcR-dependent phagocytosis of opsonized platelets by splenic macrophages (Cines et al., 2014;
Harrington et al., 1951; Shulman et al., 1965). This immune platelet destruction is associated
with a vast array of peripheral and splenic B cell and T cell defects involving, for example,
cytokine defects, Th1-skewness, regulatory T cell abnormalities and loss of tolerance (Cines et
al., 2014; McKenzie et al., 2013).
Less is known, however, about the immune mechanism(s) of reduced platelet production in
ITP although there are some early and recent clues (Dameshek and Miller, 1946; Houwerzijl et
al., 2004). Probably the first report related to reduced platelet production in ITP was in 1946
when Dameshek and Miller examined the bone marrow of patients with ITP and found
increased numbers of megakaryoblasts along with elevated numbers of degenerated
megakaryocytes and decreases in the number of mature megakaryocytes shedding platelets
(Dameshek and Miller, 1946). Houwerzijl et al. confirmed these findings by studying the fine
ultrastructural characteristics of bone marrow megakaryocytes from patients with ITP using EM
(Houwerzijl et al., 2004). Of interest, they observed apoptotic morphological abnormalities of
megakaryocytes such as cytoplasmic vacuolization and chromatin condensation and this was in
consistency with increased caspase-3 expression in the megakaryocytes (Houwerzijl et al.,
2004). The immune nature of the megakaryocyte abnormalities was first shown by two groups
that both demonstrated reduced in vitro megakaryocyte growth upon addition of anti-platelet
81
autoantibodies with various specificities e.g. anti-GPIb and anti-GPIIbIIIa (Chang et al., 2003;
McMillan et al., 2004).
More recently, it has become evident from murine models that cytotoxic T cells may also
play a significant role in megakaryocyte destruction in ITP (Chow et al., 2010; Guo et al.,
2014). However, the exact interplay between megakaryocyte dysfunction, platelet production
and the elements of both humoral and cell mediated immune systems remain ambiguous. To
better address how anti-platelet antibodies solely affect megakaryocyte numbers and
morphology in vivo, we utilize a well-characterized murine model of passive ITP and
demonstrate that while most antibodies mediate thrombocytopenia, not all are associated with in
vivo megakaryocyte effects. The results suggest that although megakaryocytes and platelets
share similar epitopes that could be recognized by anti-platelet antibodies, most antibodies do
not have direct effects on megakaryocytes in vivo. If so, the megakaryocyte abnormalities in
patients with ITP may occur due to other immune mechanisms such as T cell-mediated immune
responses.
82
Table 2.1 Details of polyclonal (serum) and monoclonal antibodies used for ITP induction
TABLE 1. Details of polyclonal (serum) and monoclonal antibodies used
for ITP induction
Name Sera/mAb Specificity Host Isotype Dose
Anti-β3 sera β3 integrin mouse IgG 200 – 250μL
RAMS sera mouse platelet rabbit IgG 5μL
MWReg30 mAb αIIb integrin rat IgG1 2-4μg
2C9.G2 mAb β3 integrin Armenian Hamster IgG1 10ug
NIT-B mAb glycoprotein Ib mouse IgG2a 1μg
NIT-E mAb glycoprotein Ib mouse IgG2b 1μg
NIT-G mAb glycoprotein Ib mouse IgG1 1μg
NIT-H1 mAb glycoprotein Ib mouse IgG1 1μg
Anti-β3=mouse anti-mouse β3 serum; RAMS=rabbit anti-mouse platelet serum.
83
2.3 Materials and Methods
2.3.1 Mice
BALB/c mice (H-2d, Charles River Laboratories, Montreal, QC, Canada) were used as
antibody recipients. BALB/c CD61 (GPIIIa, β3 integrin) knockout (KO) mice were supplied by
Dr. Heyu Ni and used as a source of mouse anti-mouse β3 sera. All mice were 8-12 weeks of
age and all studies were approved by the St. Michael’s Hospital Animal Care Committee.
2.3.2 Anti-platelet antibodies and Passive ITP
Rabbit-anti-mouse platelet serum (RAMS, Cedarlane Laboratories, Mississauga, ON) was
obtained from Dr. Alan Lazarus (St. Michael’s Hospital). Monoclonal antibodies (mAbs)
MWReg30 and 2C9.G2 were obtained from BD Biosciences (Cat No. 553847, 553344).
Monoclonal antibodies NIT-B, NIT-E, NIT-G, and NIT-H1 were obtained from Dr. Heyu Ni
(St. Michael’s Hospital). Mouse-anti-mouse β3 sera (i.e. anti- CD61, or anti-GPIIIa) was
obtained from β3 KO mice immunized with syngeneic wildtype (WT) platelets which are
positive for β3 as previously described (Chow et al., 2010; Guo et al., 2014). The antigen
specificities, isotypes as well as the doses used for ITP induction of all the sera and antibodies
are listed in Table 2.1. Doses were determined according to literature and our experience in
inducing comparable thrombocytopenia in all mice (Semple, 2010). PBS was used as a sham
control injection. To passively induce ITP, all sera and monoclonal antibodies were
administered by an intraperitoneal (ip) injection. For intravenous immunoglobulin (IVIg)
treatment of ITP, IVIg was administered ip at 2 g/kg, 5 min prior to the antibody injection.
2.3.3 Platelet counts
Mice were bled via the saphenous vein and 10 μL peripheral blood was collected into PBS
with 10% CPDA. Platelet counts were measured using a Beckman Coulter Counter-LH750
hematology analyzer as previously described (Chow et al., 2010; Guo et al., 2014).
2.3.4 Bone marrow histology and megakaryocyte count
84
Mice were sacrificed 24 h after monoclonal antibody or anti-platelet serum injection.
Femurs were collected, dissected of muscle tissue and the epiphyses were cut off and the bone
shafts were placed in fixative (B+ fixative; BBC Biochemicals) for 12 hours. The bones were
further decalcified with 10% nitric acid for 2 hours and then processed and embedded in
paraffin. Sections were stained with hematoxylin and eosin (H&E). All pictures were taken
using the CellSens Standard software under a 40× objective lens (Olympus BX50), with the
numerical aperture of objective lens 0.75. Images of 10 random fields (450μm×320μm/field)
throughout each slide were taken from every mouse (Guo et al., 2014). The number of
megakaryocytes on each image was recorded for the calculation of average value.
2.3.4 Statistical analysis
Statistical analyses were performed using a computer software Prism (GraphPad Software,
Version 6.0, San Diego, CA). Data are presented as mean ± standard deviation (SD). One-way
ANOVA with Tukey’s post-hoc test and t test were used for data analysis, and p values of less
than 0.05 were regarded as significant.
85
2.4 Results
2.4.1 The severity of thrombocytopenia varies depending on the anti-platelet
antibody used
Figure 2.1 A shows the platelet counts of BALB/c mice 24 h after injection of either PBS,
or the indicated anti-platelet sera or mAbs. Compared with the PBS control, the anti-β3 sera (i.e.
anti-GPIIIa or anti-CD61 sera) and RAMS induced the most severe (p<0.0001)
thrombocytopenia in recipient mice by 24 h post-injection. All the mAbs also induced
significant thrombocytopenia in the BALB/c recipient mice (Figure 2.1 A). The severity of the
thrombocytopenia in these mice was comparable to anti-β3 sera and RAMS induced, except
NIT-E group. The thrombocytopenia induced by NIT-E was more moderate compared with
RAMS or MWReg30 (Figure 2.1 A), although all the NIT-B, NIT-E, NIT-G and NIT-H1
antibodies were of the same host and similar specificity (anti-GPIb). Treatment of the recipient
mice with 2 g/kg IVIg significantly alleviated the thrombocytopenia in the mice except for the
mice treated with mAb NIT-E (Figure 2.1 B).
2.4.2 Anti-β3 sera and mAb MWReg30 significantly increased the number of
megakaryocytes in the bone marrow
Examination of the megakaryocyte counts in the bone marrow also showed variable effects
by injection of the anti-platelet reagents. For example, although all the sera and mAbs induced
significant thrombocytopenia, only the anti-β3 sera and mAb MWReg30 significantly increased
the number of megakaryocytes as compared to controls (Figure 2.2 A). In addition, these two
anti-platelet reagents caused significant morphological abnormalities, such as pyknotic nuclei
and cytoplasmic blebbing (Figure 2.3). Among all groups, MWReg30 treated mice showed the
highest megakaryocyte count which was significantly higher compared with all of the other
groups. Megakaryocyte counts in the anti-β3 group were higher than the 2C9.G2, NIT-G and
NIT-H1 treated groups (Figure 2.2 A). In contrast, the other groups did not show any significant
impact on megakaryocytes. With IVIg treatment, megakaryocyte counts were only modified in
86
the MWReg30 group (Figure 2.2 B). There were no significant changes in the megakaryocyte
counts of the rest of the groups after IVIg treatment (Figure 2.2 B). Representative images from
control, MWReg30 treated and MWReg30 plus IVIg treatment groups are shown in Figure 2.3.
87
Figure 2.1 Platelet counts induced by the indicated anti-platelet antibodies and IVIg
effects.
PB
S
an
ti-
3
RA
MS
RA
MS
MW
Re
g3
0
MW
Re
g3
0
2C
9.G
2
2C
9.G
2
NIT
-B
NIT
-B
NIT
-E
NIT
-E
NIT
-G
NIT
-G
NIT
-H1
NIT
-H1
0
4 0 0
8 0 0
1 2 0 0
Pla
tele
t c
ou
nts
(x
10
9/L
)
IV Ig - +- - - -- - --+ + + + + +
* *
*
*
**
* *N S
(A) From left to right, BALB/c mice received: PBS, mouse-anti-mouse 3 sera (anti-3),
rabbit-anti-mouse platelet sera (RAMS), and MWReg 30, 2C9.G2, NIT-B, NIT-E, NIT-
G, NIT-H1 mAbs respectively. Comparisons among all groups were analyzed using one-
way ANOVA with a Tukey’s post-hoc test (N=3-12, *P<0.05, ****P<0.0001). All the *
labeled indicate P values when compared to Control. (B) Comparison of platelet counts
between IVIg treated (spotted bars) and untreated mice (filled bars) that received PBS or
indicated anti-platelet sera or mAbs. Student’s t test was used for the comparisons of
each pair (N=3-12, *P<0.05, **P<0.01, NS=non-significant).
PB
S
an
ti-
3
RA
MS
MW
Re
g3
0
2C
9.G
2
NIT
-B
NIT
-E
NIT
-G
NIT
-H1
0
4 0 0
8 0 0
1 2 0 0
Pla
tele
t c
ou
nts
(x
10
9/L
)
* * * ** * * *
* * * ** * * *
* * * *
**
*
* * * *
* * * *
B
A
88
Figure 2.2 MWReg30 significantly increased the megakaryocyte counts.
PB
S
an
ti-
3
RA
MS
RA
MS
MW
Re
g3
0
MW
Re
g3
0
2C
9.G
2
2C
9.G
2
NIT
-B
NIT
-B
NIT
-E
NIT
-E
NIT
-G
NIT
-G
NIT
-H1
NIT
-H1
0
1 0
2 0
3 0
MK
co
un
ts p
er f
ield
(4
0X
)
IV Ig - +- - - -- - --+ + + + + +
*
PB
S
an
ti-
3
RA
MS
MW
Re
g3
0
2C
9.G
2
NIT
-B
NIT
-E
NIT
-G
NIT
-H1
0
1 0
2 0
3 0
MK
co
un
ts p
er f
ield
(4
0X
)
** * * * * * * *
* * * ** * * *
* * * ** * * *
** *
* *
* * * **
B
(A) The megakaryocyte counts in the bone marrow of mice treated with indicated sera
or mAbs. Comparisons among all groups were analyzed using one-way ANOVA with
a Tukey’s post-hoc test (N=3-12, *P<0.05, *P<0.01, ****P<0.0001). (B) Comparison
of megakaryocyte counts between IVIg treated (spotted bars) and untreated mice
(filled bars) that received PBS or indicated anti-platelet sera or mAbs. Student’s t test
was used for the comparisons of each pair (N=3-12, *P<0.05). The megakaryocyte
counts of each mouse were calculated from the average value of 10 random fields.
A
89
Figure 2.3 Megakaryocyte morphology induced by MReg30 and IVIg effects.
Representative H&E stained bone marrows from BALB/c received
(A) PBS
(B) MWReg30
(C) IVIg and MWReg30 respectively.
Pictures were taken at 40× magnification with a 0.75 numerical aperture of the objective lens for all panels. The arrows
point to megakaryocytes.
Control MWReg30 MWReg30+IVIg
90
2.5 Discussion
In the current study, the effects of different anti-platelet antibodies on platelet and
megakaryocyte counts were examined independently of T cells in a murine passive ITP model.
While all the anti-platelet sera and monoclonal antibodies (mAbs) induced variable degrees of
significant thrombocytopenia in BALB/c mice within 24 h of administration, only two anti-
platelet reagents caused significant elevations and abnormal morphology in bone marrow
megakaryocytes. These results suggest that antibodies of different anti-platelet specificity and
isotypes have different biological effects in vivo. Most antibodies did not show any direct effect
on megakaryocytes in our current study. In addition, while IVIg administration successfully
alleviated the thrombocytopenia induced by the anti-platelet antibodies, its effect on
megakaryocyte counts and morphology was limited within 24 hours.
Megakaryocytes are a specialized cell population in the bone marrow whose primary role is
to produce platelets and they develop from CD34+CD41
+ progenitor cells to become lineage
restricted with the expression of CD61 (also known as integrin β3, GPIIIa) and CD41 (integrin
αIIb, or GPIIb) (Battinelli et al., 2007; Italiano Jr. and Hartwig, 2013; Machlus and Italiano,
2013). Their development can be divided into 3 different stages: megakaryoblasts,
promegakaryocytes, and mature megakaryocytes (Dameshek and Miller, 1946; Italiano Jr. and
Hartwig, 2013; Long and Williams, 1981) and their maturation and proliferation are critically
dependent on the megakaryocyte growth factor thrombopoietin (TPO) (Gurney et al., 1994).
Mature megakaryocytes eventually release proplatelets into the bloodstream which become
circulating platelets (Behnke, 1969; Italiano et al., 1999; Junt et al., 2007; Patel et al., 2005). In
ITP, although antibody-mediated peripheral destruction of platelets was the earliest
manifestation discovered (Harrington et al., 1951), it is now known that platelet production
defects also occur and lead to thrombocytopenia but little is known of how reduced platelet
production in the bone marrow is mediated.
Both anti-platelet antibodies and CD8+ T cells from patients with ITP have been studied in
vitro for their effects on megakaryocyte maturation and platelet production. In 2003, Chang et al
showed that plasma from ITP patients as well as purified anti-platelet monoclonal antibodies
could inhibit the differentiation of megakaryocytes from umbilical cord blood mononuclear cells
91
in vitro (Chang et al., 2003). McMillan et al had consistent findings of decreased yield of
megakaryocytes from CD34+ progenitor cells when cultured with plasma from patients with
ITP. They further showed that anti-platelet antibodies from patients with ITP could also inhibit
the maturation of megakaryocytes as indicated by the decreased ploidy of megakaryocytes in
vitro (McMillan et al., 2004). Subsequently, Balduini showed that anti-GPIbα but not anti-
GPIIbIIIa antibodies inhibited pro-platelet formation by megakaryocytes in vitro, suggesting an
additional mechanism that contributes to the thrombocytopenia in ITP (Balduini et al., 2008).
Recently, Liu et al also showed impaired megakaryocyte production induced by allogeneic anti-
platelet antibodies (Liu et al., 2015). It was also shown that CD8+ T cells could significantly
induce megakaryocyte abnormalities and impair platelet production by megakaryocytes both in
human and mouse (Chow et al., 2010; Li et al., 2007). Despite these observations, to our
knowledge, the in vivo effects of anti-platelet antibodies, independently of CD8+ T cells, on the
megakaryocytes in ITP have not been studied.
Animal models provide a valuable tool to facilitate our understanding of the pathogenesis
of ITP (Semple, 2010). Passive ITP is the most commonly used mouse ITP model and is
generated by injecting anti-platelet sera or mAb into mice (Semple, 2010). For example, in
1981, RAMS was used to induce thrombocytopenia, for the study of changes in small
acetylcholinesterase-positive cells in the bone marrow, which were recognized as
megakaryocyte progenitor cells (Kalmaz and McDonald, 1981). The earliest anti-platelet
monoclonal antibodies were probably developed in 1981 by McMichael and Newman
independently (McMichael et al., 1981; Newman et al., 1981) and in 1992, one of the first anti-
murine platelet mAbs, MWReg30 was developed by Burstein et al and has been widely used to
induce passive ITP (Burstein et al., 1992). In the current study, all the anti-platelet sera and
mAbs induced significant thrombocytopenia within one day, irrespective of being from different
hosts or IgG isotypes (Figure 2.1 and Table 2.1). This is consistent with our previous studies and
others (Crow et al., 2003b; Kapur et al., 2015a; Leontyev et al., 2012; Semple, 2010). However,
bone marrow megakaryocyte counts revealed a significant difference among the groups of
antibody-treated mice. For example, although both anti-β3 sera and RAMS induced comparable
levels of thrombocytopenia in recipient mice, the megakaryocyte count was significantly higher
only in the anti- β3sera or MWReg30 treated groups (Figure 2.2 A). This may suggests that for
92
some anti-platelet antibodies, the megakaryocyte response in ITP is not necessarily proportional
to peripheral platelet counts. The difference in megakaryocyte counts between the anti-β3 sera
and RAMS may also be related to the differences in the antigen specificities or the Fc structure
of the antibodies in the sera. Moreover, the megakaryocyte counts of the MWReg30 treated
mice were the highest among all groups while the platelet counts of these mice were
significantly reduced (Figures 2.1 and 2.2). This may suggest a distinctive effect that MWReg30
has both on platelets and megakaryocytes in murine passive ITP. Considering the antigen
specificities of the antibodies, it seems possible that the interaction between MWReg30 and its
cognate antigen CD41 could stimulate megakaryocyte progenitors to promote the production of
megakaryocytes. Support for this, at least, is that the binding of MWReg30 to CD41
significantly increases platelet activation whereas other anti-GPIIbIIIa (CD41/CD61) mAbs
actually inhibit platelet activation (Burstein et al., 1992; Di Minno et al., 1983). Alternatively, it
might also be possible that the difference between MWReg30 and other mAbs are due to an
inhibitory effect of anti-GPIb (CD42) mAbs on megakaryocyte differentiation or proliferation.
There is supportive evidence from the study by Balduini et al showing that anti-GPIb antibody
but not anti-GPIIbIIIa antibody inhibited proplatelet formation from megakaryocytes in vitro
(Balduini et al., 2008).
Comparisons of the four different anti-GPIb mAbs including NIT-B, NIT-E, NIT-G, NIT-
H1 showed comparable numbers of megakaryocytes in the bone marrow, despite a trend of
higher platelet counts in the NIT-E-treated group (Figure 2.1 A), suggesting that the number of
megakaryocytes in the bone marrow may not be sensitive to increased platelet destruction and
thrombocytopenia. Similarly, others have shown that the number of megakaryocytes and
progenitors does not significantly change within seven days after an anti-platelet serum injection
(Ebbe et al., 1987; Stenberg and Levin, 1989). However, in these studies there was an increase
of the size of megakaryocytes, which needs to be further confirmed in our mouse model (Ebbe
et al., 1987; Stenberg and Levin, 1989). Overall, the different megakaryocyte counts between
MWReg30 and the other mAbs, and the similarities among anti-GPIb antibodies suggest that the
antigen specificity of Abs may determine their effect on megakaryocyte differentiation and
proliferation. This may help explain the variability in megakaryocyte counts in ITP patients with
counts varying from normal to elevated (Dameshek and Miller, 1946; De La Fuente, 1949;
93
Houwerzijl et al., 2004). In our study, IVIg treatment successfully increased platelet counts in
all groups except mice receiving the NIT-E mAb (Figure 2.2 B). Of interest, there were no
significant changes in the megakaryocyte counts in the IVIg-treated mice, except in the
MWReg30-treated group. Our study confirms the effectiveness of IVIg therapy in the passive
ITP mouse model (Leontyev et al., 2012; Samuelsson et al., 2001; Semple, 2010), but suggests
that IVIg may not affect megakaryocyte counts in the bone marrow.
In summary, anti-platelet antibodies, independent of anti-platelet T cell-mediated
destruction, are able to induce thrombocytopenia but most do not affect megakaryocyte counts
or morphology within the bone marrow in 24 hours. Our data suggest that while the
thrombocytopenia induced by platelet antibodies is generally sensitive to IVIg treatment, in
those mice where megakaryocyte number and morphology are affected, IVIg does not
necessarily change the effect.
94
Chapter 3
Allogeneic Platelet Transfusions Prevent Murine T
Cell-mediated ITP
This chapter is modified from the following:
Guo, L., Yang, L., Speck, E.R., Aslam, R., Kim, M., McKenzie, C.G., Lazarus, A.H., Ni, H.,
Hou, M., Freedman, J., Semple, J.W.. (2014). Allogeneic platelet transfusions prevent murine T-
cell-mediated immune thrombocytopenia. Blood 123, 422-427.
95
3.1 Abstract
Platelet transfusions are lifesaving treatments for many patients with thrombocytopenia,
however, their use is generally discouraged in the autoimmune disorder immune
thrombocytopenia (ITP). We examined whether allogeneic platelet major histocompatibility
complex (MHC) class I transfusions affected anti-platelet CD61-induced ITP. BALB/c CD61
knockout (KO) mice (CD61-/H-2
d) were immunized against platelets from either wildtype
syngeneic BALB/c (CD61+/ H-2
d) or allogeneic C57BL/6 (CD61
+/ H-2
b) or C57BL/6 CD61 KO
(CD61-/ H-2
b) mice and their splenocytes were transferred into severe combined
immunodeficient (SCID) mice to induce ITP. When non-depleted splenocytes were transferred
to induce antibody-mediated ITP, both allogeneic and syngeneic CD61+ platelet immunizations
generated immunity that caused thrombocytopenia independently of allo-MHC molecules. In
contrast, when B cell-depleted splenocytes were transferred to induce T cell-mediated ITP,
transfer of allogeneic MHC-immunized splenocytes completely prevented CD61-induced ITP
development. In addition, allogeneic platelet transfusions into SCID mice with established
CD61-induced ITP rescued the thrombocytopenia. Compared with thrombocytopenic mice,
bone marrow histology in the rescued mice showed normalized megakaryocyte morphology and
in vitro CD61-specific T cell cytotoxicity was significantly suppressed. These results indicate
that antibody-mediated ITP is resistant to allogeneic platelet transfusions whereas the T cell-
mediated form of the disease is susceptible suggesting that transfusion therapy may be
beneficial in antibody-negative ITP.
96
3.2 Introduction
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by an
isolated thrombocytopenia defined as a peripheral blood platelet count of less than 100×109/L (Provan
et al., 2010; Rodeghiero et al., 2009). The pathogenesis of ITP is complex but appears to be due
primarily to IgG-mediated peripheral platelet destruction in the spleen and/or bone marrow
megakaryocyte inhibition/destruction (Chang et al., 2003; Cines et al., 2009; Harrington et al., 1951;
McMillan et al., 2004; Shulman et al., 1965). A second mechanism of immune thrombocytopenia has
been suspected, however, as early as the famous 1951 Harrington experiments where they observed
that only 16 of the 26 ITP (62%) plasma infusions into healthy volunteers caused thrombocytopenia.
Subsequently, the cumulative results of antibody studies in patients with ITP revealed that anti-platelet
antibodies can only be identified in approximately 60% of patients with ITP (Brighton et al., 1996;
Fabris et al., 2004; Kiefel et al., 1996; McMillan, 2003; Najaoui et al., 2012). In those patients with no
identifiable antibodies, Olsson et al elegantly demonstrated that cytotoxic T cell (CTL) cytotoxicity
was responsible for the thrombocytopenia and this was subsequently confirmed by other studies in
both humans and animals with ITP (Chow et al., 2010; Olsson et al., 2003; Zhang et al., 2006; Zhao et
al., 2008). The latter ITP animal model also demonstrated that compared with antibody-mediated ITP,
the T cell-mediated form of the disorder was not sensitive to platelet-sparing therapies such as
intravenous gammaglobulin (IVIg) (Chow et al., 2010).
Therapy for patients with ITP generally includes steroids and/or IVIg and if those fail, other
treatments such as rituximab, thrombopoietin (TPO) receptor agonists and/or splenectomy etc. are
available (Provan et al., 2010; Rodeghiero et al., 2009). Of interest, despite the benefit of platelet
transfusions for many thrombocytopenic conditions such as the thrombocytopenia secondary to
leukemia (Han et al., 1966; Hirsch and Gardner, 1952; Slichter, 1980), the use of allogeneic platelets
in patients with ITP has been generally withheld (Carr et al., 1986; Salama et al., 2008; Spahr and
Rodgers, 2008; Thachil, 2010). Allogeneic platelet transfusions for chronic ITP are recommended only
as emergency treatment and to be used in combination with other treatments such as IVIg (Provan et
al., 2010; Rodeghiero et al., 2009). Platelet transfusions in ITP are generally avoided due to concerns
regarding the ineffectiveness of the treatment or possible adverse reactions (Thachil, 2010). However,
97
when one examines the literature for evidence supporting a lack of benefit for platelet transfusions in
ITP, very few reports are in fact published. Those that are published are mostly case reports but more
than half of them actually show a benefit from platelet transfusions (satisfactory corrected count
increment) in patients with ITP (Carr et al., 1986; Salama et al., 2008; Spahr and Rodgers, 2008;
Thachil, 2010).
Our murine model of active CD61-specific ITP demonstrate both antibody- and T cell-mediated
thrombocytopenia and because the T cell-mediated form of thrombocytopenia was resistant to IVIg
(Chow et al., 2010), we examined whether allogeneic platelet transfusions may have a differential
effect on the two immunopathologic forms of ITP. The results show that although antibody-mediated
ITP is not affected by allogeneic platelet transfusions, the T cell-mediated form of the disorder was
alleviated by the transfusions. This suggests that platelet transfusions may be beneficial in individuals
suffering from anti-platelet antibody negative ITP.
98
3.3 Materials and Methods
3.3.1 Mice
Female BALB/c (CD61+/H-2
d), C57BL/6 (CD61
+/H-2
b) and B6.129S2-Itgb3
tm1Hyn/J CD61
knockout (KO) (CD61-/H-2
b) mice, 8-12 weeks of age, were used as platelet donors and were
obtained from The Jackson Laboratories. BALB/c CD61 KO mice (CD61-/H-2
d) were bred in
the laboratory of Dr. Heyu Ni and used as platelet recipients and the source of immune
splenocytes. Female CB.17 severe combined deficient SCID (H-2d, CB17/Icr-
Prkdcscid
/IcrIcoCrl) mice (8-12 weeks of age) were used as spleen-cell transfer recipients for
induction of ITP and were obtained from Charles River. All mice were housed in the Li Ka
Shing Knowledge Institute’s Research Vivarium and all animal studies were approved by the St.
Michael's Hospital Animal Care Committee.
3.3.2 Platelet preparation and immunization of CD61 KO mice
Leukoreduced platelets from the indicated donor mice (Table 3.1) were prepared as previously
described (Chow et al., 2010). Briefly, blood was collected from the indicated donor mice, diluted
with 1 × phosphate buffered saline (PBS) containing 10% citrate-phosphate-dextrose with adenine
(PBS/CPDA buffer) and centrifuged at 120 × g. Platelet-rich plasma was then collected and washed at
450×g. The washed platelets were resuspended in PBS, adjusted to 1×109 cells/mL and 100 uL was
transfused into BALB/c CD61 KO (CD61-/H-2
d) mice weekly for 4 weeks.
3.3.3 Serum anti-CD61 and anti-MHC class I antibody production
Serum IgG anti-CD61-specific or anti-platelet MHC class I-specific antibodies were
detected by flow cytometry. Briefly, 1×106
leukoreduced platelets from the indicated donor
mice were incubated with titrations of serum from the indicated immune BALB/c CD61 KO
mice for 30 minutes at room temperature. The platelet-serum mixtures were washed once with
PBS/CPDA buffer and then labeled with a FITC conjugated goat anti-mouse IgG antibody (Fc
specific; Caltag Laboratories, Mississauga, ON) in the dark at room temperature for 30 minutes.
99
Table 3.1 Summary of platelet populations, immune splenocytes used and expected
immune responses induced.
Platelet Source Platelet/MHC
Phenotype
Mice Immunized
(Splenocyte
Source)
Expected Immune Response
BALB/c mice CD61+/H-2
d BALB/c CD61 KO
mice
IgG anti-CD61 antibodies.
Anti-CD61 Cytotoxic T cells.
C57BL/6 mice CD61+/H-2
b BALB/c CD61 KO
mice
IgG anti-CD61 antibodies.
IgG anti-MHC antibodies.
C57BL/6 CD61
KO mice
CD61-/H-2
b BALB/c CD61 KO
mice
IgG anti-MHC antibodies.
100
The mixture was washed with PBS/CPDA buffer and acquired and analyzed on a by flow
cytometer (BD FACSort, Becton Dickinson, Mississauga, ON).
3.3.4 Preparation of splenocytes and cell depletion
Immune CD61 KO mice (CD61-/ H-2
d) were sacrificed and their spleens removed, homogenized
in RPMI-1640 medium and washed twice by centrifugation at 400 × g for 15 minutes. Splenocyte
suspensions were further treated with ammonium-chloride-potassium red blood cell (RBC) lysing
solution and washed with 1 × PBS to remove lysed RBC. Cell depletion studies were carried out using
the Stem Cell Technologies EasySep mouse CD19 positive selection kit (Catalog No. 18754),
according to the manufactures instructions. Briefly, splenocytes were combined with the labeling
reagent and PE selection cocktail and magnetic particles were added and placed within a magnetic
field to isolate CD19+ cells. The unbound CD19
- cells were resuspended in RPMI-1640 medium at the
concentration of 1.5×105/mL and CD19
depletion efficiencies were determined by flow cytometry.
CD19+ cells were less than 1% of all splenocytes after depletion.
3.3.5 ITP induction
SCID mice were pre-screened for the presence of serum IgG by an enzyme-linked
immunoabsorbent assay (ELISA) and any mouse with a serum IgG concentration greater than
20μg/mL was deemed “leaky” and excluded from study. ITP was induced as previously described
(Chow et al., 2010). Briefly, on day -1, CB.17 SCID mice were bled via the saphenous vein and pre-
treatment platelet counts were measured using a Beckman Coulter Counter-LH750 hematology
analyzer. NK cells were depleted in the mice by an ip infusion of 50 uL of a rabbit anti-asialo GM1
antibody (Wako Pure Chemical Industries, Ltd). On day 0, the mice were sub-lethally gamma
irradiated (200 cGy) and then received 100 uL (1.5 ×104 cells total) of the non-depleted or CD19-
depleted immune splenocytes (ip) from the indicated immune CD61 KO mice. Peripheral blood of
SCID mouse recipients was collected weekly from the saphenous vein and platelet counts were
measured. The protocol for ITP induction is shown in Figure 3.1 and expected immune responses are
shown in Table 3.1.
101
Figure 3.1 Protocol for the development of ITP.
1. BALB/c CD61 KO mice were transfused weekly with one of the 3 indicated platelet
populations and immunity was confirmed at day 28 by the generation of anti-CD61 and/or anti-
MHC serum antibodies (IgG+). 2. The immune KO mice were sacrificed and their splenocytes
were either transferred ip to CB.17 SCID mice and weekly platelet counts and phenotype were
monitored or they were assessed for in vitro cytotoxicity. 3. At day 28 post-splenocyte transfer,
the SCID mice were sacrificed and effector responses (in vitro cytotoxicity) and bone marrow
(BM) histology were performed.
Weekly Transfusions:
C57BL/6
Wildtype Plts.
(CD61+/H-2b
)
Or
C57BL/6 CD61 KO Plts.
(CD61-/ H-2b
)
Or
BALB/c
Wildtype Plts
(CD61+/ H-2d
)
Sacrifice,
In vitro cytotoxicity,
BM histology
Day -1 0 7 14 21 28 28
Pre
Bleed Sacrifice, Transfer 1.5x10
4
immune
(or naïve) splenocytes (+ CD19
depletion), in vitro cytotoxicity
Platelet count
1.
2.
BALB/c CD61 KO mice
Day 0 7 14 21 28
IgG+
3.
Time
SCID mice (ITP)
Time
102
3.3.6 Bone marrow histology and megakaryocyte counts
Engrafted SCID mice were sacrificed on day 28 and their femurs were removed, dissected of
muscle tissue and the epiphyses were cut off and the bones shafts were placed in fixative (B+ fixative;
BBC Biochemicals) for 12 hours. The bones were further decalcified with 10% nitric acid for 2 hours
and then processed and embedded in paraffin. Sections were stained with hematoxylin and eosin
(H&E). All pictures were taken by using CellSens Standard software under a 40× objective lens
(Olympus BX50), with the numerical aperture of objective lens 0.75. The megakaryocyte count of
each mouse was calculated as the mean value of 10 counts from 10 random fields
(450μm×320μm/field) throughout each slide.
3.3.7 Cytotoxicity assay
Anti-CD61 cytotoxic activity of splenocytes from the transfused CD61 KO mice and engrafted
SCID mice was measured using a commercial kit (7-AAD/CFSE Cell-Mediated Cytotoxicity Assay
Kit, Cayman Chemical company, item no. 600120) and the murine macrophage cell line PU5-1.8 (H-
2d) was used as a target cell (ATCC, TIB-61) as this cell line expresses both syngeneic MHC class I
and CD61 antigens (determined by flow cytometry, not shown). Briefly, target cells were stained with
5-(6)-carboxyflourescein diacetate succinimidyl ester (CFSE) for 15 min at room temperature washed
once with RPMI-1640 medium and 104 cells were incubated in 96-well V-bottom plates with the
indicated splenocytes (effectors) for 4 h at 37 ºC at different Effector:Target ratios (1.25:1, 2.5:1, 5:1
and 10:1). The plates were then centrifuged and the cells resuspended in 7-AAD staining solution to
label dead cells. The samples were analyzed by flow cytometry (MACSQuant Analyzer,
MiltenyiBiotec) and cytotoxic activity was evaluated by analyzing the percentage of dead cells within
the CFSE-labeled target cell population. To address the effects of platelets on splenic cytotoxicity, 105
splenocytes were first incubated with either BALB/c WT (CD61+/H-2d) or C57BL/6 WT (CD61+/H-
2d) platelets at a 1:15 splenocyte: platelet ratio in V-bottom plates for 4h at 37 ºC, washed twice with
RPMI-1640 medium and then used as effectors in the cytotoxicity assay with labeled PU5-1.8 target
cells.
103
3.3.8 Statistical analysis
Differences between means were analyzed with Student’s t test and P values of < .05 were
considered significant.
104
Figure 3.2 Generation of anti-platelet IgG antibodies in CD61 KO mice.
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350
100
101
102
103
104
0
88
175
263
350a. b. c.
Cell Number
Fluorescence
Representative flow cytometric histograms of anti-CD61 and/or anti-MHC class I
serum reactivity from immunized BALB/c CD61 KO mice. BALB/c CD61 KO mice
were weekly transfused (×4) with platelets from either (a) BALB/c (CD61+/ H-2d)
mice, (b) C57BL/6 (CD61+/ H-2b) mice or (c) C57BL/6 CD61 KO (CD61-/ H-2b)
mice. Serum was prepared and incubated (1:400 dilution shown) with either BALB/c
platelets (top panels), C57BL/6 platelets (middle panels) or C57BL/6 CD61 KO
platelets (lower panels) and then incubated with a FITC-conjugated goat anti-mouse
IgG secondary antibody and fluorescence was analyzed by flow cytometry. The thin
line in each histogram is the pre-treatment serum reactivity and the thick lines are
serum reactivity after 4 platelet transfusions. Splenocytes from the immune mice were
then used in the indicated experiments.
105
3.4 Results
3.4.1 Platelet MHC class I and CD61 antigens generate high titred IgG antibody
responses
When BALB/c CD61 KO mice were immunized with either (a) BALB/c (CD61+/H-2
d), (b)
C57BL/6 (CD61+/H-2
b), or (c) C57BL/6 CD61 KO (CD61
-/H-2
b) platelets, significant levels of anti-
platelet IgG antibodies against the corresponding CD61 and/or allogeneic MHC class I antigens were
detected in the sera after the fourth platelet transfusion, i.e. (a) anti-CD61 antibodies, (b) anti-CD61
and MHC antibodies and (c) anti-MHC antibodies respectively (Figure 3.2).
3.4.2 Platelet allogeneic MHC class I antigens do not affect anti-CD61 antibody-
mediated ITP
The effect of allogeneic MHC class I molecules on CD61-specific IgG-mediated ITP was
determined by transferring non-depleted splenocytes from CD61 KO mice immunized against the
three platelet populations into SCID mice and then monitoring platelet counts weekly. As described
previously (Chow et al., 2010), irradiation-induced thrombocytopenia occurred at day 7 in all mice
(Figure 3.3). Compared with SCID mice transferred with naïve mouse splenocytes, non-depleted
splenocytes from CD61 KO mice immunized against either CD61+/H-2
d or CD61
+/H-2
b platelets
induced significant thrombocytopenia throughout the 28 day protocol (day 21, 28, P<.05) (Figure
3.3a).
3.4.3 Platelet allogeneic MHC class I antigens prevent anti-CD61 T cell-mediated
ITP
To determine whether allogeneic MHC class I molecules have an effect on CD61-specific T cell-
mediated ITP, splenocytes from the CD61 KO mice immunized with the three platelet populations
were first depleted of CD19+ B cells and then transferred into SCID mice. SCID mice transferred with
CD19-depleted immune splenocytes against CD61+/H-2
d platelets developed thrombocytopenia
throughout the 28 day protocol (Figure 3.3b). However, SCID mice transferred with CD19-depleted
106
Figure 3.3 Allogeneic platelet MHC I antigens inhibit T cell-mediated ITP.
0 7 1 4 2 1 2 80
4 0 0
8 0 0
1 2 0 0
0 7 1 4 2 1 2 80
4 0 0
8 0 0
1 2 0 0
D a y s P o s t T ra n s fe r
Pla
tele
t C
ou
nt
(x 1
0-9
/L) a . b .
Platelet counts in SCID mice transferred with either (a) non-depleted or (b) CD19-
depleted splenocytes from either naïve BALB/c mice (, N=18) or from BALB/c CD61
KO mice immunized against platelets from BALB/c (CD61+/ H-2d) mice (, N=11),
C57BL/6 (CD61+/ H-2b) mice (, N=18) or C57BL/6 (CD61-/ H-2
b) CD61 KO mice
(, N=8). Results are presented as weekly platelet counts (×109/L) after splenocyte
transfer. The dotted horizontal line represents the mouse platelet cut-off counts for
thrombocytopenia. In a) ; p<0.05 for vs. or ) and in b) ; p<0.05 for vs. .
107
immune splenocytes against CD61+/H-2
b platelets were rescued from the T cell-mediated
thrombocytopenia at days 21 and 28 (P<.01) (Figure 3.3b).
3.4.4 Allogeneic platelet transfusions prevented T cell-mediated ITP in transferred
SCID mice
To ensure that allogeneic platelet transfusions would prevent an established T cell-mediated ITP,
SCID mice were first transferred with ITP-inducing CD19-depleted CD61 KO immune splenocytes
against BALB/c (CD61+/H-2
d) platelets, and then transfused weekly with either 10
8 BALB/c
syngeneic or C57BL/6 allogeneic platelets. Compared with control non-transfused mice, platelet
counts in ITP SCID mice transfused with allogeneic C57BL/6 platelets were significantly elevated
(day 28, P <.05) (Figure 3.4).
3.4.5 Allogeneic platelet MHC class I molecules reverse abnormal bone marrow
morphology and numbers in anti-CD61 T cell-mediated ITP
Compared with SCID mice transferred with naïve splenocytes (Figure 3.5a), CD19-depleted
splenocytes transferred from CD61 KO mice immunized against BALB/c (CD61+/H-2
d) platelets
caused significant abnormalities in the SCID mouse bone marrow megakaryocytes (Figure 3.5b).
Megakaryocytes in those mice had increased size, pyknotic nuclei, and irregular membranes (Figure
3.5b). In contrast, however, megakaryocytes in the SCID mice receiving CD19-depleted CD61 KO
immune splenocytes against either C57BL/6 (CD61+/H-2
b) or C57BL/6 CD61 KO (CD61
-/H-2
b)
platelets showed no difference in bone marrow histology when compared with the control mice
(Figure 3.5c and d). Cumulatively, the numbers of megakaryocytes were significantly increased in
mice exhibiting antibody-mediated ITP, whether induced by splenocytes from CD61 KO mice
immune against CD61+/H-2
d or CD61
+/H-2
b platelet populations (Figure 3.6). However, in mice with
T cell-mediated thrombocytopenia, i.e. the SCID mice that received CD19-depleted immune
splenocytes, the presence of MHC antigens on platelets significantly reduced the number of
megakaryocytes to normal levels (Figure 3.6).
108
Figure 3.4 Allogeneic platelet transfusions inhibit T cell-mediated ITP.
0 7 1 4 2 1 2 80
4 0 0
8 0 0
1 2 0 0
D a y s P o s t T ra n s fe r
Pla
tele
t C
ou
nt
(x 1
0-9
/L)
Platelet counts in SCID mice transferred with CD19-depleted CD61 KO immune
splenocytes against BALB/c (CD61+/ H-2d) platelets and then transfused weekly
with either nothing (, N=5), syngeneic BALB/c platelets (▼, N=5) or
allogeneic C57BL/6 platelets (, N=5). Results are presented as the weekly
platelet counts (×109/L) after splenocyte transfer (; p<0.05 for vs. ).
109
3.4.6 Allogeneic platelet MHC class I molecules inhibit CD61-specific T cell
cytotoxicity in vitro
To further examine the relationship between platelet counts and the anti-CD61 T cell response,
the cytotoxic activity of splenocytes from immunized CD61 KO mice and the transferred SCID mice
were examined using an in vitro cytotoxicity assay against syngeneic CD61+ target cells (PU5-1.8)
(Figure 3.7a). Splenocytes from CD61 KO mice immunized with BALB/c (CD61+/H-2
d) platelets
showed significantly elevated anti-CD61 cytotoxicity at an Effector: Target ratio of 10:1 as compared
with control naïve splenocytes (P<.01) (Figure 3.7b). This cytotoxicity was also observed in the
spleens of SCID mice receiving those immune CD61 KO splenocytes (Figure 3.7c). In contrast, the
presence of allogeneic platelet MHC molecules (CD61+/H-2
b) significantly inhibited the anti-CD61 T
cell mediated cytotoxicity to levels similar to control naïve splenocytes in both CD61KO mice and
their SCID recipients at an Effector: Target ratio of 10:1 (CD61+/H-2
b VS CD61+/H-2
d , P<.05)
(Figure 3.7 b and c). To directly address the modulatory effect of allogeneic platelets on the anti-CD61
T cell mediated cytotoxicity, BALB/c CD61 KO mice splenocytes (CD61-/H-2
d) immunized against
syngeneic WT platelets (CD61+/H-2
d) were first incubated with either syngeneic (CD61
+/H-2
d) or
allogeneic (CD61+/H-2
d) platelets and then examined for their cytotoxic activity against PU5-1.8 cells.
Compared with splenocytes incubated with syngeneic platelets, those with allogeneic platelets showed
significantly decreased cytotoxic activity (P<.05) (Figure 3.7d).
110
Figure 3.5 Allogeneic platelet MHC I antigens rescue megakaryocyte abnormalities in T
cell-mediated ITP.
Representative H&E stained bone marrows of SCID mice transferred with CD19-
depleted splenocytes from (a) a control BALB/c naïve mouse or BALB/c CD61
KO mice immunized against (b) BALB/c (CD61+/ H-2d) platelets, (c) C57BL/6
(CD61+/ H-2b) platelets or (d) C57BL/6
CD61 KO (CD61-/ H-2
b) platelets. The
bars in each panel represent 50 μm. Pictures were taken at 40× magnification
with a 0.75 numerical aperture of the objective lens for all panels. The yellow
arrows point to megakaryocytes.
111
Figure 3.6 Allogeneic platelet MHC I antigens normalize the number of megakaryocytes in
T cell-mediated ITP.
Cumulative results of megakaryocyte enumeration in the bone marrow of control
naïve SCID mice (hatched column) or SCID mice transferred with either non-
depleted (ND) splenocytes (antibody-mediated ITP) from the indicated platelet
donors (white columns) or SCID mice transferred with CD19-depleted splenocytes
(T cell-mediated ITP) from the indicated platelet donors (black columns). At least
10 fields in each bone marrow were counted and the results are presented as the
mean megakaryocyte number per field (; P<.001).
112
Figure 3.7 Effect of allogeneic platelet MHC I antigens on in vitro cytotoxicity in antibody-
and T cell-mediated ITP.
10010
210
310
410
510
0
103
104
105 15.08%
10010
210
310
410
510
0
103
104
105
18.20%
10010
210
310
410
510
0
103
104
105 52.70%
CF
-
Na e C -
C - b
0
1 0
2 0
3 0
4 0
5 0
1 .2 5 2 .5 5 1 0
0
1 0
2 0
3 0
4 0
5 0
1 .2 5 2 .5 5 1 0
E ffe c to r :T a rg e t ra t io
Pe
rc
en
t L
ys
is
b . c .
113
B A L B /c W T p lts
(C D 6 1 + /H -2d
)
C 5 7 B L /6 W T p lts
(C D 6 1 + /H -2b
)
4 0
5 0
6 0
7 0
Pe
rc
en
t L
ys
is
d .
(a) Representative flow cytometric dot plot analysis of cytotoxic killing against
CD61+ H-2d PU5-1.8 target cells by non-depleted splenocytes from either a naïve
BALB/c mouse (left panel), or from CD61 KO mice immunized either against
BALB/c (CD61+/ H-2d) platelets (middle panel) or against C57BL/6 (CD61+/ H-
2b) platelets (right panel). Splenocytes were incubated with CFSE-labeled CD61+
H-2d PU5-1.8 cells for 4 hours in vitro and then stained with the vital dye 7-AAD
and acquired on a flow cytometer. Results are shown at an Effector:Target ratio of
10:1. (b) Cumulative cytotoxicity of splenocytes from naïve mice BALB/c mice
(, N=19) or CD61 KO mice transfused weekly with 108 platelets from either
BALB/c (CD61+ H-2d, , N=10) or C57BL/6 (CD61+/ H-2
b, , N=10) mice. (c)
Cumulative cytotoxicity of splenocytes from naïve mice BALB/c mice (, N=19)
or SCID mice transferred with the corresponding CD61 KO immune splenocyte
populations as in (b) at 4 weeks post transfer. Results in b and c are expressed as
Percent Lysis at the indicated Effector:Target ratios (; p<0.01 for vs. ).
(d) Splenocytes from BALB/c CD61 KO mice immunized against platelets from
BALB/c (CD61+/H-2d) mice were incubated with either syngeneic BALB/c WT
platelets (CD61+/H-2b) or allogeneic C57 BL/6 WT platelets (CD61+/H-2
b) for 4h
in vitro and then examined for their cytotoxicity against PU5-1.8 target cells.
Results in d are expressed as Percent Lysis at a 5:1 Effector:Target Ratio (N=3);
p<0.05).
114
3.5 Discussion
Platelet transfusions are generally not indicated as a treatment for ITP although the data
supporting this contention are rather scarce in the literature. Since ITP is caused by at least two distinct
immune effector processes e.g. antibody- and/or T cell-mediated thrombocytopenia, it is possible that
these immunopathologies may have differential responses to therapy. This is supported by an animal
model of ITP that demonstrated while antibody-mediated thrombocytopenia was sensitive to IVIg
treatment, T cell-mediated thrombocytopenia was not (Chow et al., 2010). In the current murine ITP
study we have further elucidated the effect of allogeneic platelet transfusions on antibody- and T cell-
mediated ITP. Immunization by platelet transfusions containing both allogeneic MHC class I
molecules and CD61 molecules (CD61+/H-2
b) rendered the spleens of CD61 KO mice (CD61
-/H-2
d)
incapable of mediating anti-CD61-specific T cell-mediated ITP in SCID mice. This was also true if
SCID mice that had established T cell-mediated ITP and were transfused with allogeneic platelets.
These responses were correlated with a normalization of bone marrow megakaryocytes and inhibition
of in vitro T cell-mediated cytotoxicity. The results suggest that platelet transfusions may be a
beneficial therapy in those anti-platelet antibody negative individuals that exhibit T cell-mediated ITP.
In 2003, Olsson et al showed that in patients with active ITP who did not have any identifiable
anti-platelet antibodies, there was a significantly increased cytotoxic activity by peripheral blood T
cells against self platelet antigens (Olsson et al., 2003). This activity was reduced in those patients
whose platelet counts increased either due to therapy or spontaneously and this was also reproduced in
a larger clinical trial and in animal studies (Chow et al., 2010; Zhang et al., 2006; Zhao et al., 2008). In
our current murine study, analogous results were observed in that CD19-depleted CD61 KO immune
splenocytes against CD61+/H-2
d platelets induced significant thrombocytopenia when transferred into
SCID mice (Figure 3.3). This correlated to enhanced T cell cytotoxicity against CD61+ syngeneic
target cells in vitro (Figure 3.7 b and c). This indicates that murine platelet CD61 antigens can
effectively trigger CTL responses similar to patients with ITP (Chow et al., 2010; Olsson et al., 2003;
Zhang et al., 2006; Zhao et al., 2008).
115
SCID mice transferred with CD19-depleted splenocytes from CD61 KO mice (CD61-/H-2d) that
were immunized with allogeneic CD61+/H-2
b platelets did not develop thrombocytopenia (Figure 3.3).
This correlated with a loss of in vitro cytotoxicity against CD61+ target cells (Figure 3.7).
Furthermore, in SCID mice transferred with CD19-depleted splenocytes from CD61 immune KO mice
and then transfused weekly with allogeneic platelets (CD61+/H-2
b), their platelet counts were
significantly rescued compared with control ITP mice (Figure 3.4). The mechanism of how cytotoxic
T cell inhibition and a rescue of platelet counts occur is unknown but some possibilities exist. For
example, CTL function by inducing target cell cytolysis via apoptosis that is initiated by the
interaction between their T cell receptor (TCR) complex and MHC class I/peptide complexes on target
cells (Bousso and Robey, 2003; Kaech and Ahmed, 2001). Platelet MHC class I molecules are
primarily adsorbed from plasma and consist of truncated heavy chains that are unstable, indicated by
the finding that more than 80% can be eluted from the platelet surface by chloroquine diphosphate
without affecting platelet membrane integrity (Blumberg et al., 1984; Ghio et al., 1999; Kao, 1987,
1988; Kao et al., 1986; Neumuller et al., 1993). Moreover, platelet MHC class I molecules can be
partially stabilized by addition of exogenous β2-microglobulin in vitro, indicating a reduction of the
β2-microglobulin molecule on the platelet surface (Gouttefangeas et al., 2000). Because of these
features, allogeneic platelet MHC class I molecules may thus evoke a direct faulty interaction with the
CTL TCR thereby anergizing the effector cell. Support for this comes from studies that show that
allogeneic platelet MHC class I molecules cannot activate CTLs on their own (Gouttefangeas et al.,
2000) and our previous work demonstrating that allogeneic platelet transfusions can significantly
enhance donor-specific skin graft survival, an immune process that is almost exclusively elicited by
CTL (Aslam et al., 2008). Figure 3.7 d shows that the in vitro CTL cytotoxicity could be prevented by
allogeneic platelets whereas syngeneic platelets had no effect supporting the concept that T cell-
mediated ITP may be inhibited by the direct interaction of T cells with allogeneic platelets. On the
other hand, the denatured platelet MHC class I molecules may be processed within antigen presenting
cells (APCs) in a manner that does not allow for proper cross presentation of platelet allogeneic MHC
peptides to the CTL. We are currently studying this.
116
The ITP in mice, whether induced by antibodies or T cells, showed abnormal bone marrow
histology; megakaryocytes were increased in number and they showed evidence of apoptosis e.g.
pyknotic nuclei (Figure 3.5). These data support the findings in human studies that patients with ITP
have elevated numbers of megakaryocytes that exhibit apoptosis-like qualities (Houwerzijl et al.,
2004; Li et al., 2007; Yang et al., 2010) and this concurs with in vitro assays that show that anti-
platelet antibodies significantly inhibit megakaryocyte growth (Chang et al., 2003; McMillan et al.,
2004). The increased number of megakaryocyte in this model may be a compensation response to the
thrombocytopenia and immune attack causing apoptosis of megakaryocytes. Perhaps more intriguing,
however, is the observation that when mice were rescued of T cell-mediated ITP by allogeneic platelet
transfusions, their bone marrow megakaryocyte histology was similar to control mice (Figure 3.5).
This suggests that inhibiting the anti-platelet T cell cytotoxic response by allogeneic transfusions may
relieve the megakaryocytes from T cell attack and enable them to maintain platelet production.
Future applications of these murine findings in humans will require the prerequisite of screening
for anti-platelet antibodies which may pose an obstacle e.g. requirement of quick turnaround times and
potential assay sensitivity issues. Nonetheless, the results presented here suggest that in at least a
proportion of individuals that are antibody negative, allogeneic platelet transfusions may have a
beneficial role in alleviating ITP.
In summary, our study shows that anti-CD61 specific T cell-mediated immune responses can
induce thrombocytopenia in SCID mice recipients, mimicking the situation observed in antibody
negative ITP patients. The cell-mediated ITP could be abolished by transfusions of allogeneic platelet
MHC I antigens indicating a potential immunomodulatory benefit of allogeneic platelet transfusions in
ameliorating T cell-mediated ITP.
117
Chapter 4
CD20+ B Cell Depletion Therapy Suppresses
Murine CD8+ T Cell-mediated Immune
Thrombocytopenia (ITP)
This chapter is modified from the following:
Guo, L., Kapur, R., Aslam, R., Speck, E.R., Zufferey, R., Zhao, Y., Kim, M., Lazarus, A.H., Ni,
H., and Semple, J.W.. CD20+ B cell depletion therapy suppresses murine CD8
+ T cell-mediated
immune thrombocytopenia (ITP). Blood 127, 735-8.
118
4.1 Abstract
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder with a complex
pathogenesis which includes both antibody-mediated and T cell-mediated effector mechanisms.
Rituximab (an anti-human CD20 monoclonal antibody) is one of the treatments for ITP and is
known to deplete B cells but may also work by affecting the T cell compartments. Here we
investigated the outcome of B cell depletion (Bdep) therapy on CD8+ T cell-mediated ITP using
a murine model. CD61 knockout (KO) mice were immunized with CD61+ platelets and T cell-
mediated ITP was initiated by transfer of their splenocytes into severe combined
immunodeficient (SCID) mice. The CD61 KO mice were administrated an anti-mouse CD20
monoclonal antibody either before or after CD61+ platelet immunization. This resulted in
efficient Bdep in vivo, accompanied by significant increases in splenic and lymph node CD4+
and CD8+ T cells and proportional increases of FOXP3
+ in CD4
+ and CD8
+ T cells. Moreover,
Bdep therapy resulted in significantly decreased splenic CD8+ T cell proliferation in vitro that
could be rescued by interleukin-2 (IL-2). This correlated with normalization of in vivo platelet
counts in the transferred SCID mice suggesting that anti-CD20 therapy significantly reduces the
ability of CD8+ T cells to activate and mediate ITP.
119
4.2 Introduction
Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by an
isolated thrombocytopenia (<100 ×109/L) (McKenzie et al., 2013). The pathogenesis of chronic
ITP is incompletely understood and thought to be heterogeneous (McKenzie et al., 2013). For
example, approximately two thirds of patients with ITP have detectable anti-platelet antibodies
that destroy platelets primarily by Fc-mediated phagocytosis within the spleen (Brighton et al.,
1996; Harrington et al., 1951). On the other hand, patients with no detectable antibodies harbor
platelet- and megakaryocyte-specific T cells that can mediate ITP (Li et al., 2007; Olsson et al.,
2003). These effector responses are closely correlated with dysfunctional CD4+ T regulatory
cells (Tregs) suggesting T cells are critical for ITP development (Liu et al., 2007; Sakakura et
al., 2007; Stasi et al., 2008).
B cell depletion (Bdep) therapy using rituximab has been shown to be a successful second-
line treatment for patients with ITP (Stasi et al., 2001). Although B cells function to primarily
elicit humoral immunity, they also have other roles such as antigen presentation, co-stimulation
and T cell activation/cytokine production (Lund, 2008; Lund and Randall, 2010). Moreover,
patients with ITP, whether anti-platelet antibody positive or not, can respond to rituximab and in
those antibody-positive patients, their autoantibody titers do not necessarily change (Cooper et
al., 2012). This suggests that anti-CD20 can significantly affect T cell compartments and this
was originally confirmed by Stasi et al showing that rituximab normalizes the observed CD4+ T
cell abnormalities in ITP (Stasi et al., 2008; Stasi et al., 2007). To better understand how anti-
CD20 therapy affects CD8+ T cells, we investigated its effect on T cell-mediated ITP in an
established murine model of ITP (Chow et al., 2010). The data suggest that anti-CD20 Bdep
significantly inhibits IL-2 dependent CD8+ T cell proliferation which blocks their ability to
mediate ITP.
120
4.3 Materials and Methods
4.3.1 Mice
BALB/c mice (H-2d, CD61
+, Charles River Laboratories, Montreal, QC, Canada) were
used as platelet donors and BALB/c CD61 (GPIIIa) knockout (KO) mice were supplied by Dr.
Heyu Ni and used as a source of immune splenocytes. CB.17 severe combined immune
deficient (SCID) mice (H-2d, CB17/Icr-Prkdc
scid/IcrIcoCrl, Charles River Laboratories) were
used as splenocyte transfer recipients for induction of ITP. All mice were 8-12 weeks of age and
all studies were approved by the St. Michael’s Hospital Animal Care Committee.
4.3.2 Reagents
The reagents used in our current study include: anti-Mouse CD3 antibody Purified,
eBioscience, Cat No. 15-5773-80A; anti-Mouse CD28 Purified, eBioscience, Cat No. 16-0281-
85; Mouse IL-2 recombinant protein, eBioscience, Cat No. 14-8021-64; Goat anti-mouse IgG,
FITC, Fc specific, Caltag Laboratories, Cat No. M35001; anti-Mouse CD19 Alexa fluor 647,
Biolegend, Cat No. 115522; anti-Mouse CD45R/B220 PE, PharMingen, Cat No. 553089; anti-
Mouse CD3 PerCP/Cy5.5, Biolegend, Cat No. 100218; anti-Mouse CD4 eFluor450,
eBioscience, Cat No. 48-0041-82; anti-Mouse CD4 Antibody Brilliant Violet 510, Biolegend,
Cat No. 100559; anti-Mouse CD8 FITC, eBioscience, Cat No. 11-0081-85; anti-Mouse CD8a
Antibody Alexa Fluor 647, Biolegend, Cat No. 100724; anti-Mouse/Rat FOXP3 PE-Cy5,
eBioscience, Cat No. 15-5773-80A; anti-Human/mouse Granzyme B Antibody Pacific Blue,
Biolegend, Cat No. 515407; anti-Mouse CD44 antibody FITC, ThermoFisher Scientific, Cat
No. MA5-17879; Mouse CD19 positive selection kit, Stem Cell Technologies, Cat No. 18754;
Cell Stimulation Cocktail (plus protein transport inhibitors) (500×), eBioscience, Cat. No. 00-
4975-03.
4.3.3 Platelet preparation and immunization of CD61 KO mice
Leukoreduced platelets were prepared from platelet rich plasma as previously described
(Chow et al., 2010). Briefly, blood was collected into phosphate-buffered saline (PBS)
121
containing citrate-phosphate-dextrose-adenine. The blood was centrifuged at 120 × g for 15
minutes, and the platelet rich plasma (PRP) was collected. The PRP was washed and then
resuspended in PBS at 1×109
platelets/mL. For the immunization of CD61 KO mice, recipient
mice were bled 24 h prior to the first transfusion and then injected weekly for 4 weeks with 100
μL (1×108) of fresh platelets via the tail vein.
4.3.4 Cell preparation and staining
Spleen, thymus and mesenteric lymph nodes were harvested from sacrificed mice and
homogenized in RPMI-1640 medium and washed by centrifugation at 400 × g for 15 minutes.
Cells were then ammonium-chloride-potassium (ACK) treated to remove red blood cells,
enumerated, and adjusted to a concentration of 1×107 cells/mL for antibody staining (Aslam et
al., 2012; Guo et al., 2014). For direct cell surface staining, each indicated antibody was used at
0.5-1μL/100 μL, incubated for 30 min at room temperature, washed and resuspended in staining
buffer. For FOXP3 staining, as described previously (Aslam et al., 2012), lymphocytes from
thymus, spleen or mesenteric lymph nodes were first stained with anti-mouse CD4 eFluor450
and anti-mouse CD8 FITC antibodies for 45 min at 4°C, washed, and then fixed and
permeabilized using the FOXP3 staining buffer. The cell preparations were then subsequently
stained with anti-mouse FOXP3 PE-Cy5 antibody or isotype IgG control. For Granzyme B
staining, purified CD8+ T cells from the proliferation cultures (see below) were collected and
stained for anti-mouse CD3 PerCP/Cy5.5 and anti-mouse CD8 APC antibodies, fixed and
permeabilized, and then stained with anti-mouse Granzyme B Pacific Blue antibody or isotype
IgG control. Cells were analyzed using a BD LSRFortessa X-20 flow cytometer (BD
Biosciences, USA) or a MACSQuant Analyzer (Miltenyi Biotec).
4.3.5 Cell depletion and isolation in vitro
For in vitro CD19+ depletion and CD8
+ isolation, viable nucleated cells from homogenized
spleens were purified based on density using Lympholyte-M cell separation media following the
manufacture instructions (Cedarlane, CL5030).
122
Figure 4.1 Overview of the experimental design.
Schematic overview of the entire experimental design depicted in a timeline, based on
the murine model of ITP, including timing of anti-CD20 monoclonal antibody
injections, platelet transfusions, organ harvestings, in vitro analysis and platelet count
measurements in the various groups.
123
The CD19+ depletion for adoptive transfer into SCID mice was carried out using the Stem
Cell Technologies EasySep kits according to the manufactures instructions (mouse CD19
positive selection kit, no. 18754). Briefly, splenocytes were combined with a CD19 labeling
reagent and magnetic particles, and then placed within a magnetic field to remove unbound cell
populations. The unbound CD19−
cells were collected and resuspended in RPMI-1640 Medium
at the concentration of 1.5×105/mL for adoptive transfer into SCID mice.
For the purification of the CD8+ T cells for proliferation assays in vitro, splenocytes were
labeled with anti-CD3, anti-CD8 antibodies and a viability marker 7-AAD, and sorted on a BD
FACS Aria III SORP cell sorter. CD3+CD8
+ 7AAD
− T cells were collected.
Depletion and isolation purity was examined by flow cytometry. After magnetic depletion,
CD19+ cells were less than 1% of all splenocytes. CD8
+ T cell sorting purities were more than
95%.
4.3.6 B cell depletion therapy
To deplete B cells in vivo, a monoclonal mouse anti-mouse CD20 IgG2a antibody
(αCD20) (Biogen Idec Inc) were administered to the indicated CD61 KO mice at indicated time
points intravenously (i.v.), at a dose of 250ug/100uL per mouse (Figure 4.1) (Xiu et al., 2008).
The efficiency of B cell depletion after the anti-CD20 antibody injection was evaluated by the
examination of residual CD19+CD220
+ B cell percentage in the blood after 24h, 48h, and then
weekly by flow cytometry, as well as in the spleen and lymph nodes when the mice were
sacrificed on day 21.
4.3.7 Serum anti-CD61 antibody production
To determine anti-CD61 antibody production in the immunized CD61 KO mice with or
without B cell depletion therapy, sera was collected from weekly and detected for the presence
of anti-CD61 IgG antibodies. Briefly, platelets from syngeneic WT mice were used as a positive
anti-CD61 binding control and platelets from naïve CD61 KO mice were used as the negative
control. The platelets (1×106) were incubated with titrations (from 1:100 to 1:12800) of serum
124
for 30 minutes in a total volume of 200uL at room temperature. The platelet-serum mixtures
were washed once with PBS and then labeled with a FITC conjugated goat anti-mouse IgG
(FITC-GAM, Fc specific; Caltag Laboratories, Mississauga, ON) in the dark at room
temperature for 30 minutes. The mixture was washed with PBS and then analyzed by flow
cytometry (BD FACSort flow cytometer, Becton Dickinson).
4.3.8 CD8+
T cell proliferation assay in vitro
CD8+ T cells were purified as mentioned earlier using a BD LSRFortessa X-20 flow
cytometer. Purified CD8+ T cells were washed with PBS twice and resuspended at a
concentration of 1 ×106/mL and Violet Proliferation Dye 450 (V450) (BD Biosciences, Cat
No.562158) was added at a final concentration of 1µM. The tubes were then incubated at 37°C
for 15 min following the manufacturer’s instructions. The labeled CD8+ T cells were washed
twice with RPMI-1640 medium and resuspended finally in complete RPMI-1640 medium
(supplemented with 10% fetal bovine serum (FBS), 500 U penicillin/0.5 mg streptomycin and
100 µM 2-mercaptoethanol). A 96-well flat-bottom plate was precoated with 1µg/mL purified
anti-mouse CD3 antibody at 4°C overnight and washed with cold PBS before use. CD8+ T cells
were added to the 96-well plate at 105 cells/well in triplicate. In the indicated groups, purified
anti-mouse CD28 antibody was added at a final concentration of 2µg/mL, and IL-2 was added at
0.05µg/mL. The cells were incubated for 72h at 37°C and then analyzed by flow cytometry.
Division of CD8+ T cells was reflected by the decrease of fluorescence intensity. CD8
+ T cells
from BALB/c naïve mice cultured without anti-mouse CD3/anti-CD28/IL-2 were considered the
as baseline with minimum proliferation. The average of the triplicate proliferating CD8+ T cell
wells was recorded.
4.3.9 Murine model of ITP
A murine ITP model was used as previously described (Chow et al., 2010). CD61 KO mice
were immunized against CD61+ BALB/c platelets and their non-depleted (ND) splenocytes
were transferred into SCID mice to initiate ITP (Chow et al., 2010). Splenocytes from some
CD61 KO mice were first depleted of CD19+ B cells in vitro before transfer to initiate CD8
+ T
125
cell-mediated ITP (Bdep in vitro). Other KO mice were B cell depleted (Bdep) in vivo by 2
intravenous injections of an anti-mouse CD20 monoclonal IgG2a antibody (Biogen, Cambridge,
MA, USA, 250 ug/mouse) either before or after platelet immunization (to mimic Bdep therapy).
CB.17 SCID mice were pre-screened for the presence of IgG by an enzyme-linked
immunoabsorbent assay (ELISA) and any mouse with a serum IgG concentration greater than
20μg/mL was deemed “leaky” and excluded. On day -1, the SCID mice were bled via the
saphenous vein and pre-treatment platelet counts were measured using a Beckman Coulter
Counter-LH750 hematology analyzer. Then, NK cells were depleted by an ip infusion of 50 uL
of a rabbit anti-asialo GM1 antibody (Wako Pure Chemical Industries, Ltd). On day 0, the SCID
mice were sub-lethally gamma irradiated (2 cGy) and then received an intraperitoneal injection
(100 uL) of 1.5 ×104 splenocytes from the indicated CD61 KO mice. Some SCID mice received
and ip injection of PBS and were negative controls. The peripheral blood of SCID recipients
was then collected weekly from the saphenous vein and platelet counts were measured. On day
21, the SCID mice were sacrificed and their lymphoid tissues were used in the indicated assays.
Details of the in vitro experimentation and the experimental design are shown in Figure 4.1.
4.3.10 Statistical analysis
Data are expressed as mean±SD, and were analyzed using GraphPad Prism 6.02 software
for Windows (GraphPad Software, San Diego, CA).
126
4.4 Results
4.4.1 Anti-CD20 antibody efficiently depletes B cells and increases the T cell
percentages in vivo.
Murine monoclonal anti-CD20 antibodies derived from immunization of CD20 KO mice were
first reported by Uchida et al and induce significant B cell depletion in vivo (Uchida et al.,
2004a; Uchida et al., 2004b). In our hands, all the anti-CD20 antibody-treated mice were
depleted of B cells in the peripheral blood, spleen and mesenteric lymph nodes (Figure 4.2 A
and B). In CD61KO mice Bdep before platelet immunization, a significant reduction in IgG
anti-platelet antibody production was also observed (Figure 4.2 C and D). Bdep therapy in
immune mice significantly increased the percentages of peripheral blood, splenic and lymph
node CD3+CD4
+, CD3
+CD8
+ T cells and FOXP3
+ Treg subpopulations proportionally (Figure
4.3 and 4.4), consistent with previous studies showing that Bdep therapy increases the
proportion of non-B cell populations within immune compartments (Yu et al., 2012).
4.4.2 Bdep therapy suppresses CD8+ T cell proliferation in vitro.
B cells have been previously shown to be involved in CD8+ T cell maintenance and memory cell
formation and patients with ITP treated with rituximab have significant alterations of CD8+ T
cells, including increased cytotoxicity of splenic CD8+
T cells in rituximab non-responders
(Audia et al., 2013; Brodie et al., 2008; Shen et al., 2003; Stasi et al., 2007; Thomsen et al.,
1996). Therefore we examined the proliferation potential of splenic CD8+ T cells from non-
depleted (ND) or Bdep immune CD61 KO mice. Purified splenic CD8+ T cells were stained
with V450 and cultured with anti-CD3 antibody together with either anti-CD28 antibody and/or
recombinant IL-2 for 72 hrs and the fluorescence intensity per cell was analyzed (Figure 4.5 A).
Compared with CD8+ T cells from either naïve BALB/c or ND CD61 immune KO mice, CD8
+
T cells from the Bdep KO mice showed a significantly reduced proliferation upon anti-CD3 and
anti-CD28 stimulation (Figure 4.5 B). The deficient CD8+ T cell proliferation could be rescued
by addition of recombinant IL-2 to the cultures (Figure 4.5 C). Further examination of the
127
splenic CD8+ T cells revealed increased intracellular Granzyme B expression after Bdep therapy
(Figure 4.6).
4.4.3 Bdep therapy induces normalization of platelet counts in a murine
model of ITP.
We further examined the effect of Bdep therapy on the development of ITP. SCID mice were
transferred with splenocytes from immune CD61 KO mice that were either non-depleted (ND)
or depleted of CD19+ B cells in vitro to initiate antibody- and T cell-mediated ITP respectively.
As previously described (Chow et al., 2010), both splenocyte populations induced significant
thrombocytopenia when transferred into SCID mice (Figure 4.7, columns 1-3). In contrast,
however, if the CD61 KO mice were Bdep in vivo either before or after platelet immunization,
the ability of their splenocytes to induce ITP was completely prevented (Figure 4.7, columns 4-
5).
128
Figure 4.2 Anti-CD20 antibody efficiently depletes B cells in vivo.
-102
103
104
105
-102
102
103
104
105
1.17% 26.68%
71.72% 0.43%
-102
103
104
105
-102
102
103
104
105
0.05%96.70%
0.83%2.42%
-10210
010
210
310
410
5
-102
-101
102
103
104
105
0.01%99.80%
0.01%0.18%
ND Bdep 24h Bdep Day 27 A.
C 9
B 0
C.
D.
0
2 0
4 0
6 0
CD
19
+%
(Blo
od
)
********
0
2 0
4 0
6 0
CD
19
+%
(S
ple
en
) ********
Bc n
aiv
e
Bc B
dep
KO
ND
KO
Bd
ep
0
2 0
4 0
6 0
CD
19
+%
(L
N) ****
***
0
1 0 0
2 0 0
3 0 0
4 0 0
T itra t io n s
MF
I
N D
B d e p
1 :2 0 0 1 :4 0 0 1 : 8 0 0 1 :1 6 0 0
*
B.
129
(A) The percentage of CD19
+B220
+ B cells (top-right quadrant) in peripheral blood of
immune CD61 KO mice without Bdep therapy (ND; left panel), 24 h after first anti-
CD20 antibody injection (Bdep 24 h, middle panel), and at the end of the experiment
(Bdep Day 27, right panel). (B) The percentage of CD19+ B cells in the blood (upper
panel), spleen (middle panel) and mesenteric lymph nodes (LN; lower panel) of
BALB/c naïve mice (Bc naïve), BALB/c mice depleted of B cells in vivo (Bc Bdep)
and CD61 KO mice after CD61+ platelet immunization without/with B cell depletion
in vivo (KO ND, KO Bdep, respectively). The data shown were at 24 h after anti-
CD20 antibody administration. Spleen and mesenteric lymph nodes were taken after
sacrifice on day 28. Data were analyzed using one-way ANOVA with Tukey’s post-
hoc test. (C) Representative flow cytometric histogram of anti-platelet IgG antibody in
immune CD61 KO serum at a dilution factor of 1/400. Top panel was sera from ND
and bottom panel was from Bdep pre immune CD61 KO mice (B cell depletion was
performed before platelet immunization), with overlays of control prebleed
(background) sera depicted in dashed lines. (D) The mean fluorescent intensity (MFI)
of individual serum samples from both ND and Bdep immune CD61 KO at serial
titrations from 1:200 to 1:1600. Group comparison between ND and Bdep was
analyzed using a two-way ANOVA, *P<0.05, *** P<0.001, **** P<0.0001.
130
Figure 4.3 B cell depletion therapy increases both CD4+ and CD8
+ T cells in the spleen and
lymph nodes.
Spleen (A, C, E) and mesenteric lymph nodes (B, D, F) from mice, treated as
indicated in the figure, were examined for CD4+ and CD8
+ T cells in both total
lymphocyte populations (A-D) and CD4/CD8 ratios (E-F). Data were analyzed using
a one-way ANOVA with a Tukey’s post hoc test. N=5-12. *P<0.05, **P<0.01,
***P<0.001, ****P<0.0001.
0
2 0
4 0
6 0
8 0
1 0 0
% C
D3
+C
D4
+ C
ell
s (
Sp
lee
n)
******
0
2 0
4 0
6 0
8 0
1 0 0
% C
D3
+C
D4
+ C
ell
s (
LN
)
********
0
1 0
2 0
3 0
4 0
% C
D3
+C
D8
+ C
ell
s (
Sp
lee
n)
****
**** ****
0
1 0
2 0
3 0
4 0
% C
D3
+C
D8
+ C
ell
s (
LN
)
*******
A B
C D
Bc n
aiv
e
Bc B
dep
KO
ND
KO
Bd
ep
0
1
2
3
4
5
CD
3+
CD
4+
/CD
3+
CD
8+
(S
ple
en
)
****
Bc n
aiv
e
Bc B
dep
KO
ND
KO
Bd
ep
0
1
2
3
4
5
CD
3+
CD
4+
/CD
3+
CD
8+
(L
N)
*
E F
131
Figure 4.4 The increases of FOXP3+ Tregs were proportional to the increase of CD4
+ and
CD8+
T cells in the spleen and lymph nodes.
0
5
10
15
CD
4+
FO
XP
3+
% (
Sp
lee
n)
****
0
3
6
9
12
15
CD
4+
FO
XP
3+
% (
LN
)
*****
Bc
naive
Bc
Bdep
KO N
D
KO B
dep0.0
0.5
1.0
1.5
CD
8+
FO
XP
3+
% (
Sp
lee
n)
***
Bc
naive
Bc
Bdep
KO N
D
KO B
dep0.0
0.5
1.0
1.5
CD
8+
FO
XP
3+
% (
LN
)
*
A B
C D
0
5
1 0
1 5
2 0
2 5
FO
XP
3+
/CD
4%
(S
ple
en
)
0
5
1 0
1 5
2 0
2 5
FO
XP
3+
/CD
4%
(L
N)
Bc n
aiv
e
Bc B
dep
KO
ND
KO
Bd
ep
0
2
4
6
FO
XP
3+
/CD
8%
(S
ple
en
)
Bc n
aiv
e
Bc B
dep
KO
ND
KO
Bd
ep
0
2
4
6
FO
XP
3+
/CD
8%
(L
N)
E F
G H
Spleen (A, C, E, G) and mesenteric lymph nodes (B, D, F, H) from indicated mice
were examined for CD4+FOXP3
+ and CD8
+FOXP3
+ Tregs in total lymphocyte
populations (A-D) and the proportion of FOXP3+ in CD4
+ and CD8
+ cells,
respectively. Data were analyzed using a one-way ANOVA with a Tukey’s post hoc
test. N=5-12. *P<0.05, **P<0.01, ****P<0.0001.
132
Figure 4.5 B cell depletion inhibits CD8+ T cell proliferation in vitro.
A
Bc n
aiv
e
KO
ND
KO
Bd
ep
0
2 0
4 0
6 0
8 0
1 0 0****
***
CD
8+
T c
ell
Pro
life
ra
tio
n (
%)
Bc n
aiv
e
KO
ND
KO
Bd
ep
0
2 0
4 0
6 0
8 0
1 0 0 N S
N SB C
a n ti-C D 3 /a n ti-C D 2 8 a n ti-C D 3 /IL -2
133
CD8+ T cells were purified from the spleens of BALB/c naïve mice (Bc naïve), platelet
immunized CD61 KO mice (KO ND) and platelet immunized CD61 KO mice that
received Bdep therapy during immunization (KO Bdep) and stained with the proliferation
dye V450 and cultured in vitro with anti-CD3 ± anti-CD28/IL-2 for 72h. (A)
Representative flow cytometric dot plot analysis of CD8+ T cell proliferation when either
not stimulated (top panels) or stimulated with anti-CD3/CD28 antibodies for 72 hours. A
cell division cycle is characterized by sequential halving of the V450 fluorescence.
Cumulative data of (B) CD8+ splenic T cells stimulated with anti-CD3/anti-CD28 and (C)
anti-CD3/IL-2. Data in B and C are expressed as the mean ± SD of percent CD8+ T cells
proliferating. Data were analyzed using one-way ANOVA with a Tukey’s post-hoc test
(N=5-8. ***P<0.001, ****P<0.0001, NS, non-significant).
134
Figure 4.6 B cell depletion therapy increases Granzyme B expression levels within
CD3+CD8
+ T cells.
-0 .5
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
Gra
nz
ym
e B
+/C
D8
+%
****
0
2 0
4 0
6 0
8 0
IFN
+
/CD
8+
%
Bc n
aiv
e
KO
ND
KO
Bd
ep
0
2 0
4 0
6 0
8 0
CD
44
hig
h/C
D8
+%
A
B
C
CD3+CD8
+ T cells in the spleen of the indicated mice were examined for Granzyme
B, IFN-γ and CD44 expression levels. Data were analyzed using one-way ANOVA
with a Tukey’s post hoc test. N=5-9. **P<0.01.
135
Figure 4.7 In vivo B cell depletion results in a normalization of platelet counts in a murine
model of T cell-mediated ITP.
Platelet counts in transferred recipient SCID mice after 21 days post engraftment of 3×104
splenocytes. SCID mice were either transferred with PBS or with non-depleted splenocytes from
platelet-immunized CD61 KO mice (ND), splenocytes from ND KO mice but depleted of
CD19+ B cells in vitro (ND Bdep in vitro) or splenocytes from platelet-immunized CD61 KO
mice depleted in vivo with anti-CD20 antibody before platelet immunization (B cell dep in vivo,
pre) or after platelet immunization (B cell dep in vivo, post). Each data point represents one
SCID mouse. Data were analyzed using one-way ANOVA with a Tukey’s post test (*P<0.05,
***P<0.001).
PB
SN
D
ND
Bd
ep
in
vit
ro
B c
ell d
ep
in
viv
o, p
re
B c
ell d
ep
in
viv
o, p
ost
0
3 0 0
6 0 0
9 0 0
1 2 0 0
*
*
*
* * *
Pla
tele
t c
ou
nts
(x 1
09/L
)
136
4.5 Discussion
In our current study, anti-CD20 therapy significantly depleted B cells in vivo, and
suppressed CD8+ T cell proliferation in vitro in an IL-2 dependent way. Anti-CD20 therapy lead
to a rescue of CD8+ T cell-mediated ITP in established murine model, which suggests an
alternative explanation independent of suppression of antibody-mediated ITP for the therapeutic
effect of rituximab for patients with ITP.
The increased the percentages of peripheral blood, splenic and lymph node CD3+CD4
+,
CD3+CD8
+ T cells and FOXP3
+ Treg subpopulations after anti-CD20 therapy was proportional
(Figures 4.3, 4.4). This is consistent with previous studies showing that Bdep therapy increases
the proportion of non-B cell populations within the immune compartments (Yu et al., 2012).
We found that CD8+ T cells purified from Bdep immune CD61 splenocytes had impaired
proliferation as compared to CD8+ T cells from non-depleted mice, which could be rescued by
addition of recombinant IL-2 to the cultures (Figure 4.5 C). This could be explained by at least
two different mechanisms. First, B cells may serve as APCs and present self platelet antigens to
CD4+ and CD8
+ T cells in a proinflammatory manner and promote the CD8
+ T cell mediated
ITP (Kambayashi and Laufer, 2014). The removal of B cells by anti-CD20 antibody thus
dampens the stimulation of T cell responses. Second, CD20dim/−
regulatory B cells (Bregs) may
be upregulated after B cell depletion therapy and play an important role in the suppression of
CD8+ T cell mediated ITP. For example, an increased number of CD20
dim regulatory B cells
(Bregs) have been shown capable of suppressing CD4+ and CD8
+ T cell proliferation in vitro
(Bodogai et al., 2013; Lee-Chang et al., 2014). Using the experimental autoimmune
encephalomyelitis (EAE) mouse model, Shen et al showed an important role of IL-35 producing
and IL-10 producing plasma B cells which population are usually negative for CD20−, in the
suppression of autoimmune T cell responses (Reff et al., 1994; Shen et al., 2014). In our current
study, further examination of the splenic CD8+ T cells revealed increased intracellular
Granzyme B expression after Bdep therapy (Figure 4.6 A). This may be related to how
CD20low
4-1BBL+ B regulatory cells regulate Granzyme B expression in CD8
+ T cells (Bodogai
137
et al., 2013; Lee-Chang et al., 2014). Although Granzyme B expression in CD8+ T cells was
found to be increased in follicular lymphoma patients at diagnosis, which was associated with
better prognosis, it appeared unchanged in patients with ITP (Audia et al., 2013; Laurent et al.,
2011). Taken together, our results suggest that in vivo Bdep therapy interferes with in vitro IL-
2-dependent CD8+ T cell activation but more research is required to characterize how this
phenomenon occurs.
We further examined the effect of Bdep therapy on the development of ITP. When the
CD61 KO mice were Bdep in vivo either before or after platelet immunization, we found that
the ability of their splenocytes to induce ITP was completely prevented.
Mechanistically, Bdep therapy in vivo may actively suppress or exhaust the proliferative
potential of pathogenic CD8+ T cells upon activation and thereby limit their ability to induce
ITP. Support for this possibility comes from studies showing that T cells, in the absence of B
cells, have proliferation and memory development defects (Brodie et al., 2008; Shen et al.,
2003; Thomsen et al., 1996). In addition, although B cell depletion in vivo may modulate the
balance between pro- and anti-inflammatory T cell-derived cytokines (Stasi et al., 2007), we did
not observe any significant differences in IFN-γ production by CD8+ T cells (Figure 4.6 B) or
intracellular expression of IFN-γ/IL-4 in CD4+ T cells (N=5-8, data not shown) after Bdep
therapy. Furthermore, in accordance with previous studies, the activation marker CD44 was not
increased on the CD8+ T cells after Bdep therapy (Figure 4.6 C) (Brodie et al., 2008; Shen et al.,
2003; Xiu et al., 2008). Of interest, interruption of the B and T cell interactions by, for example,
CD40L antibody, shows a similar therapeutic effect as rituximab in ITP suggesting a direct
interaction between B cells and T cells is essential for ITP induction (Cooper et al., 2012). More
studies are required, however, to further elucidate these mechanisms.
In summary, our study suggests that the effectiveness of anti-CD20 therapy is due to
induction of a significant CD8+ T cell activation/proliferation defect via IL-2 blockade that
correlates with their inability to induce thrombocytopenia. This may provide an additional
explanation for the therapeutic effects of rituximab in T cell-mediated ITP.
138
Chapter 5
Discussion And Future Directions
139
5.1 Discussion
This thesis aimed to characterize the effects of different therapies on antibody-mediated
and T cell-mediated immune thrombocytopenia (ITP) separately in murine models. Current ITP
treatment options are not satisfactory due to adverse reactions, low long-term response rates and
costs (Provan et al., 2010). Understanding the pathogenesis of the disease is essential to the
future development of effective and safe therapies. Understanding the mechanisms of current
therapies could not only broaden our knowledge of the pathogenesis of the disease but also help
us discover new therapies. Animal models of ITP are useful tools that allow us to examine the
pathogenesis of the disease and action mechanisms of the therapies.
To achieve my goals, first, I studied the effects of different anti-platelet antibodies on
megakaryocytes in a passive ITP model and the impact of IVIg therapy. Most of the anti-platelet
antibodies did not significantly change the number or morphology of megakaryocytes and
neither did IVIg therapy, suggesting limited effects on megakaryocytes. Second, I evaluated the
effectiveness of allogeneic platelet transfusion therapy and its effect on megakaryocytes in ITP
using the laboratory’s active murine ITP model. I found an inhibitory effect of the transfusion
therapy on T cell-mediated ITP associated with ameliorated megakaryocyte abnormalities. I
further characterized the interaction between allogeneic platelets and T cells in vivo and in vitro.
Third, I investigated the interaction between B cells and T cells in ITP by examining T cell-
mediated ITP development after B cell depletion therapy and found that T cell-mediated ITP
was abolished without B cells and this was associated with a decreased proliferation of the
CD8+
T cells.
Different antibodies have different effects on megakaryocytes
Megakaryocyte abnormalities in ITP were first reported by Dameshek et al in 1946,
although the reason was not clear at that time. Anti-platelet antibodies were the first immune
140
factors discovered to be responsible for the pathogenesis of ITP and these studies dated back to
the 1950s-1960s (Harrington et al., 1951; Shulman et al., 1965). Currently, it is known that
about two thirds of patients with ITP have anti-platelet antibodies that primarily target platelet
GPIIbIIIa and GPIb molecules (Chan et al., 2003; Karpatkin et al., 1972). However, the effect of
anti-platelet antibodies on megakaryocytes was not linked together in the pathogenesis of ITP
until the recognition that megakaryocytes and platelets express all the platelet lineage markers
(Michelson, 2013). In the last decade, using cultured megakaryocytes derived from
hematopoietic stem cells in vitro, it has been shown that both anti-platelet serum and T cells
from patients with ITP could inhibit megakaryocyte proliferation and maturation in vitro, as
indicated by the number, size, ploidy and apoptosis of the megakaryocytes and the number of
proplatelets/platelets produced by megakaryocytes (Balduini et al., 2008; Chang et al., 2003; Li
et al., 2007; McMillan et al., 2004). Since patients with ITP have both B and T cell
abnormalities, the effect of the antibodies independent of T cells on megakaryocytes in vivo is
unknown.
In our study, we fulfilled Aim 1 by examining the effects of various anti-platelet antibodies
on megakaryocytes in a 24 h passive ITP model. We used both anti-platelet sera which
contained polyclonal antibodies, and monoclonal antibodies with either GPIIbIIIa or GPIb
specificities. Although all the sera and antibodies induced significant thrombocytopenia in mice
(passive ITP) within 24 h, only an anti-GPIIIa serum and an anti-GPIIb monoclonal antibody
(MWReg30) significantly increased the megakaryocyte counts. These data suggest that most
anti-platelet antibodies with different specificities significantly affect platelet counts,
presumably through peripheral splenic destruction, but few affect megakaryocyte counts or
morphology. However, further studies are needed to confirm this. In addition, we evaluated the
therapeutic effect of IVIg on ITP induced by these different antibodies and on megakaryocytes
in vivo. In the majority of passive ITP groups, although IVIg could significantly ameliorate
thrombocytopenia, it did not significantly change the number of megakaryocytes as compared
with non-treated control groups. This suggests that IVIg may not directly regulate the
thrombopoiesis in ITP.
141
In this study, due to limited time and resources, there are several experiments that we have
planned but have not carried out. Previous electron microscopy (EM) studies in patients with
ITP have shown abnormal apoptotic changes in megakaryocytes such as cytoplasmic
vacuolization, distended DMS and condensed chromatin (Houwerzijl et al., 2004). Besides the
EM study findings, there is also evidence of antibody-induced increased apoptosis of cultured
human megakaryocytes in vitro (Balduini et al., 2008; Chang et al., 2003; Li et al., 2007;
McMillan et al., 2004). We also need to examine the ultrastructure of the megakaryocytes in the
passive ITP model by EM, and examine the apoptotic markers of the megakaryocytes using
immunohistochemistry staining or flow cytometry techniques. Moreover, our lab has recently
developed a protocol to isolate and culture primary megakaryocytes from mouse bone marrow.
Thus, in combination with other techniques just mentioned, I could further examine the direct
effect of antibodies on megakaryocytes and whether the antibodies trigger the thrombopoiesis
signaling pathways of megakaryocytes in vitro. Nonetheless, it appears from this chapter that
while most anti-platelet antibodies can induce thrombocytopenia independently of T cells, they
generally fail to induce changes in megakaryocyte numbers or morphology in the bone marrow.
This may suggest that megakaryocyte abnormalities observed in ITP may be due primarily to T
cells and I attempted to address this in the following chapter.
Allogeneic platelet transfusions benefit patients with T cell-mediated ITP
Historically, allogeneic platelet transfusions were not recommended for the management of
ITP (Ahn and Harrington, 1977; Slichter, 1980; Sprague et al., 1952). However, some clinical
evidence emerged to challenge this dogma by showing that allogeneic platelet transfusions
might in fact benefit patients with ITP (Carr et al., 1986; Spahr and Rodgers, 2008). However,
to date, there is still a lack of substantial investigations on this subject.
Allogeneic platelets have been shown to have transfusion-related immunomodulation
(TRIM) effects and our previous work has found significantly improved allogeneic skin graft
survival after allogeneic platelet transfusions (Aslam et al., 2008). We found this TRIM effect
was associated with the MHC class I molecules on platelets. MHC class I on platelets is
142
denatured and lacks 2 microglobulin when compared to MHC class I on nucleated cells. We
and others have shown that allogeneic platelets cannot stimulate CD8+ T cell responses
(Gouttefangeas et al., 2000; Kao, 1987; Kao et al., 1986; Semple et al., 1995). Moreover, the
immune functions of transfused platelets could also potentially regulate the immune responses
in the recipients, for example by releasing proinflammatory or anti-inflammatory cytokines
(Kapur et al., 2015b).
We examined whether this TRIM effect would affect antibody- or T cell-mediated platelet
and megakaryocyte destruction in our active ITP mouse model. We fulfilled Aim 2 by showing
that while allogeneic platelet transfusions did not affect antibody-mediated ITP, they completely
prevented the development of T cell-mediated ITP. Both T cell-mediated platelet destruction in
the spleen and increased megakaryocyte number in the bone marrow were normalized by
allogeneic platelet transfusions. Since CD8+ CTLs are recognized as the major cell population
responsible for T cell-mediated ITP in human, then we wondered: could this TRIM effect be
related to an inhibition of CTL cytotoxicity? In vitro cytotoxicity function assays were
carried out to address this question. In an ideal scenario, to test anti-CD61 specific cytotoxicity,
target cells that express CD61 peptide through intact syngeneic MHC class I complexes should
be used. However, we could not find target cells that met the criteria. None of the cell-lines with
syngeneic MHC class I were CD61 positive. Therefore we picked a macrophage cell-line PU5-
1.8 and pulsed them with CD61+ platelets, and used these cells as target cells after confirming
that they became weakly CD61 positive. Subsequent cytotoxicity assays showed increased anti-
CD61 T cell-mediated cytotoxicity in immune CD61 KO mice and SCID adoptively transferred
with immune splenocytes. In contrast, CD61 mice transfused with allogeneic CD61+ platelets
showed no cytotoxicity against target cells. We further cultured immunized CD61 KO
splenocytes in vitro together with either syngeneic or allogeneic CD61+ platelets and the latter
incubations consistently showed a significantly reduced cytotoxicity level. This suggests that
allogeneic platelets inhibit the anti-CD61 specific CTL responses (Aim 2). Furthermore,
weekly allogeneic platelet transfusions rescued pre-existing T cell-mediated ITP, suggesting a
therapeutic effect of allogeneic platelet transfusions. Based on these observations, we concluded
143
that ‘the cell-mediated ITP could be abolished by transfusions of allogeneic platelet MHC I
antigens’, ‘indicating a potential immunomodulatory benefit of allogeneic platelet transfusions
in ameliorating T cell-mediated ITP’ (Guo et al., 2014). Of interest, there was significant
increases in megakaryocyte numbers and abnormal morphology in the SCID mice with active T
cell-mediated ITP and these abnormalities were abolished by the allogeneic platelet
transfusions. These results support our claim in Chapter 2 that antibodies alone do not
necessarily induce megakaryocyte abnormalities but only when in conjunction with anti-platelet
CTL’s are megakaryocyte abnormalities observed.
A concern in this study is MHC class I polymorphisms in different mouse strains. In other
words, are the allogeneic platelet transfusion effects only seen when H-2b platelets are
transfused into H-2d recipients. In our current study, most of the work shown was carried out in
BALB/c (H-2d) mice. To exclude the possibility that the TRIM effects observed were not H-2
d
specific, we replicated the same experiments in reverse where C57BL/6 CD61 KO mice (H-2b)
were used as transfusion recipients of BALB/c (H-2d) platelets. Thus it appears that our
transfusion effect holds for different mouse strains with MHC incompatibilities.
Although we found that allogeneic platelet transfusions could inhibit the anti-CD61
specific T cell-mediated ITP, we did not further examine whether this was due to a direct
interaction between allogeneic platelets and CTLs, or an indirect effect with the involvement of
other cells such as antigen presenting cells. Overall, the novel finding that allogeneic platelet
transfusions have an inhibitory effect on T cell-mediated ITP provides an explanation for the
observation of continuous increments in platelet counts after cessation of platelet transfusions in
patients with ITP, and supports the usage of allogeneic platelet transfusions in the management
of patients with ITP.
B cells help CD8+ T cell responses in ITP
Since the initial success of raising platelet counts of patients with ITP using rituximab,
there has been an increasing interest in understanding the mechanisms of rituximab in
ameliorating the disease (Stasi et al., 2001). The initial rationale was that rituximab inhibits
144
antibody-mediated platelet destruction by removal of B cells. However, anti-platelet antibody
titers did not necessarily decrease in responders and anti-platelet antibody-negative patients
could also respond to the therapy suggesting antibody-independent mechanism(s) may exist
(Cooper et al., 2012). The effectiveness of rituximab in other CD8+ T cell mediated diseases
such as diabetes also suggests immunomodulatory effects of B cell depletion therapy on T cell
responses (Hu et al., 2007). Therefore we hypothesized that B cell depletion therapy could
regulate CD8+ T cell-mediated ITP.
B cells could regulate CD8+ T cell-mediated immune responses in several ways. An
antigen specific CD8+ T cell immune response could be divided into at least three different
stages: a) the initial activation, b) clonal expansion (extensive proliferation) and antigen
clearance, and c) apoptosis of most effector cells and differentiation of a small population into
memory CD8+ T cells (Jenkins, 2013). The CD8
+ T cell responses are regulated by APCs, CD4
+
helper T cells and cytokine patterns (Fooksman, 2014; Jenkins, 2013; Rosette et al., 2001). In
the first stage, B cells do not seem to play an important role. This is supported by the
observation that in B cell deficient mice or B cell depleted mice, antigen specific CD8+ T cell
immune response could be elicited (Lykken et al., 2014; Thomsen et al., 1996). In our study, the
proportion of CD8+ T cells in the total T cell population was also comparable between B cell
depleted and non-depleted immune CD61 KO mice. However, B cell depletion may regulate the
proliferation of CD8+ T cells, as suggested by our CD8
+ T cell proliferation in vitro experiment.
Consistently, Lykken et al also showed decreased CD8+ T cell number 7 days after initial
LCMV infection in B cell depleted mice (Lykken et al., 2014). Moreover Thomsen et al found
impaired CD8+ T cell responses in B cell deficient mice after chronic LCMV infection
(Thomsen et al., 1996). In addition, B cell deficiency may also impair the formation of CD8+ T
cell memory. This was proposed by Thomsen et al as they did not find a CD8+ T cell memory
response upon secondary antigen stimulation (Thomsen et al., 1996). Later Shen et al confirmed
a positive role for B cells in CD8+ T cell memory formation (Shen et al., 2003). In addition to
the direct effect on CD8+ T cell responses, B cell depletion could also indirectly regulate the
CD8+ T cell responses through, for example, downregulation of CD4
+ helper T cells and
145
upregulation of Tregs (Lund and Randall, 2010). Cytokines produced by B cells may also
impact the CD8+ T cell responses, for example IL-2, IL-10, IL-6 and IFN- γ (Lund, 2008).
In 2007, Stasi et al reported normalized Th1/Th2 (IFN-γ+/IL-4
+ of CD4
+ T cells) and
Tc1/Tc2 (IFN-γ+/IL-4
+ of CD8
+ T cells) ratios in the peripheral blood of rituximab responders
but not in patients with active ITP nor in rituximab non-responders suggesting an
immunomodulatory effect of rituximab on both CD4+ and CD8
+ T cell responses (Stasi et al.,
2007). Similarly, Nazi et al reported a decreased number of IFN-γ+-producing T cells in patients
with ITP upon exogenous antigen stimulation after rituximab administration (Nazi et al., 2013).
Subsequently, further examination of the CD4+ T cells showed restored number and function of
CD4+ T regulatory cells (Tregs) after rituximab therapy which may help explain the restored
balance within CD4+ and CD8
+ T cells (Stasi et al., 2008). In contrast with rituximab
responders, spleens from patients who failed rituximab therapy still showed decreased Tregs and
increased memory CD8+
T cells (Audia et al., 2011; Audia et al., 2013).
Beyond the changes in numbers and frequencies of different cell populations, our
knowledge of how B cell depletion therapy can regulate CD4+ and CD8
+ T cell responses and
restore Tregs in ITP still remains a matter of debate. One limitation is the impossibility of spleen
sample acquisition from responder patients for more detailed studies. In addition, although it is
well accepted that in patients with ITP, there are both antibody-mediated and CD8+ T cell-
mediated mechanisms, it has been difficult to sort out the exact contributions of one pathway
over the other.
In Chapter 4, our murine active ITP model was used for the study of the B cell depletion
therapy in T cell-mediated ITP. We initially tested a number of commercially available anti-
CD20 mAb but none of them caused complete depletion of B cells in vivo. We obtained a novel
anti-mouse CD20 mAb from Biogen and this antibody proved to be an almost total depletion
antibody and we used this antibody for our Chapter 4 studies.
We examined the B cell percentages in the blood, spleen, and mesenchymic lymph nodes,
as well as determination of the anti-platelet antibody titers in the blood (Aim 3). The anti-mouse
146
CD20 IgG2a antibody used in our study successfully depleted 80-99% of the B cells in vivo.
Next, we established a mouse model of B cell depletion therapy in CD8+ T cell-mediated ITP
using this antibody (Aim 3). By collecting immune splenocytes from CD61 KO mice, depleting
the splenic B cells in vitro and then transferring the depleted cells into SCID mice, CD8+ T cell-
mediated ITP independent of B cells could be induced (Chow et al., 2010). To further fulfill
Aim 3, the anti-CD20 mAb was administrated into the CD61 KO mice before or after platelet
immunization (Bdep pre and post respectively), followed by transferring their splenocytes into
SCID mice. Analyzing the platelet counts in SCID mice showed that B cell depletion therapy,
whether before or after platelet immunization, significantly ameliorated the T cell-mediated ITP.
We further investigated potential mechanisms through which B cells could regulate the CD8+ T
cell-mediated ITP. The CD8+ T cell proliferation assay in vitro revealed that CD8
+ T cells
isolated from the immune CD61 KO spleens without B cells proliferated significantly less
compared with T cells from intact (non-depleted) spleens and this appeared to be due to a lack
of IL-2 production. These data suggest an important role of B cells for CD8+ T cell immune
responses upon immune challenges and depletion of B cells leads to an impaired CD8+ T cell
response which could no longer elicit thrombocytopenia in our mice. This may provide a novel
explanation for the antibody-independent therapeutic effect of rituximab in humans.
To better understand the role of B cells on CD8+ T cell immune responses in ITP, we
further characterized the CD8+ T cells from the immune CD61 KO mice treated with B cell
depletion therapy by flow cytometry, including the CD4/CD8 T cell ratio, percentages of CD8+
Tregs, the granzyme B, IFN- and CD44 expression levels. We only observed significantly
increased expression of granzyme B with B cell depletion therapy, indicating that the regulation
of CD8+ T cell immune responses by B cells are quite specific and probably not due to any
broad secondary responses.
Although the anti-CD20 antibody in our study successfully depleted most of the B cells
in vivo, there were still a small population of B cells remaining in the spleen and lymph nodes.
Theoretically, these residual B cells might still be able to mediate some effects on the T cells.
To better address whether the ameliorated CD8+ T cell-mediated ITP is due to the removal of
147
the majority of B cells, or due to the modulation of residual B cells by anti-CD20 antibody,
experiments could be carried out using pepsin digested anti-CD20 antibody instead of intact
antibody. Pepsin cuts the antibody into F(ab’)2 fragments (Murphy et al., 2012; Paul, 2013) that
can still bind to B cells but would not be able to deplete B cells in vivo through Fc-receptor
mediated phagocytosis or ADCC. In this away, comparison of the immune CD61 KO mice that
received pepsin digested or intact antibody will reveal the potential regulatory effect, if there are
any, by residual anti-CD20 coated B cells on the CD8+ T cell-mediated ITP.
Overall, the inhibition of CD8+ T cell proliferation and prevention of the development of
ITP by a mouse anti-CD20 mAb suggest a novel role for B cells in maintaining CD8+ T cell
responses. This provides us with another explanation for the therapeutic effect of B cell
depletion therapy for ITP and potentially for other T cell-mediated autoimmune diseases as
well.
Animal models of ITP are useful tools with limitations
There are several critical challenges in human studies on ITP. First of all, due to ethical
reasons, access to immune organs and tissues is restricted. For example, while spleen is the
major lymphoid site for adaptive immune responses against platelets to initiate, it is impossible
to examine and compare spleens from treatment responders (not splenectomized) versus
nonresponders. Second, although it is clear that both B cells and T cells are involved in the
pathogenesis of the disease, their relationship in ITP remains ambiguous. Patients with
detectable autoreactive anti-platelet antibodies are usually considered to possess B cell dominant
pathological immune responses and patients without are considered as T cell dominant.
However, patients may actually have both B cells and T cells that mediated thrombocytopenia in
vivo and it is difficult to examine the individual roles of B cells or T cells separately. Third,
although the studies by Harrington made fundamental discoveries, transfer of potential
pathological components from patients with ITP into healthy volunteers are not ethical. In
addition, clinical trials of new therapies are restrictively regulated and the recruitment of
adequate number of patients is a challenge considering the low incidence of the disease.
148
In contrast, animal models of ITP are useful tools that can easily overcome the
limitations mentioned above and allow us to further understand the disease and the therapies.
Using the passive and active ITP mouse models, we were able to examine the role of anti-
platelet B cells and T cells separately. For instance, in our active ITP model, by transferring
either B cell depleted or CD8+ T cell depleted splenocytes from the immune CD61 KO into
SCID mice, we could examine the effect of allogeneic platelet transfusions on B cell-mediated
ITP and T cell-mediated ITP separately (Guo et al., 2014). Examination of the T cells in B cell
depletion therapy treated mice also revealed a novel mechanism of the anti-CD20 antibody - the
decreased CD8+ T cell proliferation after B cell depletion therapy, which could help to explain
the antibody-independent therapeutic effect of rituximab in patients (Guo et al., 2016). In our
previous work, we also found abnormal Treg development in the thymus which may contribute
to the pathogenesis of ITP but remains unclear in humans with ITP (Aslam et al., 2012).
There are, however, limitations with animal studies. Firstly, our findings in mice may not
necessarily represent the situation in humans. In our active ITP mouse model, the
thrombocytopenia in SCID mice was induced by anti-CD61 (GPIIIa) specific immune
responses. We choose CD61 KO mice because anti-GPIIbIIIa antibodies are the most frequent
detectable antibodies in patients with ITP. However, most patients have polyvalent autoreactive
antibodies that may be reactive with multiple receptors on platelets (Liel et al., 2014; van
Leeuwen et al., 1982). Secondly, in our study, B cell depletion therapy significantly alleviated T
cell-mediated ITP, providing a novel explanation of antibody-independent mechanism of the
therapy. In humans, however, only some of the anti-platelet antibody negative patients respond
to B cell depletion therapy, indicating a more complex situation in patients than in our mouse
model (Cooper et al., 2012).
149
5.2 Conclusion
I examined both antibody-mediated and T cell-mediated ITP in our mouse models and
evaluated three different treatments for ITP including IVIg, allogeneic platelet transfusions and
B cell depletion therapy. I found that IVIg could inhibit antibody-mediated thrombocytopenia
but did not necessarily affect megakaryocytes in the bone marrow. In contrast, allogeneic
platelet transfusions significantly inhibited both T cell-mediated thrombocytopenia and bone
marrow MK abnormalities. Similar findings were also observed with B cell depletion therapy in
its ability to rescue T cell-mediated ITP through inhibiting CD8+ T cell proliferation.
In general, I was able to address my primary research questions and achieved my goals.
Using the passive and active ITP mouse models, I discovered that megakaryocytes may be
resistant to the effects of antibodies in ITP and novel immunomodulatory mechanisms of both
allogeneic platelet transfusion therapy and B cell depletion therapy exist in ITP. My work has
challenged some prevailing theories, one of which believes that antibodies not only destroy
platelets peripherally but also inhibit platelet production in the bone marrow and another of
which thinks that platelet transfusions are effective as an ITP treatment. Nonetheless, I am still
cautious of my claims due to the limitations of animal studies and of potential discrepancies
between my findings and those in human ITP. It is my hope that my work can lay a knowledge
foundation for the development of new therapies that may be curative and enable patients to
maintain a good quality of life.
150
5.3 Future Directions
Megakaryocyte morphology and viability evaluations in the antibody-mediated passive
ITP model
In Chapter 2, I studied the number and morphology of megakaryocytes with H&E staining
and found discrepant effects of the different antibodies. In particular, I found increased numbers
of megakaryocytes in ITP mice which were induced by MWReg30, an anti-CD41 (anti-GPIIb)
antibody. The rest of the monoclonal antibodies did not seem to affect the megakaryocytes
number nor morphology. Our in vivo findings contradicted several in vitro studies on cultured
human megakaryocytes that showed anti-platelet antibodies from patients with ITP significantly
increased megakaryocyte apoptosis growth in vitro (Balduini et al., 2008; Chang et al., 2003; Li
et al., 2007; McMillan et al., 2004). Thus, to better address the effect of anti-platelet antibodies
on megakaryocytes in vivo, further morphology as well as viability analyses of megakaryocytes
are planned using the passive ITP model. For example, 24 hours after injection of anti-platelet
antibodies, the mice will be sacrificed and their femurs collected for the following experiments.
First, to evaluate whether the anti-platelet antibodies affect megakaryocyte integrity and
intracellular structures, bone marrow will be collected and megakaryocytes will be separated
from the bone marrow and processed for EM examinations. Second, to clarify whether anti-
platelet antibodies induce megakaryocyte apoptosis in 24h in vivo, the expression level of
caspase-3 and annexin V will be examined in the megakaryocytes by flow cytometry and
fluorescence microscopy (Josefsson et al., 2011; Lev et al., 2014; Leytin et al., 2006; Li et al.,
2007).
The effect of MWReg 30 on megakaryocytes
In Chapter 2, we showed significantly increased numbers of megakaryocytes in the bone
marrow in MWReg30 treated mice within 24 hours. The total megakaryocyte number
calculations in our study included both mature megakaryocytes and immature megakaryocytes
including promegakaryoblasts, megakaryoblasts and promegakaryocytes. It is not clear whether
MWReg30 stimulated the increase of megakaryocytes at different stages proportionally or
151
mainly regulated megakaryocytes at a certain stage. One way to separate megakaryocytes of
different stages is by their ploidy, which increases from 2N to up to 32N through maturation
(Italiano Jr. and Hartwig, 2013). Therefore we will examine the ploidy of megakaryocytes
from the passive ITP mice using an intracellular nucleic acid stain propidium iodide
followed by flow cytometry and fluorescence microscopy analysis. In addition, although all
the monoclonal antibodies we used in Chapter 2 could induce thrombocytopenia, MWReg30
showed a distinctive in vivo effect on megakaryocytes. It is possible that MWReg30 triggers
signaling pathways which upregulate megakaryocyte maturation and proliferation. One of the
most important pathways in megakaryocyte maturation and proliferation is the JAK/STAT
pathway (Pallard et al., 1995; Sattler et al., 1995). We are planning to examine whether the
binding of MWReg30 to megakaryocytes activates the JAK/STAT pathway by examining
the transcription levels of JAK2 and STAT5 mRNAs and the phosphorylation of JAK2 and
STAT5 proteins.
A potential role of megakaryocytes in triggering CD8+ T cell immune responses in ITP
In the literature, most of the studies on megakaryocytes and CD8+ T cells in ITP have being
focusing on the pathological role of autoreactive CD8+ T cells in mediating megakaryocyte
destruction (Chang et al., 2003; Li et al., 2007; McMillan et al., 2004; Olsson et al., 2003). As
mentioned in Chapter 1, both megakaryocytes and platelets have immunomodulatory roles and
dysfunction of megakaryocytes and platelets may lead to altered immunity. Recently, some
preliminary work of my colleagues suggests that megakaryocytes can function as potent APCs
that can directly activate antigen specific CD8+ T cell responses. Thus, we hypothesize that
megakaryocytes may not only be the target of immune T cells in ITP but also an initiator
of the autoreactive CD8+ T cell responses in ITP. We would test our hypothesis by pulsing
megakaryocytes from BALB/c CD61 KO mice with syngeneic WT platelets in vitro and then
culture them with naïve or immune CD8+ T cells to examine whether megakaryocytes can
stimulate anti-CD61 CD8+ T cell immune responses. The CD8
+ T cell responses would be
monitored by intracellular IL-2 and IFN-γ production and the proliferation and cytotoxicity
assays in vitro.
152
The therapeutic potential of allogeneic platelet transfusions in patients with ITP
In Chapter 3, we found an inhibitory effect from allogeneic platelet transfusions on T cell-
mediated ITP. With the evidence that allogeneic MHC class I molecules on platelets are
denatured and disable the CD8+ T cell mediated platelet destruction, we look forward to
continuing our studies using human samples. Whether human allogeneic platelets could
inhibit the CD8+ T cell mediated platelet destruction and CD8
+ T cell proliferation in vitro
will be examined. Patients with ITP as well as healthy volunteers will be recruited and their
peripheral blood will be collected and CD8+ T cells will be isolated. The cytotoxic activity of
the CD8+ T cells from patients with ITP or healthy volunteers will be evaluated by first
incubating the CD8+ T cells with autologous or allogeneic platelets and then examining the
apoptosis level of platelets. The proliferation potential of the human CD8+ T cells in vitro will
be examined similarly as in our mouse studies in Chapter 4. The isolated CD8+ T cells will be
labeled with the fluorescent Violet Proliferation Dye 450 (V450) and cultured with either
autologous or allogeneic platelets, and with anti-human CD3 antibody/anti-human
CD28/recombinant IL-2 stimulation. The proliferation of CD8+ T cells will be analyzed after
72h by flow cytometry.
Characterization of how B cells regulate CD8+ T cell responses
In Chapter 4, we found decreased CD8+ T cell proliferation after B cell depletion therapy.
Although there is evidence of impaired CD8+ T responses in B cell deficient or depleted mice,
our study provides the first mechanistic explanation (Brodie et al., 2008; Shen et al., 2003;
Thomsen et al., 1996). Further studies on how B cells promote CD8+ T cell proliferation in vivo
may not only improve our understanding of ITP as well as other T cell-mediated immune
responses, but may also provide potential novel therapeutic strategies for T cell-dominant
diseases. We can further characterize the CD8+ T cells including the expression level of
surface receptors, intracellular cytokines and transcription factors that are involved in
CD8+ T cell proliferation and memory T cell formation, such as CD28, CTLA-4, CD40L
153
(CD154), programmed death-1 receptors, IL-2, activation and nuclear localization of nuclear
factor NFκB, CCR7, CD62L.
The development of thymic regulatory T cells (Tregs) in our mouse model
In addition, in our active ITP mouse model, we found decreased FOXP3+ Tregs in the
periphery, associated with an increase of Tregs in the thymus (Aslam et al., 2012). We also
found an increase of Tregs in the periphery with the amelioration of the disease after treatment
(data not show). We need to further study the potential mechanisms responsible for the
retention of Tregs in the thymus during ITP, and potential strategies to promote their
release from the thymus as a future treatment for ITP.
The role of natural killer (NK) cells in ITP
As mentioned in Chapter 1, one mechanism of the antibody induced immunological
destruction is through the antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells
(Paul, 2013). However, in the case of ITP, discrepant findings have been documented
concerning whether NK cells are activated and involved in the pathogenesis of ITP (Breunis et
al., 2008; Engelhard et al., 1986; Garcia-Suarez et al., 1993; Semple et al., 1991; Semple and
Freedman, 1994). This question has not been well addressed so far and the role of NK cells in
ITP remains mysterious. We will examine whether NK cells are involved in ITP using our
active ITP mouse model. We will first deplete NK cells in BALB/c CD61 KO mice in vivo
using the anti-Asialo GM1 antibody on the day prior to initial platelet immunization. Rabbit
immunoglobulin will be used as an isotype control. On the following day, CD61 KO mice in
both groups will be immunized with syngeneic WT platelets and continue to be immunized
weekly. The efficiency of NK cell depletion will be further examined by detecting the
percentage of CD56+CD3
- NK cells in peripheral blood weekly and in the spleen and lymph
nodes at week 4. After four weeks, immune CD61 KO mice will be sacrificed and their
splenocytes will be further transferred either as non-depleted or in vitro CD56+CD3
- NK cell
depleted into SCID mice at a dose of 3×104 cells/mouse and the platelet counts of the SCID
154
recipient mice will be monitored. This will allow for an initial analysis of whether NK cells play
a role in murine ITP.
We hope the outlined experiments will not only improve our understanding of ITP, but
also shed light on new therapeutic strategies with better targeted action and less side effects
culminating in a more individualized therapeutic approach to ITP.
155
REFERENCES
Adly, A.A., Ragab, I.A., Ismail, E.A., and Farahat, M.M. (2015). Evaluation of the immature
platelet fraction in the diagnosis and prognosis of childhood immune thrombocytopenia.
Platelets 26, 645-650.
Afzali, B., Lombardi, G., Lechler, R.I., and Lord, G.M. (2007). The role of T helper 17 (Th17)
and regulatory T cells (Treg) in human organ transplantation and autoimmune disease. Clinical
and experimental immunology 148, 32-46.
Ahn, Y.S., and Harrington, W.J. (1977). Treatment of idiopathic thrombocytopenic purpura
(ITP). Annual review of medicine 28, 299-309ENG.
Andersson, P.O., Olsson, A., and Wadenvik, H. (2002). Reduced transforming growth factor-
beta1 production by mononuclear cells from patients with active chronic idiopathic
thrombocytopenic purpura. British journal of haematology 116, 862-867.
Andonegui, G., Kerfoot, S.M., McNagny, K., Ebbert, K.V., Patel, K.D., and Kubes, P. (2005).
Platelets express functional Toll-like receptor-4. Blood 106, 2417-2423.
Arnold, D.M., Nazi, I., Toltl, L.J., Ross, C., Ivetic, N., Smith, J.W., Liu, Y., and Kelton, J.G.
(2015). Antibody binding to megakaryocytes in vivo in patients with immune
thrombocytopenia. European journal of haematology 95, 532-537.
Asahi, A., Nishimoto, T., Okazaki, Y., Suzuki, H., Masaoka, T., Kawakami, Y., Ikeda, Y., and
Kuwana, M. (2008). Helicobacter pylori eradication shifts monocyte Fcgamma receptor balance
toward inhibitory FcgammaRIIB in immune thrombocytopenic purpura patients. The Journal of
clinical investigation 118, 2939-2949.
Aslam, R., Hu, Y., Gebremeskel, S., Segel, G.B., Speck, E.R., Guo, L., Kim, M., Ni, H.,
Freedman, J., and Semple, J.W. (2012). Thymic retention of CD4+CD25+FoxP3+ T regulatory
156
cells is associated with their peripheral deficiency and thrombocytopenia in a murine model of
immune thrombocytopenia. Blood 120, 2127-2132.
Aslam, R., Speck, E.R., Kim, M., Crow, A.R., Bang, K.W., Nestel, F.P., Ni, H., Lazarus, A.H.,
Freedman, J., and Semple, J.W. (2006). Platelet Toll-like receptor expression modulates
lipopolysaccharide-induced thrombocytopenia and tumor necrosis factor-alpha production in
vivo. Blood 107, 637-641.
Aslam, R., Speck, E.R., Kim, M., Freedman, J., and Semple, J.W. (2008). Transfusion-related
immunomodulation by platelets is dependent on their expression of MHC Class I molecules and
is independent of white cells. Transfusion 48, 1778-1786.
Assinger, A. (2014). Platelets and infection - an emerging role of platelets in viral infection.
Frontiers in immunology 5, 649.
Audia, S., Samson, M., Guy, J., Janikashvili, N., Fraszczak, J., Trad, M., Ciudad, M., Leguy, V.,
Berthier, S., Petrella, T., et al. (2011). Immunologic effects of rituximab on the human spleen in
immune thrombocytopenia. Blood 118, 4394-4400.
Audia, S., Samson, M., Mahevas, M., Ferrand, C., Trad, M., Ciudad, M., Gautheron, A.,
Seaphanh, F., Leguy, V., Berthier, S., et al. (2013). Preferential splenic CD8(+) T-cell activation
in rituximab-nonresponder patients with immune thrombocytopenia. Blood 122, 2477-2486.
Auerbach, D.J., Lin, Y., Miao, H., Cimbro, R., Difiore, M.J., Gianolini, M.E., Furci, L., Biswas,
P., Fauci, A.S., and Lusso, P. (2012). Identification of the platelet-derived chemokine
CXCL4/PF-4 as a broad-spectrum HIV-1 inhibitor. Proceedings of the National Academy of
Sciences of the United States of America 109, 9569-9574.
Balduini, A., Pallotta, I., Malara, A., Lova, P., Pecci, A., Viarengo, G., Balduini, C.L., and
Torti, M. (2008). Adhesive receptors, extracellular proteins and myosin IIA orchestrate
proplatelet formation by human megakaryocytes. Journal of thrombosis and haemostasis : JTH
6, 1900-1907.
157
Baldwin, T.A., Sandau, M.M., Jameson, S.C., and Hogquist, K.A. (2005). The timing of TCR
alpha expression critically influences T cell development and selection. The Journal of
experimental medicine 202, 111-121.
Ballem, P.J., Segal, G.M., Stratton, J.R., Gernsheimer, T., Adamson, J.W., and Slichter, S.J.
(1987). Mechanisms of thrombocytopenia in chronic autoimmune thrombocytopenic purpura.
Evidence of both impaired platelet production and increased platelet clearance. The Journal of
clinical investigation 80, 33-40.
Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, Y.J., Pulendran, B., and
Palucka, K. (2000). Immunobiology of dendritic cells. Annual review of immunology 18, 767-
811.
Bao, W., Bussel, J.B., Heck, S., He, W., Karpoff, M., Boulad, N., and Yazdanbakhsh, K. (2010).
Improved regulatory T-cell activity in patients with chronic immune thrombocytopenia treated
with thrombopoietic agents. Blood 116, 4639-4645.
Barsam, S.J., Psaila, B., Forestier, M., Page, L.K., Sloane, P.A., Geyer, J.T., Villarica, G.O.,
Ruisi, M.M., Gernsheimer, T.B., Beer, J.H., et al. (2011). Platelet production and platelet
destruction: assessing mechanisms of treatment effect in immune thrombocytopenia. Blood 117,
5723-5732.
Battinelli, E.M., Hartwig, J.H., and Italiano, J.E., Jr. (2007). Delivering new insight into the
biology of megakaryopoiesis and thrombopoiesis. Current opinion in hematology 14, 419-426.
Behnke, O. (1968). An electron microscope study of the megacaryocyte of the rat bone marrow.
I. The development of the demarcation membrane system and the platelet surface coat. Journal
of ultrastructure research 24, 412-433.
Behnke, O. (1969). An electron microscope study of the rat megacaryocyte. II. Some aspects of
platelet release and microtubules. Journal of ultrastructure research 26, 111-129.
158
Bennett, C.L., Christie, J., Ramsdell, F., Brunkow, M.E., Ferguson, P.J., Whitesell, L., Kelly,
T.E., Saulsbury, F.T., Chance, P.F., and Ochs, H.D. (2001). The immune dysregulation,
polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3.
Nature genetics 27, 20-21.
Bennett, D., Hodgson, M.E., Shukla, A., and Logie, J.W. (2011). Prevalence of diagnosed adult
immune thrombocytopenia in the United Kingdom. Advances in therapy 28, 1096-1104.
Bettelli, E., Carrier, Y., Gao, W., Korn, T., Strom, T.B., Oukka, M., Weiner, H.L., and Kuchroo,
V.K. (2006). Reciprocal developmental pathways for the generation of pathogenic effector
TH17 and regulatory T cells. Nature 441, 235-238.
Bizzozero, G. (1881). Über einer neuen formbestandteil des blutes. Arch Pathol Anat Physiol
90, 261.
Blair, P., and Flaumenhaft, R. (2009). Platelet alpha-granules: basic biology and clinical
correlates. Blood reviews 23, 177-189.
Blanchette, M., and Freedman, J. (1998). The history of idiopathic thrombocytopenic purpura
(ITP). Transfusion science 19, 231-236.
Blomgren, H., and Larsson, E.L. (1978). Regulation of lymphocyte proliferation by soluble
products released by stimulated human lymphocytes. International archives of allergy and
applied immunology 57, 15-21.
Blumberg, N., Masel, D., Mayer, T., Horan, P., and Heal, J. (1984). Removal of HLA-A,B
antigens from platelets. Blood 63, 448-450.
Bodogai, M., Lee Chang, C., Wejksza, K., Lai, J., Merino, M., Wersto, R.P., Gress, R.E., Chan,
A.C., Hesdorffer, C., and Biragyn, A. (2013). Anti-CD20 antibody promotes cancer escape via
enrichment of tumor-evoked regulatory B cells expressing low levels of CD20 and CD137L.
Cancer research 73, 2127-2138.
159
Boehm, T. (2008). Thymus development and function. Current opinion in immunology 20, 178-
184.
Boise, L.H., Minn, A.J., Noel, P.J., June, C.H., Accavitti, M.A., Lindsten, T., and Thompson,
C.B. (1995). CD28 costimulation can promote T cell survival by enhancing the expression of
Bcl-XL. Immunity 3, 87-98.
Bolton-Maggs, P., Tarantino, M.D., Buchanan, G.R., Bussel, J.B., George, J.N., and American
Society of Pediatric, H.O. (2004). The child with immune thrombocytopenic purpura: is
pharmacotherapy or watchful waiting the best initial management? A panel discussion from the
2002 meeting of the American Society of Pediatric Hematology/Oncology. Journal of pediatric
hematology/oncology 26, 146-151.
Bolton-Maggs, P.H., and Moon, I. (1997). Assessment of UK practice for management of acute
childhood idiopathic thrombocytopenic purpura against published guidelines. Lancet 350, 620-
623.
Borkowsky, W., and Karpatkin, S. (1984). Leukocyte migration inhibition of buffy coats from
patients with autoimmune thrombocytopenic purpura when exposed to normal platelets:
modulation by transfer factor. Blood 63, 83-87.
Boshkov, L.K., Kelton, J.G., and Halloran, P.F. (1992). HLA-DR expression by platelets in
acute idiopathic thrombocytopenic purpura. British journal of haematology 81, 552-557.
Bottazzi, B., Doni, A., Garlanda, C., and Mantovani, A. (2010). An integrated view of humoral
innate immunity: pentraxins as a paradigm. Annual review of immunology 28, 157-183.
Bousso, P., and Robey, E. (2003). Dynamics of CD8+ T cell priming by dendritic cells in intact
lymph nodes. Nature immunology 4, 579-585.
Bozzacco, L., Trumpfheller, C., Siegal, F.P., Mehandru, S., Markowitz, M., Carrington, M.,
Nussenzweig, M.C., Piperno, A.G., and Steinman, R.M. (2007). DEC-205 receptor on dendritic
160
cells mediates presentation of HIV gag protein to CD8+ T cells in a spectrum of human MHC I
haplotypes. Proceedings of the National Academy of Sciences of the United States of America
104, 1289-1294.
Branehog, I., Kutti, J., Ridell, B., Swolin, B., and Weinfeld, A. (1975). The relation of
thrombokinetics to bone marrow megakaryocytes in idiopathic thrombocytopenic purpura (ITP).
Blood 45, 551-562.
Branehog, I., Kutti, J., and Weinfeld, A. (1974). Platelet survival and platelet production in
idiopathic thrombocytopenic purpura (ITP). British journal of haematology 27, 127-143.
Breunis, W.B., van Mirre, E., Bruin, M., Geissler, J., de Boer, M., Peters, M., Roos, D., de
Haas, M., Koene, H.R., and Kuijpers, T.W. (2008). Copy number variation of the activating
FCGR2C gene predisposes to idiopathic thrombocytopenic purpura. Blood 111, 1029-1038.
Briggs, C., Kunka, S., Hart, D., Oguni, S., and Machin, S.J. (2004). Assessment of an immature
platelet fraction (IPF) in peripheral thrombocytopenia. British journal of haematology 126, 93-
99.
Brighton, T.A., Evans, S., Castaldi, P.A., Chesterman, C.N., and Chong, B.H. (1996).
Prospective evaluation of the clinical usefulness of an antigen-specific assay (MAIPA) in
idiopathic thrombocytopenic purpura and other immune thrombocytopenias. Blood 88, 194-201.
Brodie, G.M., Wallberg, M., Santamaria, P., Wong, F.S., and Green, E.A. (2008). B-cells
promote intra-islet CD8+ cytotoxic T-cell survival to enhance type 1 diabetes. Diabetes 57, 909-
917.
Brown, W.H. (1913). The Histogenesis of Blood Platelets. The Journal of experimental
medicine 18, 278-286.
Brunkow, M.E., Jeffery, E.W., Hjerrild, K.A., Paeper, B., Clark, L.B., Yasayko, S.A.,
Wilkinson, J.E., Galas, D., Ziegler, S.F., and Ramsdell, F. (2001). Disruption of a new
161
forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the
scurfy mouse. Nature genetics 27, 68-73.
Buchanan, G.R., and Adix, L. (2002). Grading of hemorrhage in children with idiopathic
thrombocytopenic purpura. The Journal of pediatrics 141, 683-688.
Buckner, J.H. (2010). Mechanisms of impaired regulation by CD4(+)CD25(+)FOXP3(+)
regulatory T cells in human autoimmune diseases. Nature reviews Immunology 10, 849-859.
Burdach, S.E., Evers, K.G., and Geursen, R.G. (1986). Treatment of acute idiopathic
thrombocytopenic purpura of childhood with intravenous immunoglobulin G: comparative
efficacy of 7S and 5S preparations. The Journal of pediatrics 109, 770-775.
Burstein, S.A., Friese, P., Downs, T., and Mei, R.L. (1992). Characteristics of a novel rat anti-
mouse platelet monoclonal antibody: application to studies of megakaryocytes. Experimental
hematology 20, 1170-1177.
Bussel, J.B., Kimberly, R.P., Inman, R.D., Schulman, I., Cunningham-Rundles, C., Cheung, N.,
Smithwick, E.M., O'Malley, J., Barandun, S., and Hilgartner, M.W. (1983). Intravenous
gammaglobulin treatment of chronic idiopathic thrombocytopenic purpura. Blood 62, 480-486.
Cao, J., Chen, C., Li, L., Ling-yu, Z., Zhen-yu, L., Zhi-ling, Y., Wei, C., Hai, C., Sang, W., and
Kai-lin, X. (2012). Effects of high-dose dexamethasone on regulating interleukin-22 production
and correcting Th1 and Th22 polarization in immune thrombocytopenia. Journal of clinical
immunology 32, 523-529.
Cao, J., Chen, C., Zeng, L., Li, L., Li, X., Li, Z., and Xu, K. (2011). Elevated plasma IL-22
levels correlated with Th1 and Th22 cells in patients with immune thrombocytopenia. Clinical
immunology 141, 121-123.
162
Cardarelli, P.M., Quinn, M., Buckman, D., Fang, Y., Colcher, D., King, D.J., Bebbington, C.,
and Yarranton, G. (2002). Binding to CD20 by anti-B1 antibody or F(ab')(2) is sufficient for
induction of apoptosis in B-cell lines. Cancer immunology, immunotherapy : CII 51, 15-24.
Carr, J.M., Kruskall, M.S., Kaye, J.A., and Robinson, S.H. (1986). Efficacy of platelet
transfusions in immune thrombocytopenia. The American journal of medicine 80, 1051-1054.
Catani, L., Fagioli, M.E., Tazzari, P.L., Ricci, F., Curti, A., Rovito, M., Preda, P., Chirumbolo,
G., Amabile, M., Lemoli, R.M., et al. (2006). Dendritic cells of immune thrombocytopenic
purpura (ITP) show increased capacity to present apoptotic platelets to T lymphocytes.
Experimental hematology 34, 879-887.
Catani, L., Sollazzo, D., Ricci, F., Polverelli, N., Palandri, F., Baccarani, M., Vianelli, N., and
Lemoli, R.M. (2011). The CD47 pathway is deregulated in human immune thrombocytopenia.
Experimental hematology 39, 486-494.
Catani, L., Sollazzo, D., Trabanelli, S., Curti, A., Evangelisti, C., Polverelli, N., Palandri, F.,
Baccarani, M., Vianelli, N., and Lemoli, R.M. (2013). Decreased expression of indoleamine 2,3-
dioxygenase 1 in dendritic cells contributes to impaired regulatory T cell development in
immune thrombocytopenia. Annals of hematology 92, 67-78.
Chaipan, C., Soilleux, E.J., Simpson, P., Hofmann, H., Gramberg, T., Marzi, A., Geier, M.,
Stewart, E.A., Eisemann, J., Steinkasserer, A., et al. (2006). DC-SIGN and CLEC-2 mediate
human immunodeficiency virus type 1 capture by platelets. Journal of virology 80, 8951-8960.
Chan, H., Moore, J.C., Finch, C.N., Warkentin, T.E., and Kelton, J.G. (2003). The IgG
subclasses of platelet-associated autoantibodies directed against platelet glycoproteins IIb/IIIa in
patients with idiopathic thrombocytopenic purpura. British journal of haematology 122, 818-
824.
163
Chang, M., Nakagawa, P.A., Williams, S.A., Schwartz, M.R., Imfeld, K.L., Buzby, J.S., and
Nugent, D.J. (2003). Immune thrombocytopenic purpura (ITP) plasma and purified ITP
monoclonal autoantibodies inhibit megakaryocytopoiesis in vitro. Blood 102, 887-895.
Chaplin, D.D. (2010). Overview of the immune response. The Journal of allergy and clinical
immunology 125, S3-23.
Chapman, J.F., Murphy, M.F., Berney, S.I., Ord, J., Metcalfe, P., Amess, J.A., and Waters, A.H.
(1987). Post-transfusion purpura associated with anti-Baka and anti-PIA2 platelet antibodies and
delayed haemolytic transfusion reaction. Vox sanguinis 52, 313-317.
Chen, Z., Guo, Z., Ma, J., Ma, J., Liu, F., and Wu, R. (2014). Foxp3 methylation status in
children with primary immune thrombocytopenia. Human immunology 75, 1115-1119.
Cheung, A.L., Krishnan, M., Jaffe, E.A., and Fischetti, V.A. (1991). Fibrinogen acts as a
bridging molecule in the adherence of Staphylococcus aureus to cultured human endothelial
cells. The Journal of clinical investigation 87, 2236-2245.
Chieffi, O., Benedetti, P.A., Lecchini, L., and Rossolini, A. (1964). [Action of cortisone on
neonatal thrombopenia provoked in the guinea pig with heterologous immune serum]. Atti della
Accademia dei fisiocritici in Siena Sezione medico-fisica 13, 463-470.
Chong, B.H., and Ho, S.J. (2005). Autoimmune thrombocytopenia. Journal of thrombosis and
haemostasis : JTH 3, 1763-1772.
Chow, A., Toomre, D., Garrett, W., and Mellman, I. (2002). Dendritic cell maturation triggers
retrograde MHC class II transport from lysosomes to the plasma membrane. Nature 418, 988-
994.
Chow, L., Aslam, R., Speck, E.R., Kim, M., Cridland, N., Webster, M.L., Chen, P., Sahib, K.,
Ni, H., Lazarus, A.H., et al. (2010). A murine model of severe immune thrombocytopenia is
164
induced by antibody- and CD8+ T cell-mediated responses that are differentially sensitive to
therapy. Blood 115, 1247-1253.
Cines, D.B., Bussel, J.B., Liebman, H.A., and Luning Prak, E.T. (2009). The ITP syndrome:
pathogenic and clinical diversity. Blood 113, 6511-6521.
Cines, D.B., Cuker, A., and Semple, J.W. (2014). Pathogenesis of immune thrombocytopenia.
Presse medicale 43, e49-59.
Cines, D.B., and Schreiber, A.D. (1979). Immune thrombocytopenia. Use of a Coombs
antiglobulin test to detect IgG and C3 on platelets. The New England journal of medicine 300,
106-111.
Clancy, R. (1972). Cellular immunity to autologous platelets and serum-blocking factors in
idiopathic thrombocytopenic purpura. Lancet 1, 6-9.
Clark, S.R., Ma, A.C., Tavener, S.A., McDonald, B., Goodarzi, Z., Kelly, M.M., Patel, K.D.,
Chakrabarti, S., McAvoy, E., Sinclair, G.D., et al. (2007). Platelet TLR4 activates neutrophil
extracellular traps to ensnare bacteria in septic blood. Nature medicine 13, 463-469.
Clarkson, S.B., Bussel, J.B., Kimberly, R.P., Valinsky, J.E., Nachman, R.L., and Unkeless, J.C.
(1986). Treatment of refractory immune thrombocytopenic purpura with an anti-Fc gamma-
receptor antibody. The New England journal of medicine 314, 1236-1239.
Clemetson, K.J., Clemetson, J.M., Proudfoot, A.E., Power, C.A., Baggiolini, M., and Wells,
T.N. (2000). Functional expression of CCR1, CCR3, CCR4, and CXCR4 chemokine receptors
on human platelets. Blood 96, 4046-4054.
Clough, L.E., Wang, C.J., Schmidt, E.M., Booth, G., Hou, T.Z., Ryan, G.A., and Walker, L.S.
(2008). Release from regulatory T cell-mediated suppression during the onset of tissue-specific
autoimmunity is associated with elevated IL-21. Journal of immunology 180, 5393-5401.
165
Cohen, S.B., Emery, P., Greenwald, M.W., Dougados, M., Furie, R.A., Genovese, M.C.,
Keystone, E.C., Loveless, J.E., Burmester, G.R., Cravets, M.W., et al. (2006). Rituximab for
rheumatoid arthritis refractory to anti-tumor necrosis factor therapy: Results of a multicenter,
randomized, double-blind, placebo-controlled, phase III trial evaluating primary efficacy and
safety at twenty-four weeks. Arthritis and rheumatism 54, 2793-2806.
Coopamah, M.D., Garvey, M.B., Freedman, J., and Semple, J.W. (2003). Cellular immune
mechanisms in autoimmune thrombocytopenic purpura: An update. Transfusion medicine
reviews 17, 69-80.
Cooper, N., Stasi, R., Cunningham-Rundles, S., Cesarman, E., McFarland, J.G., and Bussel, J.B.
(2012). Platelet-associated antibodies, cellular immunity and FCGR3a genotype influence the
response to rituximab in immune thrombocytopenia. British journal of haematology 158, 539-
547.
Coulson, B.S., Londrigan, S.L., and Lee, D.J. (1997). Rotavirus contains integrin ligand
sequences and a disintegrin-like domain that are implicated in virus entry into cells. Proceedings
of the National Academy of Sciences of the United States of America 94, 5389-5394.
Cross, A.H., Stark, J.L., Lauber, J., Ramsbottom, M.J., and Lyons, J.A. (2006). Rituximab
reduces B cells and T cells in cerebrospinal fluid of multiple sclerosis patients. Journal of
neuroimmunology 180, 63-70.
Crow, A.R., Amash, A., and Lazarus, A.H. (2015). CD44 antibody-mediated amelioration of
murine immune thrombocytopenia (ITP): mouse background determines the effect of
FcgammaRIIb genetic disruption. Transfusion 55, 1492-1500.
Crow, A.R., Song, S., Freedman, J., Helgason, C.D., Humphries, R.K., Siminovitch, K.A., and
Lazarus, A.H. (2003a). IVIg-mediated amelioration of murine ITP via FcgammaRIIB is
independent of SHIP1, SHP-1, and Btk activity. Blood 102, 558-560.
166
Crow, A.R., Song, S., Semple, J.W., Freedman, J., and Lazarus, A.H. (2001). IVIg inhibits
reticuloendothelial system function and ameliorates murine passive-immune thrombocytopenia
independent of anti-idiotype reactivity. British journal of haematology 115, 679-686.
Crow, A.R., Song, S., Semple, J.W., Freedman, J., and Lazarus, A.H. (2003b). IVIG induces
dose-dependent amelioration of ITP in rodent models. Blood 101, 1658-1659; author reply
1659.
Cui, W., and Kaech, S.M. (2010). Generation of effector CD8+ T cells and their conversion to
memory T cells. Immunological reviews 236, 151-166.
Dameshek, W., and Miller, E.B. (1946). The megakaryocytes in idiopathic thrombocytopenic
purpura, a form of hypersplenism. Blood 1, 27-50.
Dassin, E., Bourebia, J., Najean, Y., and Rosset, A.M. (1983). Partial purification of a
thrombocytopoiesis stimulating factor present in the serum of thrombocytopenic rats. Acta
haematologica 69, 249-253.
De La Fuente, V. (1949). Megakaryocytes in normal and in thrombocytopenic individuals; with
introduction of a new system of differential count for megakarocytes. Blood 4, 614-630.
de Larouziere, V., Brouland, J.P., Souni, F., Drouet, L., and Cramer, E. (1998). Inverse
immunostaining pattern for synthesized versus endocytosed alpha-granule proteins in human
bone marrow megakaryocytes. British journal of haematology 101, 618-625.
Delfraissy, J.F., Tchernia, G., Laurian, Y., Wallon, C., Galanaud, P., and Dormont, J. (1985).
Suppressor cell function after intravenous gammaglobulin treatment in adult chronic idiopathic
thrombocytopenic purpura. British journal of haematology 60, 315-322.
den Ottolander, G.J., Gratama, J.W., de Koning, J., and Brand, A. (1984). Long-term follow-up
study of 168 patients with immune thrombocytopenia. Implications for therapy. Scandinavian
journal of haematology 32, 101-110.
167
Denys, J. (1887). Éttudes sur la coagulation du sang dans un cas de purpura avec diminution
considérable des plaquettes. Cellule 3, 445-462.
Deppermann, C., Cherpokova, D., Nurden, P., Schulz, J.N., Thielmann, I., Kraft, P., Vogtle, T.,
Kleinschnitz, C., Dutting, S., Krohne, G., et al. (2013). Gray platelet syndrome and defective
thrombo-inflammation in Nbeal2-deficient mice. The Journal of clinical investigation 123,
3331-3342.
Deutsch, H.F., Petermann, M.L., and Williams, J.W. (1946). Biophysical studies of blood
plasma proteins; the pepsin digestion and recovery of human gamma-globulin. The Journal of
biological chemistry 164, 93-107.
Di Minno, G., Thiagarajan, P., Perussia, B., Martinez, J., Shapiro, S., Trinchieri, G., and
Murphy, S. (1983). Exposure of platelet fibrinogen-binding sites by collagen, arachidonic acid,
and ADP: inhibition by a monoclonal antibody to the glycoprotein IIb-IIIa complex. Blood 61,
140-148.
Diggs, L.W., and Hewlett, J.S. (1948). A study of the bone marrow from 36 patients with
idiopathic hemorrhagic, thrombopenic purpura. Blood 3, 1090-1104.
Dixon, R., Rosse, W., and Ebbert, L. (1975). Quantitative determination of antibody in
idiopathic thrombocytopenic purpura. Correlation of serum and platelet-bound antibody with
clinical response. The New England journal of medicine 292, 230-236.
Ebbe, S. (1976). Biology of megakaryocytes. Progress in hemostasis and thrombosis 3, 211-229.
Ebbe, S., Levin, J., Miller, K., Yee, T., Levin, F., and Phalen, E. (1987). Thrombocytopoietic
response to immunothrombocytopenia in nude mice. Blood 69, 192-198.
Eckly, A., Heijnen, H., Pertuy, F., Geerts, W., Proamer, F., Rinckel, J.Y., Leon, C., Lanza, F.,
and Gachet, C. (2014). Biogenesis of the demarcation membrane system (DMS) in
megakaryocytes. Blood 123, 921-930.
168
Eisenlohr, L.C., Huang, L., and Golovina, T.N. (2007). Rethinking peptide supply to MHC class
I molecules. Nature reviews Immunology 7, 403-410.
Elzey, B.D., Sprague, D.L., and Ratliff, T.L. (2005). The emerging role of platelets in adaptive
immunity. Cellular immunology 238, 1-9.
Engelhard, D., Waner, J.L., Kapoor, N., and Good, R.A. (1986). Effect of intravenous immune
globulin on natural killer cell activity: possible association with autoimmune neutropenia and
idiopathic thrombocytopenia. The Journal of pediatrics 108, 77-81.
Etzioni, A., and Pollack, S. (1989). High dose intravenous gammaglobulins in autoimmune
disorders: mode of action and therapeutic uses. Autoimmunity 3, 307-315.
Fabris, F., Scandellari, R., Ruzzon, E., Randi, M.L., Luzzatto, G., and Girolami, A. (2004).
Platelet-associated autoantibodies as detected by a solid-phase modified antigen capture ELISA
test (MACE) are a useful prognostic factor in idiopathic thrombocytopenic purpura. Blood 103,
4562-4564.
Faustman, D.L., and Davis, M. (2009). The primacy of CD8 T lymphocytes in type 1 diabetes
and implications for therapies. Journal of molecular medicine 87, 1173-1178.
Fehr, J., Hofmann, V., and Kappeler, U. (1982). Transient reversal of thrombocytopenia in
idiopathic thrombocytopenic purpura by high-dose intravenous gamma globulin. The New
England journal of medicine 306, 1254-1258.
Feng, X., Scheinberg, P., Samsel, L., Rios, O., Chen, J., McCoy, J.P., Jr., Ghanima, W., Bussel,
J.B., and Young, N.S. (2012). Decreased plasma cytokines are associated with low platelet
counts in aplastic anemia and immune thrombocytopenic purpura. Journal of thrombosis and
haemostasis : JTH 10, 1616-1623.
Fillatreau, S., Sweenie, C.H., McGeachy, M.J., Gray, D., and Anderton, S.M. (2002). B cells
regulate autoimmunity by provision of IL-10. Nature immunology 3, 944-950.
169
Fisher, G.H., Rosenberg, F.J., Straus, S.E., Dale, J.K., Middleton, L.A., Lin, A.Y., Strober, W.,
Lenardo, M.J., and Puck, J.M. (1995). Dominant interfering Fas gene mutations impair
apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81, 935-946.
Flaujac, C., Boukour, S., and Cramer-Borde, E. (2010). Platelets and viruses: an ambivalent
relationship. Cellular and molecular life sciences : CMLS 67, 545-556.
Fogarty, P.F., Rick, M.E., Zeng, W., Risitano, A.M., Dunbar, C.E., and Bussel, J.B. (2003). T
cell receptor VB repertoire diversity in patients with immune thrombocytopenia following
splenectomy. Clinical and experimental immunology 133, 461-466.
Fooksman, D.R. (2014). Organizing MHC Class II Presentation. Frontiers in immunology 5,
158.
Frank, E. (1915). Die essentielle thrombopenie. Berl Klin Wochenschr 52, 454-458.
Freedman, J. (2003). ITP: an overview of the conference and future directions with an
abbreviated ITP history. Journal of pediatric hematology/oncology 25 Suppl 1, S77-84.
Freedman, J., and Blanchette, M. (1998). Idiopathic thrombocytopenic purpura (ITP): a
historical odyssey. Acta paediatrica 424, 3-6.
Frelinger, A.L., 3rd, Grace, R.F., Gerrits, A.J., Berny-Lang, M.A., Brown, T., Carmichael, S.L.,
Neufeld, E.J., and Michelson, A.D. (2015). Platelet function tests, independent of platelet count,
are associated with bleeding severity in ITP. Blood 126, 873-879.
Fridman, W.H. (1991). Fc receptors and immunoglobulin binding factors. FASEB journal :
official publication of the Federation of American Societies for Experimental Biology 5, 2684-
2690.
Fukaya, T., Murakami, R., Takagi, H., Sato, K., Sato, Y., Otsuka, H., Ohno, M., Hijikata, A.,
Ohara, O., Hikida, M., et al. (2012). Conditional ablation of CD205+ conventional dendritic
170
cells impacts the regulation of T-cell immunity and homeostasis in vivo. Proceedings of the
National Academy of Sciences of the United States of America 109, 11288-11293.
Furie, B., and Furie, B.C. (2008). Mechanisms of thrombus formation. The New England
journal of medicine 359, 938-949.
Garcia-Suarez, J., Prieto, A., Reyes, E., Manzano, L., Merino, J.L., and Alvarez-Mon, M.
(1993). Severe chronic autoimmune thrombocytopenic purpura is associated with an expansion
of CD56+ CD3- natural killer cells subset. Blood 82, 1538-1545.
Ghio, M., Contini, P., Mazzei, C., Brenci, S., Barberis, G., Filaci, G., Indiveri, F., and Puppo, F.
(1999). Soluble HLA class I, HLA class II, and Fas ligand in blood components: a possible key
to explain the immunomodulatory effects of allogeneic blood transfusions. Blood 93, 1770-
1777.
Gilliet, M., Cao, W., and Liu, Y.J. (2008). Plasmacytoid dendritic cells: sensing nucleic acids in
viral infection and autoimmune diseases. Nature reviews Immunology 8, 594-606.
Gimmi, C.D., Freeman, G.J., Gribben, J.G., Sugita, K., Freedman, A.S., Morimoto, C., and
Nadler, L.M. (1991). B-cell surface antigen B7 provides a costimulatory signal that induces T
cells to proliferate and secrete interleukin 2. Proceedings of the National Academy of Sciences
of the United States of America 88, 6575-6579.
Godfrey, V.L., Wilkinson, J.E., Rinchik, E.M., and Russell, L.B. (1991). Fatal lymphoreticular
disease in the scurfy (sf) mouse requires T cells that mature in a sf thymic environment:
potential model for thymic education. Proceedings of the National Academy of Sciences of the
United States of America 88, 5528-5532.
Golay, J., Zaffaroni, L., Vaccari, T., Lazzari, M., Borleri, G.M., Bernasconi, S., Tedesco, F.,
Rambaldi, A., and Introna, M. (2000). Biologic response of B lymphoma cells to anti-CD20
monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell
lysis. Blood 95, 3900-3908.
171
Goodnow, C.C., Crosbie, J., Jorgensen, H., Brink, R.A., and Basten, A. (1989). Induction of
self-tolerance in mature peripheral B lymphocytes. Nature 342, 385-391.
Goodnow, C.C., and Ohashi, P.S. (2013). Chapter 32 Immunologic tolerance. In Fundamental
immunology, W.E. Paul, ed. (Philadelphia: Wolters Kluwer Health/Lippincott Williams &
Wilkins), pp. xviii, 1283 p.
Gouttefangeas, C., Diehl, M., Keilholz, W., Hornlein, R.F., Stevanovic, S., and Rammensee,
H.G. (2000). Thrombocyte HLA molecules retain nonrenewable endogenous peptides of
megakaryocyte lineage and do not stimulate direct allocytotoxicity in vitro. Blood 95, 3168-
3175.
Greer, J.P. (2014). Wintrobe's clinical hematology (Philadelphia: Wolters Kluwer, Lippincott
Williams & Wilkins Health), pp. xxvii, 2,278 p.
Grewal, P.K., Uchiyama, S., Ditto, D., Varki, N., Le, D.T., Nizet, V., and Marth, J.D. (2008).
The Ashwell receptor mitigates the lethal coagulopathy of sepsis. Nature medicine 14, 648-655.
Gudbrandsdottir, S., Birgens, H.S., Frederiksen, H., Jensen, B.A., Jensen, M.K., Kjeldsen, L.,
Klausen, T.W., Larsen, H., Mourits-Andersen, H.T., Nielsen, C.H., et al. (2013). Rituximab and
dexamethasone vs dexamethasone monotherapy in newly diagnosed patients with primary
immune thrombocytopenia. Blood 121, 1976-1981.
Guo, L., Kapur, R., Aslam, R., Speck, E.R., Zufferey, A., Zhao, Y., Kim, M., Lazarus, A.H., Ni,
H., and Semple, J.W. (2016). CD20+ B-cell depletion therapy suppresses murine CD8+ T-cell-
mediated immune thrombocytopenia. Blood 127, 735-738.
Guo, L., Yang, L., Speck, E.R., Aslam, R., Kim, M., McKenzie, C.G., Lazarus, A.H., Ni, H.,
Hou, M., Freedman, J., et al. (2014). Allogeneic platelet transfusions prevent murine T-cell-
mediated immune thrombocytopenia. Blood 123, 422-427.
172
Guo, Y.L., Liu, D.Q., Bian, Z., Zhang, C.Y., and Zen, K. (2009). Down-regulation of platelet
surface CD47 expression in Escherichia coli O157:H7 infection-induced thrombocytopenia.
PloS one 4, e7131.
Gurcan, H.M., Keskin, D.B., Stern, J.N., Nitzberg, M.A., Shekhani, H., and Ahmed, A.R.
(2009). A review of the current use of rituximab in autoimmune diseases. International
immunopharmacology 9, 10-25.
Gurney, A.L., Carver-Moore, K., de Sauvage, F.J., and Moore, M.W. (1994).
Thrombocytopenia in c-mpl-deficient mice. Science 265, 1445-1447.
Haas, W., Pereira, P., and Tonegawa, S. (1993). Gamma/delta cells. Annual review of
immunology 11, 637-685.
Hagihara, M., Higuchi, A., Tamura, N., Ueda, Y., Hirabayashi, K., Ikeda, Y., Kato, S.,
Sakamoto, S., Hotta, T., Handa, S., et al. (2004). Platelets, after exposure to a high shear stress,
induce IL-10-producing, mature dendritic cells in vitro. Journal of immunology 172, 5297-5303.
Hamann, D., Baars, P.A., Rep, M.H., Hooibrink, B., Kerkhof-Garde, S.R., Klein, M.R., and van
Lier, R.A. (1997). Phenotypic and functional separation of memory and effector human CD8+ T
cells. The Journal of experimental medicine 186, 1407-1418.
Han, T., Stutzman, L., Cohen, E., and Kim, U. (1966). Effect of platelet transfusion on
hemorrhage in patients with acute leukemia. An autopsy study. Cancer 19, 1937-1942.
Hanabuchi, S., Koyanagi, M., Kawasaki, A., Shinohara, N., Matsuzawa, A., Nishimura, Y.,
Kobayashi, Y., Yonehara, S., Yagita, H., and Okumura, K. (1994). Fas and its ligand in a
general mechanism of T-cell-mediated cytotoxicity. Proceedings of the National Academy of
Sciences of the United States of America 91, 4930-4934.
173
Handagama, P.J., George, J.N., Shuman, M.A., McEver, R.P., and Bainton, D.F. (1987).
Incorporation of a circulating protein into megakaryocyte and platelet granules. Proceedings of
the National Academy of Sciences of the United States of America 84, 861-865.
Handin, R.I., and Stossel, T.P. (1974). Phagocytosis of antibody-coated platelets by human
granulocytes. The New England journal of medicine 290, 989-993.
Hansen, T.H., and Roche, P.A. (2013). Chapter 22 Antigen Processing and Presentation. In
Fundamental immunology, W.E. Paul, ed. (Philadelphia: Wolters Kluwer Health/Lippincott
Williams & Wilkins), pp. xviii, 1283 p.
Hardy, R.R. (2003). B-cell commitment: deciding on the players. Current opinion in
immunology 15, 158-165.
Hardy, R.R., and Hayakawa, K. (2001). B cell development pathways. Annual review of
immunology 19, 595-621.
Harker, L.A. (1970). Thrombokinetics in idiopathic thrombocytopenic purpura. British journal
of haematology 19, 95-104.
Harker, L.A., and Finch, C.A. (1969). Thrombokinetics in man. The Journal of clinical
investigation 48, 963-974.
Harrington, W.J., Minnich, V., Hollingsworth, J.W., and Moore, C.V. (1951). Demonstration of
a thrombocytopenic factor in the blood of patients with thrombocytopenic purpura. The Journal
of laboratory and clinical medicine 38, 1-10.
Hartwig, J.H., and Italiano, J.E., Jr. (2006). Cytoskeletal mechanisms for platelet production.
Blood cells, molecules & diseases 36, 99-103.
Hayashi, T., and Hirayama, F. (2015). Advances in alloimmune thrombocytopenia: perspectives
on current concepts of human platelet antigens, antibody detection strategies, and genotyping.
Blood transfusion = Trasfusione del sangue 13, 380-390.
174
Hedman, A., Henter, J.I., Hedlund, I., and Elinder, G. (1997). Prevalence and treatment of
chronic idiopathic thrombocytopenic purpura of childhood in Sweden. Acta Paediatr 86, 226-
227.
Heino, M., Peterson, P., Kudoh, J., Nagamine, K., Lagerstedt, A., Ovod, V., Ranki, A., Rantala,
I., Nieminen, M., Tuukkanen, J., et al. (1999). Autoimmune regulator is expressed in the cells
regulating immune tolerance in thymus medulla. Biochemical and biophysical research
communications 257, 821-825.
Henn, V., Slupsky, J.R., Grafe, M., Anagnostopoulos, I., Forster, R., Muller-Berghaus, G., and
Kroczek, R.A. (1998). CD40 ligand on activated platelets triggers an inflammatory reaction of
endothelial cells. Nature 391, 591-594.
Heyns Adu, P., Badenhorst, P.N., Lotter, M.G., Pieters, H., Wessels, P., and Kotze, H.F. (1986).
Platelet turnover and kinetics in immune thrombocytopenic purpura: results with autologous
111In-labeled platelets and homologous 51Cr-labeled platelets differ. Blood 67, 86-92.
Hirsch, E.O., and Dameshek, W. (1951). Idiopathic thrombocytopenia; review of eighty-nine
cases with particular reference to the differentiation and treatment of acute (self-limited and
chronic types). AMA archives of internal medicine 88, 701-728.
Hirsch, E.O., and Gardner, F.H. (1952). The transfusion of human blood platelets with a note on
the transfusion of granulocytes. The Journal of laboratory and clinical medicine 39, 556-569.
Houwerzijl, E.J., Blom, N.R., van der Want, J.J., Esselink, M.T., Koornstra, J.J., Smit, J.W.,
Louwes, H., Vellenga, E., and de Wolf, J.T. (2004). Ultrastructural study shows morphologic
features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic
thrombocytopenic purpura. Blood 103, 500-506.
Hu, C.Y., Rodriguez-Pinto, D., Du, W., Ahuja, A., Henegariu, O., Wong, F.S., Shlomchik, M.J.,
and Wen, L. (2007). Treatment with CD20-specific antibody prevents and reverses autoimmune
diabetes in mice. The Journal of clinical investigation 117, 3857-3867.
175
Hu, Y., Li, H., Zhang, L., Shan, B., Xu, X., Li, H., Liu, X., Xu, S., Yu, S., Ma, D., et al. (2012).
Elevated profiles of Th22 cells and correlations with Th17 cells in patients with immune
thrombocytopenia. Human immunology 73, 629-635.
Hu, Y., Ma, D.X., Shan, N.N., Zhu, Y.Y., Liu, X.G., Zhang, L., Yu, S., Ji, C.Y., and Hou, M.
(2011). Increased number of Tc17 and correlation with Th17 cells in patients with immune
thrombocytopenia. PloS one 6, e26522.
Huppa, J.B., and Davis, M.M. (2013). The interdisciplinary science of T-cell recognition.
Advances in immunology 119, 1-50.
Hymes, K., Schur, P.H., and Karpatkin, S. (1980). Heavy-chain subclass of round antiplatelet
IgG in autoimmune thrombocytopenic purpura. Blood 56, 84-87.
Imbach, P., Barandun, S., d'Apuzzo, V., Baumgartner, C., Hirt, A., Morell, A., Rossi, E.,
Schoni, M., Vest, M., and Wagner, H.P. (1981). High-dose intravenous gammaglobulin for
idiopathic thrombocytopenic purpura in childhood. Lancet 1, 1228-1231.
Imbach, P., Kuhne, T., Muller, D., Berchtold, W., Zimmerman, S., Elalfy, M., and Buchanan,
G.R. (2006). Childhood ITP: 12 months follow-up data from the prospective registry I of the
Intercontinental Childhood ITP Study Group (ICIS). Pediatric blood & cancer 46, 351-356.
Imbach, P., Wagner, H.P., Berchtold, W., Gaedicke, G., Hirt, A., Joller, P., Mueller-Eckhardt,
C., Muller, B., Rossi, E., and Barandun, S. (1985). Intravenous immunoglobulin versus oral
corticosteroids in acute immune thrombocytopenic purpura in childhood. Lancet 2, 464-468.
Isailovic, N., Daigo, K., Mantovani, A., and Selmi, C. (2015). Interleukin-17 and innate
immunity in infections and chronic inflammation. Journal of autoimmunity 60, 1-11.
Italiano, J.E., Jr., Lecine, P., Shivdasani, R.A., and Hartwig, J.H. (1999). Blood platelets are
assembled principally at the ends of proplatelet processes produced by differentiated
megakaryocytes. The Journal of cell biology 147, 1299-1312.
176
Italiano Jr., J.E., and Hartwig, J.H. (2013). Chapter 2 Megakaryocyte development and platelet
formation. In Platelets, A.D. Michelson, ed. (London ; Waltham, MA: Academic Press), pp.
xliv, 1353 p.
Jacobs, P., Wood, L., and Dent, D.M. (1986). Results of treatment in immune
thrombocytopenia. The Quarterly journal of medicine 58, 153-165.
Jansen, P.H., Renier, W.O., de Vaan, G., Reekers, P., Vingerhoets, D.M., and Gabreels, F.J.
(1987). Effect of thymectomy on myasthenia gravis and autoimmune thrombocytopenic purpura
in a 13-year-old girl. European journal of pediatrics 146, 587-589.
Jenkins, M.K. (2013). Chapter 14 Peripheral T lymphocyte responses and function. In
Fundamental immunology, W.E. Paul, ed. (Philadelphia: Wolters Kluwer Health/Lippincott
Williams & Wilkins), pp. xviii, 1283 p.
Ji, L., Zhan, Y., Hua, F., Li, F., Zou, S., Wang, W., Song, D., Min, Z., Chen, H., and Cheng, Y.
(2012). The ratio of Treg/Th17 cells correlates with the disease activity of primary immune
thrombocytopenia. PloS one 7, e50909.
Johnson, P., Gagnon, J., Barclay, A.N., and Williams, A.F. (1985). Purification, chain
separation and sequence of the MRC OX-8 antigen, a marker of rat cytotoxic T lymphocytes.
The EMBO journal 4, 2539-2545.
Jolicoeur, C., Hanahan, D., and Smith, K.M. (1994). T-cell tolerance toward a transgenic beta-
cell antigen and transcription of endogenous pancreatic genes in thymus. Proceedings of the
National Academy of Sciences of the United States of America 91, 6707-6711.
Jones, H., and Tocantins, L. (1933). The history of purpura hemorrhagica. Ann Med Hist 5, 349-
359.
Josefsson, E.C., James, C., Henley, K.J., Debrincat, M.A., Rogers, K.L., Dowling, M.R., White,
M.J., Kruse, E.A., Lane, R.M., Ellis, S., et al. (2011). Megakaryocytes possess a functional
177
intrinsic apoptosis pathway that must be restrained to survive and produce platelets. The Journal
of experimental medicine 208, 2017-2031.
Joseph, M., Auriault, C., Capron, A., Vorng, H., and Viens, P. (1983). A new function for
platelets: IgE-dependent killing of schistosomes. Nature 303, 810-812.
Junt, T., Schulze, H., Chen, Z., Massberg, S., Goerge, T., Krueger, A., Wagner, D.D., Graf, T.,
Italiano, J.E., Jr., Shivdasani, R.A., et al. (2007). Dynamic visualization of thrombopoiesis
within bone marrow. Science 317, 1767-1770.
Kaech, S.M., and Ahmed, R. (2001). Memory CD8+ T cell differentiation: initial antigen
encounter triggers a developmental program in naive cells. Nature immunology 2, 415-422.
Kahr, W.H., Hinckley, J., Li, L., Schwertz, H., Christensen, H., Rowley, J.W., Pluthero, F.G.,
Urban, D., Fabbro, S., Nixon, B., et al. (2011). Mutations in NBEAL2, encoding a BEACH
protein, cause gray platelet syndrome. Nature genetics 43, 738-740.
Kahr, W.H., Lo, R.W., Li, L., Pluthero, F.G., Christensen, H., Ni, R., Vaezzadeh, N., Hawkins,
C.E., Weyrich, A.S., Di Paola, J., et al. (2013). Abnormal megakaryocyte development and
platelet function in Nbeal2(-/-) mice. Blood 122, 3349-3358.
Kalmanson, G.M., and Bronfenbrenner, J. (1942). The Reversal of Pneumococcus Quellung by
Digestion of the Antibody with Papain. Science 96, 21-22.
Kalmaz, G.D., and McDonald, T.P. (1981). Effects of antiplatelet serum and thrombopoietin on
the percentage of small acetylcholinesterase-positive cells in bone marrow of mice.
Experimental hematology 9, 1002-1010.
Kambayashi, T., and Laufer, T.M. (2014). Atypical MHC class II-expressing antigen-presenting
cells: can anything replace a dendritic cell? Nature reviews Immunology 14, 719-730.
Kamphuis, M.M., Paridaans, N., Porcelijn, L., De Haas, M., Van Der Schoot, C.E., Brand, A.,
Bonsel, G.J., and Oepkes, D. (2010). Screening in pregnancy for fetal or neonatal alloimmune
178
thrombocytopenia: systematic review. BJOG : an international journal of obstetrics and
gynaecology 117, 1335-1343.
Kamphuis, M.M., Paridaans, N.P., Porcelijn, L., Lopriore, E., and Oepkes, D. (2014). Incidence
and consequences of neonatal alloimmune thrombocytopenia: a systematic review. Pediatrics
133, 715-721.
Kaneko, Y., Nimmerjahn, F., and Ravetch, J.V. (2006). Anti-inflammatory activity of
immunoglobulin G resulting from Fc sialylation. Science 313, 670-673.
Kao, K.J. (1987). Plasma and platelet HLA in normal individuals: quantitation by competitive
enzyme-linked immunoassay. Blood 70, 282-286.
Kao, K.J. (1988). Selective elution of HLA antigens and beta 2-microglobulin from human
platelets by chloroquine diphosphate. Transfusion 28, 14-17.
Kao, K.J., Cook, D.J., and Scornik, J.C. (1986). Quantitative analysis of platelet surface HLA
by W6/32 anti-HLA monoclonal antibody. Blood 68, 627-632.
Kapur, R., Heitink-Polle, K.M., Porcelijn, L., Bentlage, A.E., Bruin, M.C., Visser, R., Roos, D.,
Schasfoort, R.B., de Haas, M., van der Schoot, C.E., et al. (2015a). C-reactive protein enhances
IgG-mediated phagocyte responses and thrombocytopenia. Blood 125, 1793-1802.
Kapur, R., Zufferey, A., Boilard, E., and Semple, J.W. (2015b). Nouvelle Cuisine: Platelets
Served with Inflammation. Journal of immunology 194, 5579-5587.
Karpatkin, S., Fotino, M., Gibofsky, A., and Winchester, R.J. (1979). Association of HLA-
DRw2 with autoimmune thrombocytopenic purpura. The Journal of clinical investigation 63,
1085-1088.
Karpatkin, S., Strick, N., Karpatkin, M.B., and Siskind, G.W. (1972). Cumulative experience in
the detection of antiplatelet antibody in 234 patients with idiopathic thrombocytopenic purpura,
179
systemic lupus erythematosus and other clinical disorders. The American journal of medicine
52, 776-785.
Kay, H.D., Bonnard, G.D., West, W.H., and Herberman, R.B. (1977). A functional comparison
of human Fc-receptor-bearing lymphocytes active in natural cytotoxicity and antibody-
dependent cellular cytotoxicity. Journal of immunology 118, 2058-2066.
Kaznelson, P. (1916). Verschwinden der Wimorrhagischen diathese bei einem fable von
``essentieller thrombopenie'' (Frank) nach Milzexstirpation: Splenogene thrombolytische
purpura. Wiener klinische Wochenschrift 29, 1451-1454.
Kekomaki, R., Kauppinen, H.L., Penttinen, K., and Myllyla, G. (1977). Interactions of immune
complexes and platelets in rabbits immunized with hapten-carrier conjugates. Acta pathologica
et microbiologica Scandinavica Section C, Immunology 85, 207-214.
Kelso, A., Costelloe, E.O., Johnson, B.J., Groves, P., Buttigieg, K., and Fitzpatrick, D.R.
(2002). The genes for perforin, granzymes A-C and IFN-gamma are differentially expressed in
single CD8(+) T cells during primary activation. International immunology 14, 605-613.
Kernoff, L.M., Blake, K.C., and Shackleton, D. (1980). Influence of the amount of platelet-
bound IgG on platelet survival and site of sequestration in autoimmune thrombocytopenia.
Blood 55, 730-733.
Kerr, J.F., Wyllie, A.H., and Currie, A.R. (1972). Apoptosis: a basic biological phenomenon
with wide-ranging implications in tissue kinetics. British journal of cancer 26, 239-257.
Khan, W.I., Motomura, Y., Blennerhassett, P.A., Kanbayashi, H., Varghese, A.K., El-Sharkawy,
R.T., Gauldie, J., and Collins, S.M. (2005). Disruption of CD40-CD40 ligand pathway inhibits
the development of intestinal muscle hypercontractility and protective immunity in nematode
infection. American journal of physiology Gastrointestinal and liver physiology 288, G15-22.
180
Khellaf, M., Charles-Nelson, A., Fain, O., Terriou, L., Viallard, J.F., Cheze, S., Graveleau, J.,
Slama, B., Audia, S., Ebbo, M., et al. (2014). Safety and efficacy of rituximab in adult immune
thrombocytopenia: results from a prospective registry including 248 patients. Blood 124, 3228-
3236.
Kickler, T.S., Herman, J.H., Furihata, K., Kunicki, T.J., and Aster, R.H. (1988). Identification of
Bakb, a new platelet-specific antigen associated with posttransfusion purpura. Blood 71, 894-
898.
Kiefel, V., Freitag, E., Kroll, H., Santoso, S., and Mueller-Eckhardt, C. (1996). Platelet
autoantibodies (IgG, IgM, IgA) against glycoproteins IIb/IIIa and Ib/IX in patients with
thrombocytopenia. Annals of hematology 72, 280-285.
Kim, J.I., Rothstein, D.M., and Markmann, J.F. (2015). Role of B cells in tolerance induction.
Current opinion in organ transplantation 20, 369-375.
King, I.L., and Segal, B.M. (2005). Cutting edge: IL-12 induces CD4+CD25- T cell activation
in the presence of T regulatory cells. Journal of immunology 175, 641-645.
Kirkham, P.M., and Schroeder, H.W., Jr. (1994). Antibody structure and the evolution of
immunoglobulin V gene segments. Seminars in immunology 6, 347-360.
Kobayashi, S., Teramura, M., Sugawara, I., Oshimi, K., and Mizoguchi, H. (1993). Interleukin-
11 acts as an autocrine growth factor for human megakaryoblastic cell lines. Blood 81, 889-893.
Koylu, A., Pamuk, G.E., Uyanik, M.S., Demir, M., and Pamuk, O.N. (2015). Immune
thrombocytopenia: epidemiological and clinical features of 216 patients in northwestern Turkey.
Annals of hematology 94, 459-466.
Krauss, E. (1883). Überpurpura. (Heidelberg).
Kuhne, T., Buchanan, G.R., Zimmerman, S., Michaels, L.A., Kohan, R., Berchtold, W., Imbach,
P., Intercontinental Childhood, I.T.P.S.G., and Intercontinental Childhood, I.T.P.S.G. (2003). A
181
prospective comparative study of 2540 infants and children with newly diagnosed idiopathic
thrombocytopenic purpura (ITP) from the Intercontinental Childhood ITP Study Group. The
Journal of pediatrics 143, 605-608.
Kuhne, T., Imbach, P., Bolton-Maggs, P.H., Berchtold, W., Blanchette, V., Buchanan, G.R., and
Intercontinental Childhood, I.T.P.S.G. (2001). Newly diagnosed idiopathic thrombocytopenic
purpura in childhood: an observational study. Lancet 358, 2122-2125.
Kunert, S., Meyer, I., Fleischhauer, S., Wannack, M., Fiedler, J., Shivdasani, R.A., and Schulze,
H. (2009). The microtubule modulator RanBP10 plays a critical role in regulation of platelet
discoid shape and degranulation. Blood 114, 5532-5540.
Kuwana, M., Kaburaki, J., Kitasato, H., Kato, M., Kawai, S., Kawakami, Y., and Ikeda, Y.
(2001). Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in
patients with immune thrombocytopenic purpura. Blood 98, 130-139.
Kuwana, M., Kaburaki, J., Pandey, J.P., Murata, M., Kawakami, Y., Inoko, H., and Ikeda, Y.
(2000). HLA class II alleles in Japanese patients with immune thrombocytopenic purpura.
Associations with anti-platelet glycoprotein autoantibodies and responses to splenectomy.
Tissue antigens 56, 337-343.
Kuwana, M., Kawakami, Y., and Ikeda, Y. (2003). Suppression of autoreactive T-cell response
to glycoprotein IIb/IIIa by blockade of CD40/CD154 interaction: implications for treatment of
immune thrombocytopenic purpura. Blood 101, 621-623.
Laky, K., and Fowlkes, B.J. (2007). Presenilins regulate alphabeta T cell development by
modulating TCR signaling. The Journal of experimental medicine 204, 2115-2129.
Lamb, J.R., Skidmore, B.J., Green, N., Chiller, J.M., and Feldmann, M. (1983). Induction of
tolerance in influenza virus-immune T lymphocyte clones with synthetic peptides of influenza
hemagglutinin. The Journal of experimental medicine 157, 1434-1447.
182
Langer, H.F., Daub, K., Braun, G., Schonberger, T., May, A.E., Schaller, M., Stein, G.M.,
Stellos, K., Bueltmann, A., Siegel-Axel, D., et al. (2007). Platelets recruit human dendritic cells
via Mac-1/JAM-C interaction and modulate dendritic cell function in vitro. Arteriosclerosis,
thrombosis, and vascular biology 27, 1463-1470.
Larson, M.K., and Watson, S.P. (2006). Regulation of proplatelet formation and platelet release
by integrin alpha IIb beta3. Blood 108, 1509-1514.
Laurent, C., Muller, S., Do, C., Al-Saati, T., Allart, S., Larocca, L.M., Hohaus, S., Duchez, S.,
Quillet-Mary, A., Laurent, G., et al. (2011). Distribution, function, and prognostic value of
cytotoxic T lymphocytes in follicular lymphoma: a 3-D tissue-imaging study. Blood 118, 5371-
5379.
Lazarus, A.H., and Crow, A.R. (2003). Mechanism of action of IVIG and anti-D in ITP.
Transfusion and apheresis science : official journal of the World Apheresis Association : official
journal of the European Society for Haemapheresis 28, 249-255.
Leandro, M.J., Edwards, J.C., Cambridge, G., Ehrenstein, M.R., and Isenberg, D.A. (2002). An
open study of B lymphocyte depletion in systemic lupus erythematosus. Arthritis and
rheumatism 46, 2673-2677.
Lee-Chang, C., Bodogai, M., Moritoh, K., Olkhanud, P.B., Chan, A.C., Croft, M., Mattison,
J.A., Holst, P.J., Gress, R.E., Ferrucci, L., et al. (2014). Accumulation of 4-1BBL+ B cells in
the elderly induces the generation of granzyme-B+ CD8+ T cells with potential antitumor
activity. Blood 124, 1450-1459.
Lefebvre, M.L., Krause, S.W., Salcedo, M., and Nardin, A. (2006). Ex vivo-activated human
macrophages kill chronic lymphocytic leukemia cells in the presence of rituximab: mechanism
of antibody-dependent cellular cytotoxicity and impact of human serum. Journal of
immunotherapy 29, 388-397.
183
Leontyev, D., Katsman, Y., and Branch, D.R. (2012). Mouse background and IVIG dosage are
critical in establishing the role of inhibitory Fcgamma receptor for the amelioration of
experimental ITP. Blood 119, 5261-5264.
Leontyev, D., Neschadim, A., and Branch, D.R. (2014). Cytokine profiles in mouse models of
experimental immune thrombocytopenia reveal a lack of inflammation and differences in
response to intravenous immunoglobulin depending on the mouse strain. Transfusion 54, 2871-
2879.
Leung, A.Y., Hawkins, B.R., Chim, C.S., Kwong, Y.Y., and Liang, R.H. (2001). Genetic
analysis of HLA-typing in Chinese patients with idiopathic thrombocytopenic purpura.
Haematologica 86, 221-222.
Lev, P.R., Grodzielski, M., Goette, N.P., Glembotsky, A.C., Espasandin, Y.R., Pierdominici,
M.S., Contrufo, G., Montero, V.S., Ferrari, L., Molinas, F.C., et al. (2014). Impaired proplatelet
formation in immune thrombocytopenia: a novel mechanism contributing to decreased platelet
count. British journal of haematology 165, 854-864.
Leytin, V., Mykhaylov, S., Starkey, A.F., Allen, D.J., Lau, H., Ni, H., Semple, J.W., Lazarus,
A.H., and Freedman, J. (2006). Intravenous immunoglobulin inhibits anti-glycoprotein IIb-
induced platelet apoptosis in a murine model of immune thrombocytopenia. British journal of
haematology 133, 78-82.
Li, C., Li, J., Li, Y., Lang, S., Yougbare, I., Zhu, G., Chen, P., and Ni, H. (2012a). Crosstalk
between Platelets and the Immune System: Old Systems with New Discoveries. Advances in
hematology 2012, 384685.
Li, F., Ji, L., Wang, W., Hua, F., Zhan, Y., Zou, S., Yuan, L., Ke, Y., Min, Z., Song, D., et al.
(2015a). Insufficient secretion of IL-10 by Tregs compromised its control on over-activated
CD4+ T effector cells in newly diagnosed adult immune thrombocytopenia patients.
Immunologic research 61, 269-280.
184
Li, H., Ge, J., Zhao, H., Du, W., Xu, J., Sui, T., Ma, L., Zhou, Z., Qi, A., and Yang, R. (2011).
Association of cytotoxic T-lymphocyte antigen 4 gene polymorphisms with idiopathic
thrombocytopenic purpura in a Chinese population. Platelets 22, 39-44.
Li, J., Callum, J.L., Lin, Y., Zhou, Y., Zhu, G., and Ni, H. (2014). Severe platelet desialylation
in a patient with glycoprotein Ib/IX antibody-mediated immune thrombocytopenia and fatal
pulmonary hemorrhage. Haematologica 99, e61-63.
Li, J., van der Wal, D.E., Zhu, G., Xu, M., Yougbare, I., Ma, L., Vadasz, B., Carrim, N.,
Grozovsky, R., Ruan, M., et al. (2015b). Desialylation is a mechanism of Fc-independent
platelet clearance and a therapeutic target in immune thrombocytopenia. Nature
communications 6, 7737.
Li, Q., Yang, M., Xia, R., Xia, L., and Zhang, L. (2015c). Elevated expression of IL-12 and IL-
23 in patients with primary immune thrombocytopenia. Platelets 26, 453-458.
Li, S., Wang, L., Zhao, C., Li, L., Peng, J., and Hou, M. (2007). CD8+ T cells suppress
autologous megakaryocyte apoptosis in idiopathic thrombocytopenic purpura. British journal of
haematology 139, 605-611.
Li, X., Zhong, H., Bao, W., Boulad, N., Evangelista, J., Haider, M.A., Bussel, J., and
Yazdanbakhsh, K. (2012b). Defective regulatory B-cell compartment in patients with immune
thrombocytopenia. Blood 120, 3318-3325.
Li, Z., Delaney, M.K., O'Brien, K.A., and Du, X. (2010). Signaling during platelet adhesion and
activation. Arteriosclerosis, thrombosis, and vascular biology 30, 2341-2349.
Liao, W., Lin, J.X., Wang, L., Li, P., and Leonard, W.J. (2011). Modulation of cytokine
receptors by IL-2 broadly regulates differentiation into helper T cell lineages. Nature
immunology 12, 551-559.
185
Lieberman, J. (2010). Anatomy of a murder: how cytotoxic T cells and NK cells are activated,
develop, and eliminate their targets. Immunological reviews 235, 5-9.
Lieberman, J. (2013). chapter 37 Cell-mediated cytotoxicity In Fundamental immunology,
W.E. Paul, ed. (Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins), pp. xviii,
1283 p.
Liel, M.S., Recht, M., and Calverley, D.C. (2014). Chapter 47 Thrombocytopenia Caused by
Immunologic Platelet Destruction. In Wintrobe's clinical hematology, J.P. Greer, ed.
(Philadelphia: Wolters Kluwer, Lippincott Williams & Wilkins Health), pp. xxvii, 2,278 p.
Lin, A., Schildknecht, A., Nguyen, L.T., and Ohashi, P.S. (2010). Dendritic cells integrate
signals from the tumor microenvironment to modulate immunity and tumor growth.
Immunology letters 127, 77-84.
Ling, Y., Cao, X., Yu, Z., and Ruan, C. (2007). Circulating dendritic cells subsets and
CD4+Foxp3+ regulatory T cells in adult patients with chronic ITP before and after treatment
with high-dose dexamethasome. European journal of haematology 79, 310-316.
Liu, B., Zhao, H., Poon, M.C., Han, Z., Gu, D., Xu, M., Jia, H., Yang, R., and Han, Z.C. (2007).
Abnormality of CD4(+)CD25(+) regulatory T cells in idiopathic thrombocytopenic purpura.
European journal of haematology 78, 139-143.
Liu, K., and Nussenzweig, M.C. (2013). Chapter 16 Dendritic Cells. In Fundamental
immunology, W.E. Paul, ed. (Philadelphia: Wolters Kluwer Health/Lippincott Williams &
Wilkins), pp. xviii, 1283 p.
Liu, Z.J., Bussel, J.B., Lakkaraja, M., Ferrer-Marin, F., Ghevaert, C., Feldman, H.A.,
McFarland, J.G., Chavda, C., and Sola-Visner, M. (2015). Suppression of in vitro
megakaryopoiesis by maternal sera containing anti-HPA-1a antibodies. Blood 126, 1234-1236.
186
Long, M.W., and Williams, N. (1981). Immature Megakaryocytes in the Mouse: Morphology
and quantitation by acetylcholinesterase staining. Blood 58, 1032-1039.
Long, M.W., Williams, N., and Ebbe, S. (1982). Immature megakaryocytes in the mouse:
physical characteristics, cell cycle status, and in vitro responsiveness to thrombopoietic
stimulatory factor. Blood 59, 569-575.
Louwes, H., Vellenga, E., Houwerzijl, E.J., and de Wolf, J.T. (2001). Effects of prednisone and
splenectomy in patients with idiopathic thrombocytopenic purpura: only splenectomy induces a
complete remission. Annals of hematology 80, 728-732.
Luiken, G.A., McMillan, R., Lightsey, A.L., Gordon, P., Zevely, S., Schulman, I., Gribble, T.J.,
and Longmire, R.L. (1977). Platelet-associated IgG in immune thrombocytopenic purpura.
Blood 50, 317-325.
Lund, F.E. (2008). Cytokine-producing B lymphocytes-key regulators of immunity. Current
opinion in immunology 20, 332-338.
Lund, F.E., and Randall, T.D. (2010). Effector and regulatory B cells: modulators of CD4+ T
cell immunity. Nature reviews Immunology 10, 236-247.
Lusher, J.M., and Iyer, R. (1977). Idiopathic thrombocytopenic purpura in children. Seminars in
thrombosis and hemostasis 3, 175-199.
Lusher, J.M., and Zuelzer, W.W. (1966). Idiopathic thrombocytopenic purpura in childhood.
The Journal of pediatrics 68, 971-979.
Lusitanus, A. (1965). Curationum medicinalium centuriae duae tertia et quanta. . In Classic
Descriptions of Disease, 3rd edition, R.H. Major, ed. (IL: Springfield).
Lykken, J.M., DiLillo, D.J., Weimer, E.T., Roser-Page, S., Heise, M.T., Grayson, J.M.,
Weitzmann, M.N., and Tedder, T.F. (2014). Acute and chronic B cell depletion disrupts CD4+
187
and CD8+ T cell homeostasis and expansion during acute viral infection in mice. Journal of
immunology 193, 746-756.
Ma, L., Zhou, Z., Zhang, D., Wang, H., Li, H., Xue, F., and Yang, R. (2012). Bmi-1 regulates
autoreactive CD4+ T cell survival in immune thrombocytopenia patients. Journal of clinical
immunology 32, 505-513.
Machlus, K.R., and Italiano, J.E., Jr. (2013). The incredible journey: From megakaryocyte
development to platelet formation. The Journal of cell biology 201, 785-796.
Machlus, K.R., Thon, J.N., and Italiano, J.E., Jr. (2014). Interpreting the developmental dance of
the megakaryocyte: a review of the cellular and molecular processes mediating platelet
formation. British journal of haematology 165, 227-236.
Mackow, E.R., and Gavrilovskaya, I.N. (2001). Cellular receptors and hantavirus pathogenesis.
Current topics in microbiology and immunology 256, 91-115.
Mangan, P.R., Harrington, L.E., O'Quinn, D.B., Helms, W.S., Bullard, D.C., Elson, C.O.,
Hatton, R.D., Wahl, S.M., Schoeb, T.R., and Weaver, C.T. (2006). Transforming growth factor-
beta induces development of the T(H)17 lineage. Nature 441, 231-234.
Masson, D., Peters, P.J., Geuze, H.J., Borst, J., and Tschopp, J. (1990). Interaction of
chondroitin sulfate with perforin and granzymes of cytolytic T-cells is dependent on pH.
Biochemistry 29, 11229-11235.
Matsui, Y., Shapiro, H.M., Sheehy, M.J., Christenson, L., Staunton, D.E., Eynon, E.E., and
Yunis, E.J. (1986). Differential expression of T cell differentiation antigens and major
histocompatibility antigens on activated T cells during the cell cycle. European journal of
immunology 16, 248-251.
188
Matsumoto, M., Baba, A., Yokota, T., Nishikawa, H., Ohkawa, Y., Kayama, H., Kallies, A.,
Nutt, S.L., Sakaguchi, S., Takeda, K., et al. (2014). Interleukin-10-producing plasmablasts exert
regulatory function in autoimmune inflammation. Immunity 41, 1040-1051.
Mauri, C., Gray, D., Mushtaq, N., and Londei, M. (2003). Prevention of arthritis by interleukin
10-producing B cells. The Journal of experimental medicine 197, 489-501.
Mazharian, A., Mori, J., Wang, Y.J., Heising, S., Neel, B.G., Watson, S.P., and Senis, Y.A.
(2013). Megakaryocyte-specific deletion of the protein-tyrosine phosphatases Shp1 and Shp2
causes abnormal megakaryocyte development, platelet production, and function. Blood 121,
4205-4220.
McKenzie, C.G., Guo, L., Freedman, J., and Semple, J.W. (2013). Cellular immune dysfunction
in immune thrombocytopenia (ITP). British journal of haematology 163, 10-23.
McMichael, A.J., Rust, N.A., Pilch, J.R., Sochynsky, R., Morton, J., Mason, D.Y., Ruan, C.,
Tobelem, G., and Caen, J. (1981). Monoclonal antibody to human platelet glycoprotein I. I.
Immunological studies. British journal of haematology 49, 501-509.
McMillan, R. (1981). Chronic idiopathic thrombocytopenic purpura. The New England journal
of medicine 304, 1135-1147.
McMillan, R. (2003). Antiplatelet antibodies in chronic adult immune thrombocytopenic
purpura: assays and epitopes. Journal of pediatric hematology/oncology 25 Suppl 1, S57-61.
McMillan, R., Wang, L., Tomer, A., Nichol, J., and Pistillo, J. (2004). Suppression of in vitro
megakaryocyte production by antiplatelet autoantibodies from adult patients with chronic ITP.
Blood 103, 1364-1369.
Metcalf Pate, K.A., Lyons, C.E., Dorsey, J.L., Shirk, E.N., Queen, S.E., Adams, R.J., Gama, L.,
Morrell, C.N., and Mankowski, J.L. (2013). Platelet activation and platelet-monocyte aggregate
189
formation contribute to decreased platelet count during acute simian immunodeficiency virus
infection in pig-tailed macaques. The Journal of infectious diseases 208, 874-883.
Metkar, S.S., Wang, B., Aguilar-Santelises, M., Raja, S.M., Uhlin-Hansen, L., Podack, E.,
Trapani, J.A., and Froelich, C.J. (2002). Cytotoxic cell granule-mediated apoptosis: perforin
delivers granzyme B-serglycin complexes into target cells without plasma membrane pore
formation. Immunity 16, 417-428.
Michaelsen, T.E., Garred, P., and Aase, A. (1991). Human IgG subclass pattern of inducing
complement-mediated cytolysis depends on antigen concentration and to a lesser extent on
epitope patchiness, antibody affinity and complement concentration. European journal of
immunology 21, 11-16.
Michelson, A.D. (2013). Platelets (London ; Waltham, MA: Academic Press), pp. xliv, 1353 p.
Miller, E., Waight, P., Farrington, C.P., Andrews, N., Stowe, J., and Taylor, B. (2001).
Idiopathic thrombocytopenic purpura and MMR vaccine. Archives of disease in childhood 84,
227-229.
Minot, G. (1916). Studies on a case of idiopathic purpura hemorrhagica. Am J Med Science
152, 48-65.
Mohan, K.V., Rao, S.S., and Atreya, C.D. (2010). Antiviral activity of selected antimicrobial
peptides against vaccinia virus. Antiviral research 86, 306-311.
Moulis, G., Palmaro, A., Montastruc, J.L., Godeau, B., Lapeyre-Mestre, M., and Sailler, L.
(2014). Epidemiology of incident immune thrombocytopenia: a nationwide population-based
study in France. Blood 124, 3308-3315.
Mueller-Eckhardt, C., Kiefel, V., Grubert, A., Kroll, H., Weisheit, M., Schmidt, S., Mueller-
Eckhardt, G., and Santoso, S. (1989). 348 cases of suspected neonatal alloimmune
thrombocytopenia. Lancet 1, 363-366.
190
Murphy, K., Travers, P., Walport, M., and Janeway, C. (2012). Janeway's immunobiology, 8th
edn (New York: Garland Science).
Murphy, M.F., and Bussel, J.B. (2007). Advances in the management of alloimmune
thrombocytopenia. British journal of haematology 136, 366-378.
Musaji, A., Cormont, F., Thirion, G., Cambiaso, C.L., and Coutelier, J.P. (2004). Exacerbation
of autoantibody-mediated thrombocytopenic purpura by infection with mouse viruses. Blood
104, 2102-2106.
Najaoui, A., Bakchoul, T., Stoy, J., Bein, G., Rummel, M.J., Santoso, S., and Sachs, U.J. (2012).
Autoantibody-mediated complement activation on platelets is a common finding in patients with
immune thrombocytopenic purpura (ITP). European journal of haematology 88, 167-174.
Nakao, K., and Angrist, A.A. (1968). Membrane surface specialization of blood platelet and
megakaryocyte. Nature 217, 960-961.
Nakeff, A., and Roozendaal, K.J. (1975). Thrombopoietin activity in mice following immune-
induced thrombocytopenia. Acta haematologica 54, 340-344.
Nardi, M., Tomlinson, S., Greco, M.A., and Karpatkin, S. (2001). Complement-independent,
peroxide-induced antibody lysis of platelets in HIV-1-related immune thrombocytopenia. Cell
106, 551-561.
Nardi, M.A., Gor, Y., Feinmark, S.J., Xu, F., and Karpatkin, S. (2007). Platelet particle
formation by anti GPIIIa49-66 Ab, Ca2+ ionophore A23187, and phorbol myristate acetate is
induced by reactive oxygen species and inhibited by dexamethasone blockade of platelet
phospholipase A2, 12-lipoxygenase, and NADPH oxidase. Blood 110, 1989-1996.
Nazi, I., Kelton, J.G., Larche, M., Snider, D.P., Heddle, N.M., Crowther, M.A., Cook, R.J.,
Tinmouth, A.T., Mangel, J., and Arnold, D.M. (2013). The effect of rituximab on vaccine
responses in patients with immune thrombocytopenia. Blood 122, 1946-1953.
191
Negi, R.R., Bhoria, P., Pahuja, A., Saikia, B., Varma, N., Malhotra, P., Varma, S., and Luthra-
Guptasarma, M. (2012). Investigation of the possible association between the HLA antigens and
idiopathic thrombocytopenic purpura (ITP). Immunological investigations 41, 117-128.
Neumuller, J., Tohidast-Akrad, M., Fischer, M., and Mayr, W.R. (1993). Influence of
chloroquine or acid treatment of human platelets on the antigenicity of HLA and the
'thrombocyte-specific' glycoproteins Ia/IIa, IIb, and IIb/IIIa. Vox sanguinis 65, 223-231.
Neunert, C.E., Buchanan, G.R., Imbach, P., Bolton-Maggs, P.H., Bennett, C.M., Neufeld, E.J.,
Vesely, S.K., Adix, L., Blanchette, V.S., Kuhne, T., et al. (2008). Severe hemorrhage in
children with newly diagnosed immune thrombocytopenic purpura. Blood 112, 4003-4008.
Newland, A.C., Treleaven, J.G., Minchinton, R.M., and Waters, A.H. (1983). High-dose
intravenous IgG in adults with autoimmune thrombocytopenia. Lancet 1, 84-87.
Newman, P.J., Kahn, R.A., and Hines, A. (1981). Detection and characterization of monoclonal
antibodies to platelet membrane proteins. The Journal of cell biology 90, 249-253.
Nguyen, T., Ghebrehiwet, B., and Peerschke, E.I. (2000). Staphylococcus aureus protein A
recognizes platelet gC1qR/p33: a novel mechanism for staphylococcal interactions with
platelets. Infection and immunity 68, 2061-2068.
Niemann, S., Spehr, N., Van Aken, H., Morgenstern, E., Peters, G., Herrmann, M., and Kehrel,
B.E. (2004). Soluble fibrin is the main mediator of Staphylococcus aureus adhesion to platelets.
Circulation 110, 193-200.
Nomura, S., Matsuzaki, T., Ozaki, Y., Yamaoka, M., Yoshimura, C., Katsura, K., Xie, G.L.,
Kagawa, H., Ishida, T., and Fukuhara, S. (1998). Clinical significance of HLA-DRB1*0410 in
Japanese patients with idiopathic thrombocytopenic purpura. Blood 91, 3616-3622.
192
Novotny, J., Tonegawa, S., Saito, H., Kranz, D.M., and Eisen, H.N. (1986). Secondary, tertiary,
and quaternary structure of T-cell-specific immunoglobulin-like polypeptide chains.
Proceedings of the National Academy of Sciences of the United States of America 83, 742-746.
Nugent, D., McMillan, R., Nichol, J.L., and Slichter, S.J. (2009). Pathogenesis of chronic
immune thrombocytopenia: increased platelet destruction and/or decreased platelet production.
British journal of haematology 146, 585-596.
O'Shea, J.J. (2013). Chapter 29 Helper T‐cell differentiation and plasticity. In Fundamental
immunology, W.E. Paul, ed. (Philadelphia: Wolters Kluwer Health/Lippincott Williams &
Wilkins), pp. xviii, 1283 p.
Ochs, H.D., Gambineri, E., and Torgerson, T.R. (2007). IPEX, FOXP3 and regulatory T-cells: a
model for autoimmunity. Immunologic research 38, 112-121.
Ogawa, M. (1993). Differentiation and proliferation of hematopoietic stem cells. Blood 81,
2844-2853.
Ohashi, P.S., and DeFranco, A.L. (2002). Making and breaking tolerance. Current opinion in
immunology 14, 744-759.
Oldenborg, P.A., Zheleznyak, A., Fang, Y.F., Lagenaur, C.F., Gresham, H.D., and Lindberg,
F.P. (2000). Role of CD47 as a marker of self on red blood cells. Science 288, 2051-2054.
Olsson, B., Andersson, P.O., Jernas, M., Jacobsson, S., Carlsson, B., Carlsson, L.M., and
Wadenvik, H. (2003). T-cell-mediated cytotoxicity toward platelets in chronic idiopathic
thrombocytopenic purpura. Nature medicine 9, 1123-1124.
Olsson, M., Bruhns, P., Frazier, W.A., Ravetch, J.V., and Oldenborg, P.A. (2005). Platelet
homeostasis is regulated by platelet expression of CD47 under normal conditions and in passive
immune thrombocytopenia. Blood 105, 3577-3582.
193
Othman, M., Labelle, A., Mazzetti, I., Elbatarny, H.S., and Lillicrap, D. (2007). Adenovirus-
induced thrombocytopenia: the role of von Willebrand factor and P-selectin in mediating
accelerated platelet clearance. Blood 109, 2832-2839.
Page, L.K., Psaila, B., Provan, D., Michael Hamilton, J., Jenkins, J.M., Elish, A.S., Lesser,
M.L., and Bussel, J.B. (2007). The immune thrombocytopenic purpura (ITP) bleeding score:
assessment of bleeding in patients with ITP. British journal of haematology 138, 245-248.
Pallard, C., Gouilleux, F., Benit, L., Cocault, L., Souyri, M., Levy, D., Groner, B., Gisselbrecht,
S., and Dusanter-Fourt, I. (1995). Thrombopoietin activates a STAT5-like factor in
hematopoietic cells. The EMBO journal 14, 2847-2856.
Patel, S.R., Richardson, J.L., Schulze, H., Kahle, E., Galjart, N., Drabek, K., Shivdasani, R.A.,
Hartwig, J.H., and Italiano, J.E., Jr. (2005). Differential roles of microtubule assembly and
sliding in proplatelet formation by megakaryocytes. Blood 106, 4076-4085.
Patel, V.L., Mahevas, M., Lee, S.Y., Stasi, R., Cunningham-Rundles, S., Godeau, B., Kanter, J.,
Neufeld, E., Taube, T., Ramenghi, U., et al. (2012). Outcomes 5 years after response to
rituximab therapy in children and adults with immune thrombocytopenia. Blood 119, 5989-
5995.
Paul, W.E. (2013). Fundamental immunology (Philadelphia: Wolters Kluwer Health/Lippincott
Williams & Wilkins), pp. xviii, 1283 p.
Pavkovic, M., Georgievski, B., Cevreska, L., Spiroski, M., and Efremov, D.G. (2003). CTLA-4
exon 1 polymorphism in patients with autoimmune blood disorders. American journal of
hematology 72, 147-149.
Pedersen, I.M., Buhl, A.M., Klausen, P., Geisler, C.H., and Jurlander, J. (2002). The chimeric
anti-CD20 antibody rituximab induces apoptosis in B-cell chronic lymphocytic leukemia cells
through a p38 mitogen activated protein-kinase-dependent mechanism. Blood 99, 1314-1319.
194
Peerschke, E.I., Andemariam, B., Yin, W., and Bussel, J.B. (2010). Complement activation on
platelets correlates with a decrease in circulating immature platelets in patients with immune
thrombocytopenic purpura. British journal of haematology 148, 638-645.
Pescovitz, M.D., Greenbaum, C.J., Krause-Steinrauf, H., Becker, D.J., Gitelman, S.E., Goland,
R., Gottlieb, P.A., Marks, J.B., McGee, P.F., Moran, A.M., et al. (2009). Rituximab, B-
lymphocyte depletion, and preservation of beta-cell function. The New England journal of
medicine 361, 2143-2152.
Petermann, M.L. (1946). The splitting of human gamma globulin antibodies by papain and
bromelin. Journal of the American Chemical Society 68, 106-113.
Petermann, M.L., and Pappenheimer, A.M., Jr. (1941). The Action of Crystalline Pepsin on
Horse Anti-Pneumococcus Antibody. Science 93, 458.
Piessens, W.F., Wybran, J., Manaster, J., and Strijckmans, P.A. (1970). Lymphocyte
transformation induced by autologous platelets in a case of thrombocytopenic purpura. Blood
36, 421-427.
Pizzuto, J., and Ambriz, R. (1984). Therapeutic experience on 934 adults with idiopathic
thrombocytopenic purpura: Multicentric Trial of the Cooperative Latin American group on
Hemostasis and Thrombosis. Blood 64, 1179-1183.
Plow, E.F., and Ma, Y.-Q. (2007). Inside-out, outside-in: what's the difference? Blood 109,
3128-3129.
Polgar, J., Matuskova, J., and Wagner, D.D. (2005). The P-selectin, tissue factor, coagulation
triad. Journal of thrombosis and haemostasis : JTH 3, 1590-1596.
Provan, D., Stasi, R., Newland, A.C., Blanchette, V.S., Bolton-Maggs, P., Bussel, J.B., Chong,
B.H., Cines, D.B., Gernsheimer, T.B., Godeau, B., et al. (2010). International consensus report
195
on the investigation and management of primary immune thrombocytopenia. Blood 115, 168-
186.
Prussin, C., Yin, Y., and Upadhyaya, B. (2010). T(H)2 heterogeneity: Does function follow
form? The Journal of allergy and clinical immunology 126, 1094-1098.
Psaila, B., Petrovic, A., Page, L.K., Menell, J., Schonholz, M., and Bussel, J.B. (2009).
Intracranial hemorrhage (ICH) in children with immune thrombocytopenia (ITP): study of 40
cases. Blood 114, 4777-4783.
Radder, C.M., Brand, A., and Kanhai, H.H. (2003). Will it ever be possible to balance the risk
of intracranial haemorrhage in fetal or neonatal alloimmune thrombocytopenia against the risk
of treatment strategies to prevent it? Vox sanguinis 84, 318-325.
Radley, J.M., and Haller, C.J. (1982). The demarcation membrane system of the
megakaryocyte: a misnomer? Blood 60, 213-219.
Radwan, E.R., and Goda, R.L. (2015). Lack of impact of cytotoxic T-lymphocyte antigen 4
gene exon 1 polymorphism on susceptibility to or clinical course of egyptian childhood immune
thrombocytopenic purpura. Clinical and applied thrombosis/hemostasis : official journal of the
International Academy of Clinical and Applied Thrombosis/Hemostasis 21, 378-382.
Ramsdell, F., and Ziegler, S.F. (2014). FOXP3 and scurfy: how it all began. Nature reviews
Immunology 14, 343-349.
Ravetch, J.V., and Lanier, L.L. (2000). Immune inhibitory receptors. Science 290, 84-89.
Ravetch, J.V., and Nimmerjahn, F. (2013). Chapter 24 Fc receptors and their role in immune
regulation and inflammation. In Fundamental immunology, W.E. Paul, ed. (Philadelphia:
Wolters Kluwer Health/Lippincott Williams & Wilkins), pp. xviii, 1283 p.
196
Ray, A., Basu, S., Williams, C.B., Salzman, N.H., and Dittel, B.N. (2012). A novel IL-10-
independent regulatory role for B cells in suppressing autoimmunity by maintenance of
regulatory T cells via GITR ligand. Journal of immunology 188, 3188-3198.
Reff, M.E., Carner, K., Chambers, K.S., Chinn, P.C., Leonard, J.E., Raab, R., Newman, R.A.,
Hanna, N., and Anderson, D.R. (1994). Depletion of B cells in vivo by a chimeric mouse human
monoclonal antibody to CD20. Blood 83, 435-445.
Richardson, J.L., Shivdasani, R.A., Boers, C., Hartwig, J.H., and Italiano, J.E., Jr. (2005).
Mechanisms of organelle transport and capture along proplatelets during platelet production.
Blood 106, 4066-4075.
Riverius, L. (1668). On pestilential feavers, in Culpepper N (transl.): Praxis Medica or the
Compleat Practice of Physick. London: Streator, 618.
Robb-Smith, A.H. (1967). Why the platelets were discovered. British journal of haematology
13, 618-637.
Rocha, A.M., Souza, C., Rocha, G.A., de Melo, F.F., Clementino, N.C., Marino, M.C., Bozzi,
A., Silva, M.L., Martins Filho, O.A., and Queiroz, D.M. (2011). The levels of IL-17A and of the
cytokines involved in Th17 cell commitment are increased in patients with chronic immune
thrombocytopenia. Haematologica 96, 1560-1564.
Rodan, G.A., and Feinstein, M.B. (1976). Interrelationships between Ca2+ and adenylate and
guanylate cyclases in the control of platelet secretion and aggregation. Proceedings of the
National Academy of Sciences of the United States of America 73, 1829-1833.
Rodeghiero, F., Michel, M., Gernsheimer, T., Ruggeri, M., Blanchette, V., Bussel, J.B., Cines,
D.B., Cooper, N., Godeau, B., Greinacher, A., et al. (2013). Standardization of bleeding
assessment in immune thrombocytopenia: report from the International Working Group. Blood
121, 2596-2606.
197
Rodeghiero, F., Stasi, R., Gernsheimer, T., Michel, M., Provan, D., Arnold, D.M., Bussel, J.B.,
Cines, D.B., Chong, B.H., Cooper, N., et al. (2009). Standardization of terminology, definitions
and outcome criteria in immune thrombocytopenic purpura of adults and children: report from
an international working group. Blood 113, 2386-2393.
Rolovic, Z., Baldini, M., and Dameshek, W. (1970). Megakaryocytopoiesis in experimentally
induced immune thrombocytopenia. Blood 35, 173-188.
Rosenfeld, S.I., Looney, R.J., Leddy, J.P., Phipps, D.C., Abraham, G.N., and Anderson, C.L.
(1985). Human platelet Fc receptor for immunoglobulin G. Identification as a 40,000-
molecular-weight membrane protein shared by monocytes. The Journal of clinical investigation
76, 2317-2322.
Rosette, C., Werlen, G., Daniels, M.A., Holman, P.O., Alam, S.M., Travers, P.J., Gascoigne,
N.R., Palmer, E., and Jameson, S.C. (2001). The impact of duration versus extent of TCR
occupancy on T cell activation: a revision of the kinetic proofreading model. Immunity 15, 59-
70.
Rosser, E.C., and Mauri, C. (2015). Regulatory B cells: origin, phenotype, and function.
Immunity 42, 607-612.
Rothenberg, E.V., and Champhekar, A. (2013). Chapter 13 T-Lymphocyte Developmental
Biology. In Fundamental immunology, W.E. Paul, ed. (Philadelphia: Wolters Kluwer
Health/Lippincott Williams & Wilkins), pp. xviii, 1283 p.
Rumjantseva, V., Grewal, P.K., Wandall, H.H., Josefsson, E.C., Sorensen, A.L., Larson, G.,
Marth, J.D., Hartwig, J.H., and Hoffmeister, K.M. (2009). Dual roles for hepatic lectin receptors
in the clearance of chilled platelets. Nature medicine 15, 1273-1280.
Ryan, M.E., Webster, M.L., and Statler, J.D. (1996). Adverse effects of intravenous
immunoglobulin therapy. Clinical pediatrics 35, 23-31.
198
Sabri, S., Jandrot-Perrus, M., Bertoglio, J., Farndale, R.W., Mas, V.M., Debili, N., and
Vainchenker, W. (2004). Differential regulation of actin stress fiber assembly and proplatelet
formation by alpha2beta1 integrin and GPVI in human megakaryocytes. Blood 104, 3117-3125.
Sailer, T., Lechner, K., Panzer, S., Kyrle, P.A., and Pabinger, I. (2006). The course of severe
autoimmune thrombocytopenia in patients not undergoing splenectomy. Haematologica 91,
1041-1045.
Saito, A., Yokohama, A., Osaki, Y., Ogawa, Y., Nakahashi, H., Toyama, K., Mitsui, T.,
Hashimoto, Y., Koiso, H., Uchiumi, H., et al. (2012). Circulating plasmacytoid dendritic cells in
patients with primary and Helicobacter pylori-associated immune thrombocytopenia. European
journal of haematology 88, 340-349.
Sakaguchi, S., Miyara, M., Costantino, C.M., and Hafler, D.A. (2010). FOXP3+ regulatory T
cells in the human immune system. Nature reviews Immunology 10, 490-500.
Sakaguchi, S., Ono, M., Setoguchi, R., Yagi, H., Hori, S., Fehervari, Z., Shimizu, J., Takahashi,
T., and Nomura, T. (2006). Foxp3+ CD25+ CD4+ natural regulatory T cells in dominant self-
tolerance and autoimmune disease. Immunological reviews 212, 8-27.
Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., and Toda, M. (1995). Immunologic self-
tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25).
Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases.
Journal of immunology 155, 1151-1164.
Sakakura, M., Wada, H., Tawara, I., Nobori, T., Sugiyama, T., Sagawa, N., and Shiku, H.
(2007). Reduced Cd4+Cd25+ T cells in patients with idiopathic thrombocytopenic purpura.
Thrombosis research 120, 187-193.
Salama, A., Kiefel, V., Amberg, R., and Mueller-Eckhardt, C. (1984). Treatment of autoimmune
thrombocytopenic purpura with rhesus antibodies (anti-Rh0(D). Blut 49, 29-35.
199
Salama, A., Kiefel, V., and Mueller-Eckhardt, C. (1986). Effect of IgG anti-Rho(D) in adult
patients with chronic autoimmune thrombocytopenia. American journal of hematology 22, 241-
250.
Salama, A., Kiesewetter, H., Kalus, U., Movassaghi, K., and Meyer, O. (2008). Massive platelet
transfusion is a rapidly effective emergency treatment in patients with refractory autoimmune
thrombocytopenia. Thrombosis and haemostasis 100, 762-765.
Salama, A., Mueller-Eckhardt, C., and Kiefel, V. (1983). Effect of intravenous immunoglobulin
in immune thrombocytopenia. Lancet 2, 193-195.
Samuelsson, A., Towers, T.L., and Ravetch, J.V. (2001). Anti-inflammatory activity of IVIG
mediated through the inhibitory Fc receptor. Science 291, 484-486.
Santoso, S., and Kiefel, V. (2001). Human platelet alloantigens. Wiener klinische
Wochenschrift 113, 806-813.
Satoh, T., Miyazaki, K., Shimohira, A., Amano, N., Okazaki, Y., Nishimoto, T., Akahoshi, T.,
Munekata, S., Kanoh, Y., Ikeda, Y., et al. (2013). Fcgamma receptor IIB gene polymorphism in
adult Japanese patients with primary immune thrombocytopenia. Blood 122, 1991-1992.
Sattler, M., Durstin, M.A., Frank, D.A., Okuda, K., Kaushansky, K., Salgia, R., and Griffin, J.D.
(1995). The thrombopoietin receptor c-MPL activates JAK2 and TYK2 tyrosine kinases.
Experimental hematology 23, 1040-1048.
Saxena, A., Martin-Blondel, G., Mars, L.T., and Liblau, R.S. (2011). Role of CD8 T cell subsets
in the pathogenesis of multiple sclerosis. FEBS letters 585, 3758-3763.
Schietinger, A., and Greenberg, P.D. (2014). Tolerance and exhaustion: defining mechanisms of
T cell dysfunction. Trends in immunology 35, 51-60.
200
Schirrmacher, V., Halloran, P., and David, C.S. (1975). Interactions of Fc receptors with
antibodies against Ia antigens and other cell surface components. The Journal of experimental
medicine 141, 1201-1209.
Schlosser, E., Otero, C., Wuensch, C., Kessler, B., Edelmann, M., Brunisholz, R., Drexler, I.,
Legler, D.F., and Groettrup, M. (2007). A novel cytosolic class I antigen-processing pathway
for endoplasmic-reticulum-targeted proteins. EMBO reports 8, 945-951.
Schorle, H., Holtschke, T., Hunig, T., Schimpl, A., and Horak, I. (1991). Development and
function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature 352, 621-
624.
Schroeder, H.W., Jr., Ippolito, G.C., and Shiokawa, S. (1998). Regulation of the antibody
repertoire through control of HCDR3 diversity. Vaccine 16, 1383-1390.
Schroeder, H.W., Jr., Mortari, F., Shiokawa, S., Kirkham, P.M., Elgavish, R.A., and Bertrand,
F.E., 3rd (1995). Developmental regulation of the human antibody repertoire. Annals of the
New York Academy of Sciences 764, 242-260.
Schulze, H., Korpal, M., Hurov, J., Kim, S.W., Zhang, J., Cantley, L.C., Graf, T., and
Shivdasani, R.A. (2006). Characterization of the megakaryocyte demarcation membrane system
and its role in thrombopoiesis. Blood 107, 3868-3875.
Schwab, I., Biburger, M., Kronke, G., Schett, G., and Nimmerjahn, F. (2012). IVIg-mediated
amelioration of ITP in mice is dependent on sialic acid and SIGNR1. European journal of
immunology 42, 826-830.
Schwarzenberger, P., La Russa, V., Miller, A., Ye, P., Huang, W., Zieske, A., Nelson, S.,
Bagby, G.J., Stoltz, D., Mynatt, R.L., et al. (1998). IL-17 stimulates granulopoiesis in mice: use
of an alternate, novel gene therapy-derived method for in vivo evaluation of cytokines. Journal
of immunology 161, 6383-6389.
201
Scott, J.P., Nunez, D., Reardon, D., Luddington, R., Folawski, J., Evans, D.B., Higenbottam,
T.W., and Calne, R. (1987). Effect of cyclosporine on platelet aggregation in renal transplant
recipients. Transplantation proceedings 19, 4008.
Segal, J.B., and Powe, N.R. (2006). Prevalence of immune thrombocytopenia: analyses of
administrative data. Journal of thrombosis and haemostasis : JTH 4, 2377-2383.
Seiffert, M., Cant, C., Chen, Z., Rappold, I., Brugger, W., Kanz, L., Brown, E.J., Ullrich, A.,
and Buhring, H.J. (1999). Human signal-regulatory protein is expressed on normal, but not on
subsets of leukemic myeloid cells and mediates cellular adhesion involving its counterreceptor
CD47. Blood 94, 3633-3643.
Semple, J.W. (2010). Animal models of immune thrombocytopenia (ITP). Annals of
hematology 89, 37-44.
Semple, J.W., Aslam, R., Kim, M., Speck, E.R., and Freedman, J. (2007). Platelet-bound
lipopolysaccharide enhances Fc receptor-mediated phagocytosis of IgG-opsonized platelets.
Blood 109, 4803-4805.
Semple, J.W., Bruce, S., and Freedman, J. (1991). Suppressed natural killer cell activity in
patients with chronic autoimmune thrombocytopenic purpura. American journal of hematology
37, 258-262.
Semple, J.W., and Freedman, J. (1991). Increased antiplatelet T helper lymphocyte reactivity in
patients with autoimmune thrombocytopenia. Blood 78, 2619-2625.
Semple, J.W., and Freedman, J. (1994). Natural killer cell numbers and activity in patients with
chronic autoimmune thrombocytopenic purpura. Blood 83, 870-871.
Semple, J.W., Milev, Y., Cosgrave, D., Mody, M., Hornstein, A., Blanchette, V., and Freedman,
J. (1996). Differences in serum cytokine levels in acute and chronic autoimmune
202
thrombocytopenic purpura: relationship to platelet phenotype and antiplatelet T-cell reactivity.
Blood 87, 4245-4254.
Semple, J.W., Speck, E.R., Milev, Y.P., Blanchette, V., and Freedman, J. (1995). Indirect
allorecognition of platelets by T helper cells during platelet transfusions correlates with anti-
major histocompatibility complex antibody and cytotoxic T lymphocyte formation. Blood 86,
805-812.
Shah, D.K., and Zuniga-Pflucker, J.C. (2014). An overview of the intrathymic intricacies of T
cell development. Journal of immunology 192, 4017-4023.
Shao, L., Wu, Y., Zhou, H., Qin, P., Ni, H., Peng, J., and Hou, M. (2015). Successful treatment
with oseltamivir phosphate in a patient with chronic immune thrombocytopenia positive for
anti-GPIb/IX autoantibody. Platelets 26, 495-497.
Shen, H., Whitmire, J.K., Fan, X., Shedlock, D.J., Kaech, S.M., and Ahmed, R. (2003). A
specific role for B cells in the generation of CD8 T cell memory by recombinant Listeria
monocytogenes. Journal of immunology 170, 1443-1451.
Shen, P., Roch, T., Lampropoulou, V., O'Connor, R.A., Stervbo, U., Hilgenberg, E., Ries, S.,
Dang, V.D., Jaimes, Y., Daridon, C., et al. (2014). IL-35-producing B cells are critical
regulators of immunity during autoimmune and infectious diseases. Nature 507, 366-370.
Shimomura, T., Fujimura, K., Takafuta, T., Fujii, T., Katsutani, S., Noda, M., Fujimoto, T., and
Kuramoto, A. (1996). Oligoclonal accumulation of T cells in peripheral blood from patients
with idiopathic thrombocytopenic purpura. British journal of haematology 95, 732-737.
Shlomchik, M.J., and Weisel, F. (2012). Germinal center selection and the development of
memory B and plasma cells. Immunological reviews 247, 52-63.
Shortman, K., and Liu, Y.J. (2002). Mouse and human dendritic cell subtypes. Nature reviews
Immunology 2, 151-161.
203
Shulman, N.R., Aster, R.H., Leitner, A., and Hiller, M.C. (1961). Immunoreactions Involving
Platelets. V. Post-Transfusion Purpura Due to a Complement-Fixing Antibody against a
Genetically Controlled Platelet Antigen. A Proposed Mechanism for Thrombocytopenia and Its
Relevance in "Autoimmunity". The Journal of clinical investigation 40, 1597-1620.
Shulman, N.R., Marder, V.J., and Weinrach, R.S. (1965). Similarities between known
antiplatelet antibodies and the factor responsible for thrombocytopenia in idiopathic purpura.
Physiologic, serologic and isotopic studies. Annals of the New York Academy of Sciences 124,
499-542.
Simon, T.L., Collins, J., Kunicki, T.J., Furihata, K., Smith, K.J., and Aster, R.H. (1988).
Posttransfusion purpura associated with alloantibody specific for the platelet antigen, Pen(a).
American journal of hematology 29, 38-40.
Singh, M.V., Davidson, D.C., Kiebala, M., and Maggirwar, S.B. (2012). Detection of circulating
platelet-monocyte complexes in persons infected with human immunodeficiency virus type-1.
Journal of virological methods 181, 170-176.
Siragam, V., Crow, A.R., Brinc, D., Song, S., Freedman, J., and Lazarus, A.H. (2006).
Intravenous immunoglobulin ameliorates ITP via activating Fc gamma receptors on dendritic
cells. Nature medicine 12, 688-692.
Slichter, S.J. (1980). Controversies in platelet transfusion therapy. Annual review of medicine
31, 509-540.
Smyth, S.S., McEver, R.P., Weyrich, A.S., Morrell, C.N., Hoffman, M.R., Arepally, G.M.,
French, P.A., Dauerman, H.L., Becker, R.C., and Platelet Colloquium, P. (2009). Platelet
functions beyond hemostasis. Journal of thrombosis and haemostasis : JTH 7, 1759-1766.
Solomon Tsegaye, T., Gnirss, K., Rahe-Meyer, N., Kiene, M., Kramer-Kuhl, A., Behrens, G.,
Munch, J., and Pohlmann, S. (2013). Platelet activation suppresses HIV-1 infection of T cells.
Retrovirology 10, 48.
204
Sood, R., Wong, W., Gotlib, J., Jeng, M., and Zehnder, J.L. (2008). Gene expression and
pathway analysis of immune thrombocytopenic purpura. British journal of haematology 140,
99-103.
Soubrane, C., Tourani, J.M., Andrieu, J.M., Visonneau, S., Beldjord, K., Israel-Biet, D.,
Mouawad, R., Bussel, J., Weil, M., and Khayat, D. (1993). Biologic response to anti-CD16
monoclonal antibody therapy in a human immunodeficiency virus-related immune
thrombocytopenic purpura patient. Blood 81, 15-19.
Spahr, J.E., and Rodgers, G.M. (2008). Treatment of immune-mediated thrombocytopenia
purpura with concurrent intravenous immunoglobulin and platelet transfusion: a retrospective
review of 40 patients. American journal of hematology 83, 122-125.
Spits, H., van Schooten, W., Keizer, H., van Seventer, G., van de Rijn, M., Terhorst, C., and de
Vries, J.E. (1986). Alloantigen recognition is preceded by nonspecific adhesion of cytotoxic T
cells and target cells. Science 232, 403-405.
Sprague, C.C., Harrington, W.J., Lange, R.D., and Shapleigh, J.B. (1952). Platelet transfusions
and the pathogenesis of idiopathic thrombocytopenic purpura. Journal of the American Medical
Association 150, 1193-1198.
Stahl, C.P., Zucker-Franklin, D., and McDonald, T.P. (1986). Incomplete antigenic cross-
reactivity between platelets and megakaryocytes: relevance to ITP. Blood 67, 421-428.
Stasi, R., Cooper, N., Del Poeta, G., Stipa, E., Laura Evangelista, M., Abruzzese, E., and
Amadori, S. (2008). Analysis of regulatory T-cell changes in patients with idiopathic
thrombocytopenic purpura receiving B cell-depleting therapy with rituximab. Blood 112, 1147-
1150.
Stasi, R., Del Poeta, G., Stipa, E., Evangelista, M.L., Trawinska, M.M., Cooper, N., and
Amadori, S. (2007). Response to B-cell depleting therapy with rituximab reverts the
205
abnormalities of T-cell subsets in patients with idiopathic thrombocytopenic purpura. Blood
110, 2924-2930.
Stasi, R., Pagano, A., Stipa, E., and Amadori, S. (2001). Rituximab chimeric anti-CD20
monoclonal antibody treatment for adults with chronic idiopathic thrombocytopenic purpura.
Blood 98, 952-957.
Stasi, R., Stipa, E., Masi, M., Cecconi, M., Scimo, M.T., Oliva, F., Sciarra, A., Perrotti, A.P.,
Adomo, G., Amadori, S., et al. (1995). Long-term observation of 208 adults with chronic
idiopathic thrombocytopenic purpura. The American journal of medicine 98, 436-442.
Stenberg, P.E., and Levin, J. (1989). Ultrastructural analysis of acute immune thrombocytopenia
in mice: dissociation between alterations in megakaryocytes and platelets. Journal of cellular
physiology 141, 160-169.
Stoll, D., Cines, D.B., Aster, R.H., and Murphy, S. (1985). Platelet kinetics in patients with
idiopathic thrombocytopenic purpura and moderate thrombocytopenia. Blood 65, 584-588.
Stuart, M.J., Tomar, R.H., Miller, M.L., and Davey, F.R. (1978). Chronic idiopathic
thrombocytopenic purpura. A familial immunodeficiency syndrome? Jama 239, 939-942.
Sugiyama, T., Okuma, M., Ushikubi, F., Sensaki, S., Kanaji, K., and Uchino, H. (1987). A novel
platelet aggregating factor found in a patient with defective collagen-induced platelet
aggregation and autoimmune thrombocytopenia. Blood 69, 1712-1720.
Tablin, F., Castro, M., and Leven, R.M. (1990). Blood platelet formation in vitro. The role of
the cytoskeleton in megakaryocyte fragmentation. Journal of cell science 97 ( Pt 1), 59-70.
Teeling, J.L., Jansen-Hendriks, T., Kuijpers, T.W., de Haas, M., van de Winkel, J.G., Hack,
C.E., and Bleeker, W.K. (2001). Therapeutic efficacy of intravenous immunoglobulin
preparations depends on the immunoglobulin G dimers: studies in experimental immune
thrombocytopenia. Blood 98, 1095-1099.
206
Teramura, M., Kobayashi, S., Hoshino, S., Oshimi, K., and Mizoguchi, H. (1992). Interleukin-
11 enhances human megakaryocytopoiesis in vitro. Blood 79, 327-331.
Terrell, D.R., Beebe, L.A., Neas, B.R., Vesely, S.K., Segal, J.B., and George, J.N. (2012).
Prevalence of primary immune thrombocytopenia in Oklahoma. American journal of
hematology 87, 848-852.
Tesse, R., Del Vecchio, G.C., De Mattia, D., Sangerardi, M., Valente, F., and Giordano, P.
(2012). Association of interleukin-(IL)10 haplotypes and serum IL-10 levels in the progression
of childhood immune thrombocytopenic purpura. Gene 505, 53-56.
Thachil, J. (2010). The myths about platelet transfusions in immune-mediated
thrombocytopenias. British journal of haematology 150, 494-495.
Thienelt, C.D., and Calverley, D.C. (2009). Chapter 51 Thrombocytopenia Caused by
Immunologic Platelet Destruction. In Wintrobe's clinical hematology, M.M. Wintrobe, J.P.
Greer, and Ovid Technologies Inc., eds. (Philadelphia: Wolters Kluwer Health/Lippincott
Williams & Wilkins), pp. 2 v. (xxv, 2606, I-2647 p.).
Thomsen, A.R., Johansen, J., Marker, O., and Christensen, J.P. (1996). Exhaustion of CTL
memory and recrudescence of viremia in lymphocytic choriomeningitis virus-infected MHC
class II-deficient mice and B cell-deficient mice. Journal of immunology 157, 3074-3080.
Thon, J.N., and Italiano, J.E. (2010). Platelet formation. Seminars in hematology 47, 220-226.
Thornton, A.M., Donovan, E.E., Piccirillo, C.A., and Shevach, E.M. (2004). Cutting edge: IL-2
is critically required for the in vitro activation of CD4+CD25+ T cell suppressor function.
Journal of immunology 172, 6519-6523.
Tovo, P.A., Miniero, R., Fiandino, G., Saracco, P., and Messina, M. (1984). Fc-depleted vs
intact intravenous immunoglobulin in chronic ITP. The Journal of pediatrics 105, 676-677.
207
Tremblay, T., Aubin, E., Lemieux, R., and Bazin, R. (2007). Picogram doses of
lipopolysaccharide exacerbate antibody-mediated thrombocytopenia and reduce the therapeutic
efficacy of intravenous immunoglobulin in mice. British journal of haematology 139, 297-302.
Trowbridge, E.A., Martin, J.F., Slater, D.N., Kishk, Y.T., Warren, C.W., Harley, P.J., and
Woodcock, B. (1984). The origin of platelet count and volume. Clinical physics and
physiological measurement : an official journal of the Hospital Physicists' Association,
Deutsche Gesellschaft fur Medizinische Physik and the European Federation of Organisations
for Medical Physics 5, 145-170.
Tsubakio, T., Hamano, T., Shintome, M., Watanabe, M., Tomiyama, Y., Mizutani, H., Zhenjing,
J., Kurata, Y., Yonezawa, T., and Tarui, S. (1987). [Mechanism of increase in platelet count by
intravenous gammaglobulin in thrombocytopenic rats]. Nihon Ketsueki Gakkai zasshi : journal
of Japan Haematological Society 50, 1031-1037.
Uchida, J., Hamaguchi, Y., Oliver, J.A., Ravetch, J.V., Poe, J.C., Haas, K.M., and Tedder, T.F.
(2004a). The innate mononuclear phagocyte network depletes B lymphocytes through Fc
receptor-dependent mechanisms during anti-CD20 antibody immunotherapy. The Journal of
experimental medicine 199, 1659-1669.
Uchida, J., Lee, Y., Hasegawa, M., Liang, Y., Bradney, A., Oliver, J.A., Bowen, K., Steeber,
D.A., Haas, K.M., Poe, J.C., et al. (2004b). Mouse CD20 expression and function. International
immunology 16, 119-129.
Urbanus, R.T., van der Wal, D.E., Koekman, C.A., Huisman, A., van den Heuvel, D.J.,
Gerritsen, H.C., Deckmyn, H., Akkerman, J.W., Schutgens, R.E., and Gitz, E. (2013). Patient
autoantibodies induce platelet destruction signals via raft-associated glycoprotein Ibalpha and
Fc RIIa in immune thrombocytopenia. Haematologica 98, e70-72.
van Bladel, E.R., Laarhoven, A.G., van der Heijden, L.B., Heitink-Polle, K.M., Porcelijn, L.,
van der Schoot, C.E., de Haas, M., Roest, M., Vidarsson, G., de Groot, P.G., et al. (2014).
208
Functional platelet defects in children with severe chronic ITP as tested with 2 novel assays
applicable for low platelet counts. Blood 123, 1556-1563.
van Leeuwen, E.F., van der Ven, J.T., Engelfriet, C.P., and von dem Borne, A.E. (1982).
Specificity of autoantibodies in autoimmune thrombocytopenia. Blood 59, 23-26.
Veneri, D., Gottardi, M., Guizzardi, E., Zanuso, C., Krampera, M., and Franchini, M. (2002).
Idiopathic thrombocytopenic purpura, Helicobacter pylori infection, and HLA class II alleles.
Blood 100, 1925-1926; author reply 1926-1927.
Victora, G.D., and Mesin, L. (2014). Clonal and cellular dynamics in germinal centers. Current
opinion in immunology 28, 90-96.
Vignali, D.A., Collison, L.W., and Workman, C.J. (2008). How regulatory T cells work. Nature
reviews Immunology 8, 523-532.
Vogelsang, G., Kickler, T.S., and Bell, W.R. (1986). Post-transfusion purpura: a report of five
patients and a review of the pathogenesis and management. American journal of hematology 21,
259-267.
von dem Borne, A.E., van Leeuwen, E.F., von Riesz, L.E., van Boxtel, C.J., and Engelfriet, C.P.
(1981). Neonatal alloimmune thrombocytopenia: detection and characterization of the
responsible antibodies by the platelet immunofluorescence test. Blood 57, 649-656.
Wakisaka, G., Yasunaga, K., Kuramoto, A., Okuma, M., and Furukawa, H. (1964).
[Experimental Study of Immune Thrombocytopenia Induced by Quinidine]. Arerugi = [Allergy]
13, 686-689.
Wang, C.M., Sheng, G.Y., Zou, X., Bai, S.T., and Wang, L. (2009). [Levels of Toll-like
receptors-2,-4 on platelets in children with idiopathic thrombocytopenic purpura]. Zhongguo
dang dai er ke za zhi = Chinese journal of contemporary pediatrics 11, 797-801.
209
Wang, J.D., Ou, T.T., Wang, C.J., Chang, T.K., and Lee, H.J. (2010). Platelet apoptosis
resistance and increased CXCR4 expression in pediatric patients with chronic immune
thrombocytopenic purpura. Thrombosis research 126, 311-318.
Wang, T.T., Zhao, H., Ren, H., Guo, J.H., Xu, M.Q., Yang, R.C., and Han, Z.C. (2006).
[Profiles of type 1 and type 2 T cells in chronic idiopathic thrombocytopenic purpura].
Zhonghua yi xue za zhi 86, 669-673.
Ware, R.E., and Howard, T.A. (1993). Phenotypic and clonal analysis of T lymphocytes in
childhood immune thrombocytopenic purpura. Blood 82, 2137-2142.
Warkentin, T.E., and Smith, J.W. (1997). The alloimmune thrombocytopenic syndromes.
Transfusion medicine reviews 11, 296-307.
Warkentin, T.E., Smith, J.W., Hayward, C.P., Ali, A.M., and Kelton, J.G. (1992).
Thrombocytopenia caused by passive transfusion of anti-glycoprotein Ia/IIa alloantibody (anti-
HPA-5b). Blood 79, 2480-2484.
Watts, C. (1997). Capture and processing of exogenous antigens for presentation on MHC
molecules. Annual review of immunology 15, 821-850.
Webert, K.E., Smith, J.W., Arnold, D.M., Chan, H.H.W., Heddle, N.M., and Kelton, J.G.
(2014). Chapter 20 Red Cell, Platelet, and White Cell Antigens. In Wintrobe's clinical
hematology, J.P. Greer, ed. (Philadelphia: Wolters Kluwer, Lippincott Williams & Wilkins
Health), pp. xxvii, 2,278 p.
Wei, J., Long, L., Yang, K., Guy, C., Shrestha, S., Chen, Z., Wu, C., Vogel, P., Neale, G.,
Green, D.R., et al. (2016). Autophagy enforces functional integrity of regulatory T cells by
coupling environmental cues and metabolic homeostasis. Nature immunology 17, 277-285.
Werlhof, P. (1965). Opera omnia. In Classic Descriptions of Disease, 3rd edition, R. Major, ed.
(IL: Springfield).
210
White, J.G., and Escolar, G. (1993). Current concepts of platelet membrane response to surface
activation. Platelets 4, 175-189.
Wildin, R.S., Ramsdell, F., Peake, J., Faravelli, F., Casanova, J.L., Buist, N., Levy-Lahad, E.,
Mazzella, M., Goulet, O., Perroni, L., et al. (2001). X-linked neonatal diabetes mellitus,
enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nature
genetics 27, 18-20.
Willan, R. (1808). On Cutaneous Diseases. (London: Barnard, JG).
Williams, A.F., and Barclay, A.N. (1988). The immunoglobulin superfamily--domains for cell
surface recognition. Annual review of immunology 6, 381-405.
Wilson, N.J., Boniface, K., Chan, J.R., McKenzie, B.S., Blumenschein, W.M., Mattson, J.D.,
Basham, B., Smith, K., Chen, T., Morel, F., et al. (2007). Development, cytokine profile and
function of human interleukin 17-producing helper T cells. Nature immunology 8, 950-957.
Wilson, S.S., Wiens, M.E., and Smith, J.G. (2013). Antiviral mechanisms of human defensins.
Journal of molecular biology 425, 4965-4980.
Wing, K., and Sakaguchi, S. (2010). Regulatory T cells exert checks and balances on self
tolerance and autoimmunity. Nature immunology 11, 7-13.
Winiarski, J., and Holm, G. (1983). Platelet associated immunoglobulins and complement in
idiopathic thrombocytopenic purpura. Clinical and experimental immunology 53, 201-207.
Wintrobe, M.M., Cartwright, G.E., Palmer, J.G., Kuhns, W.J., and Samuels, L.T. (1951). Effect
of corticotrophin and cortisone on the blood in various disorders in man. AMA archives of
internal medicine 88, 310-336.
Wolf, S.D., Dittel, B.N., Hardardottir, F., and Janeway, C.A., Jr. (1996). Experimental
autoimmune encephalomyelitis induction in genetically B cell-deficient mice. The Journal of
experimental medicine 184, 2271-2278.
211
Wybran, J., and Fudenberg, H.H. (1972). Cellular immunity to platelets in idiopathic
thrombocytopenic purpura. Blood 40, 856-861.
Xiu, Y., Wong, C.P., Bouaziz, J.D., Hamaguchi, Y., Wang, Y., Pop, S.M., Tisch, R.M., and
Tedder, T.F. (2008). B lymphocyte depletion by CD20 monoclonal antibody prevents diabetes
in nonobese diabetic mice despite isotype-specific differences in Fc gamma R effector
functions. Journal of immunology 180, 2863-2875.
Xu, J., Huang, Y., Tao, J., Zhou, Z., Chen, Z., Ge, J., and Yang, R. (2010). An FcgammaRIIb
transmembrane polymorphism in Chinese ITP patients. Platelets 21, 479-485.
Xu, S.Q., Wang, C.Y., Zhu, X.J., Dong, X.Y., Shi, Y., Peng, J., Qin, P., Sun, J.Z., Guo, C., Ni,
H., et al. (2012). Decreased indoleamine 2,3-dioxygenase expression in dendritic cells and role
of indoleamine 2,3-dioxygenase-expressing dendritic cells in immune thrombocytopenia.
Annals of hematology 91, 1623-1631.
Yamashita, M., Kuwahara, M., Suzuki, A., Hirahara, K., Shinnaksu, R., Hosokawa, H.,
Hasegawa, A., Motohashi, S., Iwama, A., and Nakayama, T. (2008). Bmi1 regulates memory
CD4 T cell survival via repression of the Noxa gene. The Journal of experimental medicine 205,
1109-1120.
Yanabu, M., Nomura, S., Fukuroi, T., Suzuki, M., Kawakatsu, T., Kido, H., Yamaguchi, K.,
Kokawa, T., and Yasunaga, K. (1993). Platelet activation induced by an antiplatelet
autoantibody against CD9 antigen and its inhibition by another autoantibody in immune
thrombocytopenic purpura. British journal of haematology 84, 694-701.
Yang, L., Wang, L., Zhao, C.H., Zhu, X.J., Hou, Y., Jun, P., and Hou, M. (2010). Contributions
of TRAIL-mediated megakaryocyte apoptosis to impaired megakaryocyte and platelet
production in immune thrombocytopenia. Blood 116, 4307-4316.
212
Ye, X., Zhang, L., Wang, H., Chen, Y., Zhang, W., Zhu, R., Fang, C., Deng, A., and Qian, B.
(2015). The role of IL-23/Th17 pathway in patients with primary immune thrombocytopenia.
PloS one 10, e0117704.
Yeaman, M.R. (2010). Platelets in defense against bacterial pathogens. Cellular and molecular
life sciences : CMLS 67, 525-544.
Yong, M., Schoonen, W.M., Li, L., Kanas, G., Coalson, J., Mowat, F., Fryzek, J., and Kaye,
J.A. (2010). Epidemiology of paediatric immune thrombocytopenia in the General Practice
Research Database. British journal of haematology 149, 855-864.
Youssefian, T., and Cramer, E.M. (2000). Megakaryocyte dense granule components are sorted
in multivesicular bodies. Blood 95, 4004-4007.
Yu, H., Liu, Y., Han, J., Yang, Z., Sheng, W., Dai, H., Wang, Y., Xia, T., and Hou, M. (2011).
TLR7 regulates dendritic cell-dependent B-cell responses through BlyS in immune
thrombocytopenic purpura. European journal of haematology 86, 67-74.
Yu, J., Heck, S., Patel, V., Levan, J., Yu, Y., Bussel, J.B., and Yazdanbakhsh, K. (2008).
Defective circulating CD25 regulatory T cells in patients with chronic immune
thrombocytopenic purpura. Blood 112, 1325-1328.
Yu, S., Ellis, J.S., Dunn, R., Kehry, M.R., and Braley-Mullen, H. (2012). Transient depletion of
B cells in young mice results in activation of regulatory T cells that inhibit development of
autoimmune disease in adults. International immunology 24, 233-242.
Yu, S., Liu, C., Li, L., Tian, T., Wang, M., Hu, Y., Yuan, C., Zhang, L., Ji, C., and Ma, D.
(2015). Inactivation of Notch signaling reverses the Th17/Treg imbalance in cells from patients
with immune thrombocytopenia. Laboratory investigation; a journal of technical methods and
pathology 95, 157-167.
213
Zahn, A., Jennings, N., Ouwehand, W.H., and Allain, J.P. (2006). Hepatitis C virus interacts
with human platelet glycoprotein VI. The Journal of general virology 87, 2243-2251.
Zeller, B., Rajantie, J., Hedlund-Treutiger, I., Tedgard, U., Wesenberg, F., Jonsson, O.G.,
Henter, J.I., and Nopho, I.T.P. (2005). Childhood idiopathic thrombocytopenic purpura in the
Nordic countries: epidemiology and predictors of chronic disease. Acta Paediatr 94, 178-184.
Zhang, B., Lo, C., Shen, L., Sood, R., Jones, C., Cusmano-Ozog, K., Park-Snyder, S., Wong,
W., Jeng, M., Cowan, T., et al. (2011). The role of vanin-1 and oxidative stress-related
pathways in distinguishing acute and chronic pediatric ITP. Blood 117, 4569-4579.
Zhang, F., Chu, X., Wang, L., Zhu, Y., Li, L., Ma, D., Peng, J., and Hou, M. (2006). Cell-
mediated lysis of autologous platelets in chronic idiopathic thrombocytopenic purpura.
European journal of haematology 76, 427-431.
Zhang, J., Ma, D., Zhu, X., Qu, X., Ji, C., and Hou, M. (2009). Elevated profile of Th17, Th1
and Tc1 cells in patients with immune thrombocytopenic purpura. Haematologica 94, 1326-
1329.
Zhang, X., Zhang, D., Liu, W., Li, H., Fu, R., Liu, X., Xue, F., and Yang, R. (2016). Abnormal
lipid rafts related ganglioside expression and signaling in T lymphocytes in immune
thrombocytopenia patients. Autoimmunity 49, 58-68.
Zhang, X.L., Ma, J., Xu, M., Meng, F., Qu, M., Sun, J., Qin, P., Wang, L., Hou, Y., Song, Q., et
al. (2015). Imbalance between CD205 and CD80/CD86 in dendritic cells in patients with
immune thrombocytopenia. Thrombosis research 135, 352-361.
Zhao, C., Li, X., Zhang, F., Wang, L., Peng, J., and Hou, M. (2008). Increased cytotoxic T-
lymphocyte-mediated cytotoxicity predominant in patients with idiopathic thrombocytopenic
purpura without platelet autoantibodies. Haematologica 93, 1428-1430.
214
Zhong, H., Bao, W., Li, X., Miller, A., Seery, C., Haq, N., Bussel, J., and Yazdanbakhsh, K.
(2012). CD16+ monocytes control T-cell subset development in immune thrombocytopenia.
Blood 120, 3326-3335.
Zhou, F., Ciric, B., Li, H., Yan, Y., Li, K., Cullimore, M., Lauretti, E., Gonnella, P., Zhang,
G.X., and Rostami, A. (2012). IL-10 deficiency blocks the ability of LPS to regulate expression
of tolerance-related molecules on dendritic cells. European journal of immunology 42, 1449-
1458.
Zhou, H., Xu, M., Qin, P., Zhang, H.Y., Yuan, C.L., Zhao, H.G., Cui, Z.G., Meng, Y.S., Wang,
L., Zhou, F., et al. (2015). A multicenter randomized open-label study of rituximab plus rhTPO
vs rituximab in corticosteroid-resistant or relapsed ITP. Blood 125, 1541-1547.
Zhu, F., Qiao, J., Cao, J., Sun, H.Y., Wu, Q.Y., Sun, Z.T., Zhao, K., Sang, W., Qi, K.M., Zeng,
L.Y., et al. (2015). Decreased level of cytotoxic T lymphocyte antigen-4 (CTLA-4) in patients
with acute immune thrombocytopenia (ITP). Thrombosis research 136, 797-802.
215
APPENDICES
Appendix I. Definition and grading of bleeding manifestations. Modified from Rodeghiero, F., Michel, M., Gernsheimer, T.,
Ruggeri, M., Blanchette, V., Bussel, J.B., Cines, D.B., Cooper, N., Godeau, B., Greinacher, A., et al. (2013). Standardization of
bleeding assessment in immune thrombocytopenia: report from the International Working Group. Blood 121, 2596-2606.
Type of
bleeding
Definition Grade based on the worst incident episode since last visit
0 1 2 3 4
Skin(epidermis
and dermis)
Petechiae
(does not include
steroid-induced or
senile purpura)
Red (recent) or
purplish (a few days
old) discoloration in
the skin with a
diameter of 0.5-3
mm that does not
blanche with
pressure and is not
palpable
[ ] No [ ] Less than or
equal to 10 in a
patient's palm-
sized area in the
most affected
body area
[ ] Any number if
reported by the
patient
[ ] More than 10
in a patient's
palm-sized area
or more than 5 in
at least 2 patient's
palm-sized areas
located in at least
2 different body
areas,‡ one above
and one below the
belt (in the most
affected body
areas)
[ ] More than
50, if scattered
both above and
below the belt
216
Ecchymoses
(purpuric macule,
bruises, or
contusions)
Flat, rounded, or
irregular red, blue,
purplish, or
yellowish green
patch, larger than a
petechia. Elevation
indicated spreading
of an underlying
hematoma into the
superficial layers of
the skin
[ ] None or
up to 2 in the
same body
area, but
smaller than
a patient's
palm-sized
area, if (a)
spontaneous
or (b)
dispropor-
tionate to
trauma
/constriction
[ ] 3 or more in
the same body
area, but all
smaller than a
patient's palm-
sized area, if (a)
spontaneous or
(b) dispropor-
tionate to trauma
/constriction
[ ] At least 2 in
two different
body areas,
smaller than a
patient's palm-
sized area, if (a)
spontaneous or
(b) dispropor-
tionate to trauma
/constriction
[ ] Any number
and size if
reported by the
patient
[ ] From 1 to 5
larger than a
patient's palm-
sized area, if (a)
spontaneous or
(b) dispropor-
tionate to trauma
/constriction with
or without smaller
ones
[ ] More than 5
larger than a
patient's palm-
sized area, if (a)
spontaneous or
(b) dispro-
portionate to
trauma
/constriction
217
Subcutaneous
hematomas
Bulging localized
accumulation of
blood, often with
discoloration of
overlying skin
[ ] No [ ] 1 smaller than
a patient's palm-
sized area
[ ] Any number
and size if
reported by the
patient
[ ] 2 smaller than
a patient's palm-
sized area,
spontaneous
[ ] 2 smaller than
a patient's palm-
sized area,
disproportionate
to trauma
[ ] More than 2
smaller or at
least 1 larger
than a patient's
palm-sized area,
spontaneous
[ ] More than 2
smaller or at
least 1 larger
than a patient's
palm-sized area,
disproportionate
to trauma
Bleeding from
minor wounds
[ ] No [ ]Lasting ≤5 min
[ ] Any episode if
reported by the
patient
[ ]Lasting >5 min
or interfering with
daily activities
[ ]Requiring
protracted
medical
observation at
the time of this
visit
[ ]Medical
report
describing
patient's
evaluation by a
physician
Mucosa
Petechiae,
purpuric macules,
and ecchymosis
Same as for skin
218
Epistaxis Any bleeding from
the nose may be
anterior or posterior
and unilateral or
bilateral
[ ] No [ ]Lasting ≤5 min
[ ] Any episode if
reported by the
patient
[ ]Lasting >5 min
or interfering with
daily activities
[ ]Packing or
cauterization or
in-hospital
evaluation at the
time of this visit
[ ]Medical
report
describing
packing or
cauterization or
in-hospital
evaluation
[ ] RBC
transfusion
or Hb drop
>2 g/dL
Oral cavity, gum
bleeding
Any bleeding from
the gingival margins
[ ] No [ ]Lasting ≤5 min
[ ] Any episode if
reported by the
patient
[ ]Lasting >5 min
or interfering with
daily activities
[ ]Requiring
protracted
medical
observation at
the time of this
visit
[ ]Medical
report
describing
patient's
evaluation by a
physician
219
Oral cavity,
hemorrhagic
bullae or blisters
Visible raised, thin-
walled,
circumscribed lesion
containing blood.
Each bulla (>5 mm)
is larger than a
vesicle. Bullae,
vesicles, and blisters
should be counted
together as bulla
[ ] No [ ] Less than 3
[ ] Any number if
reported by the
patient
[ ] From 3 to 10
but no difficulty
with mastication
[ ] More than 10
or more than 5 if
difficulty with
mastication
Oral cavity,
bleeding from
bites to lips and
tongue or after
deciduous tooth
loss
Any localized
collection of blood
visible, palpable, or
revealed by imaging.
May dissect through
fascial planes
[ ] No [ ]Lasting ≤5 min
[ ] Any episode if
reported by the
patient
[ ]Lasting >5 min
or interfering with
daily activities
[ ] Interventions
to ensure
hemostasis or
in-hospital
evaluation at the
time of this visit
[ ]Medical
report
describing
interventions to
ensure
hemostasis or
in-hospital
evaluation
220
Subconjunctival
hemorrhage (not
due to
conjunctival
disease)
Bright red
discoloration
underneath the
conjunctiva at onset;
may assume the
appearance of an
ecchymosis over
time
[ ] No [ ]Petechiae/
hemorrhage
partially
involving 1 eye
[ ] Any episode if
reported by the
patient
[ ]Petechiae/
hemorrhage
partially
involving both
eyes, or diffuse
hemorrhage in 1
eye
[ ]Diffuse
hemorrhage in
both eyes
Organ ( and
internal mucosae)
GI bleeding not
explained by
visible mucosal
bleeding or lesion:
hematemesis,
melena,
hematochezia,
rectorrhagia
[ ] No
[ ] Any episode if
reported by the
patient
[ ]Present at the
visit
[ ]Described in a
medical report
[ ]Requiring
endoscopy or
other therapeutic
procedures or
in-hospital
evaluation at the
time of this visit
[ ]Medical
report
prescribing
endoscopy or
other therapeutic
procedures or
in-hospital
evaluation
[ ] RBC
transfusion
or Hb drop
>2 g/dL
221
Lung bleeding [ ] No
[ ] Any episode if
reported by the
patient
[ ]Present at this
visit
[ ]Requiring
bronchoscopy or
other therapeutic
procedures or
in-hospital
evaluation at the
time of this visit
[ ] RBC
transfusion
or Hb drop
>2 g/dL
Hemoptysis [ ] No
[ ]Described in a
medical report
[ ] An
equivalent
episode if
described in a
medical report
Tracheobronchial
bleeding
[ ] No [ ]Medical
report exhibited
by the patient
prescribing
endoscopy or
other procedures
or in-hospital
evaluation
222
Hematuria [ ] No
[ ] Any episode if
reported by the
patient
[ ]Microscopic
(laboratory
analysis)
[ ]Macroscopic
[ ]Described in a
medical report
[ ]Macroscopic,
and requiring
cystoscopy or
other therapeutic
procedures or
in-hospital
evaluation at the
time of this visit
[ ] An
equivalent
episode if
described in a
medical report
[ ] RBC
transfusion
or Hb drop
>2 g/dL
Menorrhagia
(compared with
pre-ITP or to a
phase of disease
with normal
platelet count)
[ ] No
[ ]Doubling
number of pads or
tampons in last
cycle compared
with pre-ITP or to
a phase of disease
with normal
platelet count
[ ]Score >100
using PBAC in
the last cycle, if
normal score in
pre-ITP cycles or
in a phase of
disease with
normal platelet
count
[ ]Changing pads
more frequently
than every 2 h or
clot and flooding
[ ]Requiring
combined
treatment with
antifibrinolytics
and hormonal
therapy or
gynecologic
investigation
(either at this visit
or described in a
medical report)
[ ] Acute
menorrhagia
requiring
hospital
admission or
endometrial
ablation (either
at this visit or
described in a
medical report)
[ ] RBC
transfusion
or Hb drop
>2 g/dL
223
Intramuscular
hematomas (only
if diagnosed by a
physician with an
objective method)
[ ] No
[ ] Post trauma,
diagnosed at this
visit, if judged
disproportionate
to trauma
[ ] An equivalent
episode if
described in a
medical report
[ ]Spontaneous,
diagnosed at this
visit
[ ] An equivalent
episode if
described in a
medical report
[ ]Spontaneous
or post trauma
(if judged
disproportionate
to trauma)
diagnosed at this
visit and
requiring
hospital
admission or
surgical
intervention,
[ ] An
equivalent
episode if
described in a
medical report
[ ] RBC
transfusion
or Hb drop
>2 g/dL
Hemarthrosis
(only if diagnosed
by a physician
with an objective
method)
[ ] No
[ ] Post trauma,
diagnosed at this
visit, function
conserved or
minimally
impaired, if
judged
disproportionate
to trauma
[ ] An equivalent
episode if
described in a
medical report
[ ]Spontaneous,
diagnosed at this
visit, function
conserved or
minimally
impaired
[ ] An equivalent
episode if
described in a
medical report
[ ]Spontaneous
or post trauma
(if judged
disproportionate
to trauma),
diagnosed at this
visit and
requiring
immobilization
or joint
aspiration
[ ] An
equivalent
episode if
described in a
[ ]
Spontaneous
or post
trauma (if
judged
disproportio
nate to
trauma)
diagnosed at
this visit and
requiring
surgical
intervention
[ ] An
equivalent
224
medical report episode if
described in
a medical
report
Ocular bleeding
(only if diagnosed
by a physician
with an objective
method)
[ ] No
[ ] Any post
trauma vitreous or
retinal
hemorrhage
involving one or
both eyes with or
without
impaired/blurred
vision present at
this visit if judged
disproportionate
to trauma
[ ] An equivalent
episode if
described in a
medical report
[ ]Spontaneous
vitreous or
retinal
hemorrhage
involving one or
both eyes with
impaired/blurred
vision present at
this visit
[ ] An
equivalent
episode if
described in a
medical report
[ ]
Spontaneous
vitreous or
retinal
hemorrhage
with loss of
vision in
one or both
eyes present
at this visit
[ ] An
equivalent
episode if
described in
a medical
report
Intracranial
bleeding
intracerebral,
intraventricular,
subarachnoidal,
subdural,
extradural (only if
diagnosed with an
objective method
at the visit or
described in a
medical report
[ ] No
[ ] Any post
trauma event
requiring
hospitalization
[ ] Any
spontaneous
event requiring
hospitalization
in the presence
of an underlying
intracranial
lesion
[ ] Any
spontaneous
event
requiring
hospitalizati
on without
an
underlying
intracranial
lesion
225
provided by the
patient)
Other internal
bleeding:
hemoperitoneum,
hemopericardium,
hemothorax,
retroperitoneal
bleeding, hepatic
and splenic
peliosis with organ
rupture, retro-
orbital bleeding
metrorrhagia etc.
[ ] Any event
requiring
hospitalization
<48 h
[ ] Any
event
requiring
hospitalizati
on >48 h
or RBC
transfusion
or
Hb drop >2
g/dL