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UMI
Regulation of Apoptosis in
CD4-CDS- aBf T Cells
Qasim Khan
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Cellular and Molecular Pathology
University of Toronto
O Qasim Khan
1997
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ABSTRACT
The Regulation of Apoptosis in CD4-CD8- (Double Negative) ap' T Cells
Master of Science, 1997
The Graduate Department of Cellular and Molecular Pathology
University of Toronto
No snidy has exarnined the regulation of apoptosis in CD4TD8- (Double
Negative (DN)) ap' T cells. In this study. a DN ap' T ce11 clone called L1Z (CD8-CD4
1 B2') was used with an in vitro T CeIl Receptor (TCR) cross-linking mode1 to study the
regulation of apoptosis in DN ap* T cells.
Unlike primary activated lB2TD8' T cells, where 70-80% died by apoptosis
upon TCR cross-linking, greater than 70% of L12 cells were resistant to apoptosis. RT-
PCR and FACS@ analysis revealed a low expression of Fas mRNA and Fas surface
expression compared to activated C D ~ ' T cells. Comparable levels of Fas-Ligand (Fas-L)
were expressed by the L12 clone cornpared to CD8' T-cells and sufficient TNF-a was
produced by L12 cells. A lower expression of TNF-Receptors (TNF-Rs), particularly the
MF-R2 isofom. was also found compared to the control L929 fibroblast cells. Western
blot analyses revealed an upregulation of the death-repressing molecules Bcl-2 and Bcl-XL
in the cross-linked cells. In contrat, no change in the level of the death-inducing
molecule Bcl-xs was observed.
Taken together, these results suggest that D ~ a p ' T cells are highly resistant to
apoptosis, possibly due to a defect in the TNF-R family and/or due to an increased
activity of Bcl-2 and Bcl-xL. Further studies are required to elucidate the relationship
between the TNF-R family and the Bcl-2 family.
. . Il
ACKNOWLEDGEMENTS
It is hard to believe that I have spent two years in the Graduate Department of
Pathology. As 1 approach the end of my brief career here, it goes without saying that 1 am
indebted to many people who have helped me dong the way in completing my Master's
degree.
First of all, 1 have to extend my deepest gratitude to my supervisor, Dr. Li Zhang.
1 cannot find any words that would describe how much her guidance, advice, patience.
support and fiiendliness have meant to me. She gave me the opportunity to learn and
improve my skills in basic science research and taught me to appreciate science like a
--real" scientist. Her confidence and belief in me helped me excel in rny work and her
professionalism has inspired me to look upon her as a role mode1 for my future
endeavours.
1 also wish to extend my thanks to my Graduate Supervisory Cornmittee. Drs.
John Chamberlain, Reginald Gorczynski. Thomas Issekutz and Jeremy Squire. Chair of
the Cornmittee, were instrumental in their guidance and support and without whom this
work could not have been completed. 1 thank them for the overwhelming advice, patience
and encouragement they have provided over the last two years. A special thanks goes to
Dr. Gorczynski. without whose constructive criticism 1 would not have become the better
scientist 1 am.
1 would like to give a big thank-you to Barb DuTemple and Liming Yang who
have always been there for me and helped in every way possible. I have learned a lot from
them both and their support and encouragement was always greatly appreciated.
I have made many more friends during my time at the University of Toronto. both
from the Department of Pathology and from the Toronto General Hospital. 1 wish to
thank them al1 (you know who you are) for being able to share in the trials and
tribulations of research and wish them al1 the very best in their hture endeavours.
There are two people who deserve a book of their own but, will have to suffice
with a few words for now. My heartiest thanks go to A n a Kulidjian who has been here
with me from the begiming and whose friendship has only gotten better and stronger. She
always found a way to lift my spirits with her "Christian" ways and made life in CCRW
2-815 al1 the more enjoyable. I am particularly grateful for having such a caring and
generous person in my life and hope she finds happiness in everything she does. Frouz
Paiwand has been a enormous source of faith and wisdom who taught me to always look
on the brighter side of life. Her unconditional support, encouragement and advice have
helped me reach my goals and for that, 1 am etemally grateful. 1 wish her every success
and hope she achieves what she wants from life.
1 would not be where 1 am today if it were not my loving, caring, supportive.
patient. and outright brilliant parents. They always had faith in me and believed in my
abilities when I did not. 1 hope 1 have been able to fùlfil their expectations and I hope 1
continue to instil their pride in me. I love them dearly and thank them for everything they
have done for me.
Last, but not Ieast, al1 this would not have been possible without God's will.
Thank-you God for giving me the strength and determination to succeed and fulfil my
li fklong drearns.
TABLE OF CONTENTS
ABSTRACT ............ ...... .................................................................................................... II
................... ..................................................... ACKNOWLEDGEMENTS ................... III
................................................... .................. LIST OF FIGURES AND TABLES ....... VI
........................ LIST OF ABBREVIATIONS .. ....................................................... VI1
..................................................................... . CHAPTER 1 INTRODUCTION... ....... 1
7 ......................................................................... 1.1.2-Activation-ind~lced Apoptosis in vivo
............................................... ................... . . 1 I 3-Acrivation-lnduced Apoptosis in vitro .... 3
. 2-PATHWAYS AND MOLECULES INVOLVED IN THE REGULATION OF APOPTOSIS .................... 5
............................... I . 2.1.WF. Receptor Fumiiy and fheir Ligands .................................... .... 5
. 1 2 . 1 1 -Fas/Fas-Ligand ............................. .., ................................................................................ 6
............................................................................................ 1.2.1.2-TNF/TNF-Receptor (TNF-R) 9
1.2.2-Bcl-2 Familv ........................ .. .............................................................................. 1 1
............................................................. 1.2.2.1 -Bcl-2 and Bcl-xL: Apoptosis repressing molecules 1 1
......... 1.2.2.2-Bad, Bak. Bax and Bcl-xS: Apoptosis inducing molecuIes ............................ ............ 12
1.7.3-lnterleukin- /P Converting Enzyme (ICE) Family of Prote~ses .................................. 13
.................................................................................................. 1 &DOUBLE NEGATIVE CELLS 2 I
1.3. I - Descript ion ................................................................................................................... 2 1
7 7 I.3.2.Fztnction ......................................................................................................................
......................................................................................................... 1 .+OUR P ~ ~ v r o u s WORK 24
1.4.1 -Description of2C Transgenic Mottse Systern .............................................................. 24
............................................................................. 1.4.2.Donor Specrfic Transfirsion fDST) 3
........................................................................ 1 -5-HYPOTHES~S AND THE AIM OF THIS STUDY 27
CHAPTER 2-MATERIALS AND METHODS ................... ................................... 28
2.I.Generarion and Maintenance of L" Specijic T Cell Clones ......................................... 28
LIST OF FIGURES AND TABLES
FIGURE 1 : SCHEMATIC DIAGRAM OF APOPTOTIC PATHWAY ............................................ 16
FIGURE 2: SURFACE MARKER EXPRESSION OF L 12 CLONE ................................................ 36
FIGCRE 3: RESISTANCE OF DNaPi T CELL CLONE L12 TO TCR-CROSS-LINKING-INDUCED
APOPTOSIS ................................................................................................................... 38
.................. FIGURE 4: TCR CROSS-L~NKNG INDUCED CELL DEATH IS DUE TO APOPTOSIS 40
FIGURE 5 : EXPRESSION OF FAS AND FAS-L BY L 12 CELLS .................................................. 44
FIGUKE 6: ADDITION OF AGONIST ANTI-FAS AB INCREASES CELL DEXTH A N D DECREASES
CELL VIABILITY IN CROSS-LINKED CELLS ..................................................................... 46
FIGURE 7: CROSS-LINKED L 12 CELLS PRODUCE HIGHER LEVELS OF IXF-(r ........................ 51
-* FIGURE 8: ADDITION OF EXOGENOUS TNF-cc DID NOT INCREASE CELL DEATH .................... JJ
- - FIGURE 9: EXPRESSION OF TNF-RECEPTORS ON L12 CLONE .............................................. 33
FIGURE 1 O: EXPRESSION OF BCL-3, BCL-xL AND BCL-xs IN L 12 CELLS .............................. 62
FIGURE 1 1 : ABSENCE OF IL-4 INCREASES CELL DEATH ....................................................... 64
TABLE 1 : A D D ~ I O N OF ..~\NTI.TNF.R 1. XNTI-TNF-R3 OR BOTH ABS TOGETHER DECREASES
................................................................................................................ CELL DEATH 57
TABLE 2 ADDITION OF ANTI.TNF.RI, ANTI-TNF-EU OR BOTH A B S TOGETHER INCREASES
CELL VIABILITY ........................................................................................................... 57
vii
LIST OF ABBREVIATIONS
Ab
Abs
AIA
Con A
crmA
CTL
DD
DED
DISC
DNaP'
DST
FADD
Fas-L
FITC
FLICE
gld
GVHD
ICE
IFN-y
IL
Antibody
Antibodies
Activation-Induced Apoptosis
Concanavalin A
Cytokine Response Modifier A
Cytotoxic T Lymphocyte
Death Domain
Death Effector Domain
Death Inducing Signalling Complex
Double Negative ap' T ce11
Donor Specific Transfusion
Fas-Associating protein with a Death Domain
Fas-Ligand
Fluorescein IsoThioCyanate
FADD-Like ICE
Generalised Lymphoproliferative Disorder
Graft versus Host Disease
Interleukin- 1 P Converting Enzyme
Interleukin
a..
Vl l l
L929
I P ~
mAb
MHC
MLR
PAEU'
RT-PCR
SP
TCR
TGF-P
TNF
TNF-a
TNF-R
TNF-RI
TNF-R.2
TNF-RS
TRADD
Murine Fibroblast Cell Line
Lymphoproliferative disease
monoc~onal antibody
Major Histocompatibility Complex
Mixed Lymphocyte Reaction
poly-(ADP)-Ribose Polymerase
Reverse Transcriptase-Polymerase Chain Reaction
Single Positive
T Ce11 Receptor
Transforming Growth Factor-P
Tumour Necrosis Factor
Tumour Necrosis Factor-a
Tumour Necrosis Factor Receptor
Tumour Necrosis Factor Receptor 1 ( 55 kDa)
Tumour Necrosis Factor Receptor 2 (75 kDa)
Tumour Necrosis Factor Receptors
TNF-R-Associated protein with a Death Domain
CHAPTER 1 - INTRODUCTION
In 1972. Kerr and his colleagues identified the process of programmsd ce11 death
as apoprosis (1). This word, denved from archaic Greek to describe leaves "falling off'
trees. was used to descnbe the ce11 loss that was necessary for the host's survival. More
than twenty-five years later. the process of apoptosis is still being actively studird.
Apoptosis. as opposed to necrosis. is believed to be a regulated event that plays a
large part in the homeostasis of many physiological systems or processes. including
differentiation. development, ce11 maturation. and immunologie function (2.3). .A ournber
of characteristics dishnguish apoptosis from necrosis. These are usually classified into
three events; i) morphological : nuclear condensation. membrane ' blebbing' . c d 1
shrinkage, ii) molecular: fragmentation of nuclear DNA into units of 180-200 base pairs.
by endonucleases. and iii) cellular: protein phosphorylation, changes in the leveis of
intracel lular calcium (4.5). The most widely used ' hallmark' is intemucleosomal
fragmentation which can easily be identified as DNA "ladders" following DNA gel
electrophoresis. The final stages of an apoptotic process include the engulfment of
apoptotic bodies by macrophages, neutrophils and eosinophils (6). This process prevents
the surrounding tissue fiorn being damaged by the potential intracellular contents released
by the dying cell.
1
The process of apoptosis is cntically regulated by the extrinsic and intrinsic
signals received by the ce11 (7.8). Any malfimction in the delicate balance between ce11
proliferation and ce11 dearh c m lead to a number of pathologic conditions. such as
neoplasia. ischemic injury. or neurodegenerative disorders (9). Apart from pathologic
conditions. apoptosis also plays a vital role in the homeostatic regulation of the immune
system. For example. negative selection of T cells in the thymus takes place via
apoptosis. B ce11 development is regulated by apoptosis and peripheral deletion of
activated lymphocytes also takes place by apoptosis (6). Similarly. the immune systern
can be subject to many pathologic diseases baçed on increased or decreased apoptosis.
e-g., AiDS or autoimmune disease. respectively (9).
1.1.2-Activation-Induced Apoptosis in vivo
The peripheral lymphoid organs are home to quiescent T cells that have the
potential to respond to specific antigens. When these cells recognise an antigen, they
proliferate into a cional population of cells that mounts a strong immune response against
the invading antigen. However, when the antigen begins to disappear from the periphery
of the organism, a surplus of activated lymphocytes remain. To re-establish the immune
repertoire following an immune response, most of the activated lymphocytes are triggered
to die by a process terrned Activation-Induced Apoptosis (AIA). This process has been
shown to occur in both CD4- and CD8' T cells in vivo. For example, when bacterial
superantigens are administered, there is an expansion, followed by apoptosis in the
responding cells with the appropriate VP T Ce11 Receptors (TCRs) ( 10-13). Similady.
when allogeneic cells are infused into a recipient. the donor-reactive cells undergo clonal
expansion. and then proceed to die by apoptosis (14.15). In most cases. about 50-80% of
T cells are deleted within one week afier encountering antigen. This process of clonal
deletion, different fiom the clonal deletion seen in negative selection in thymocytes (16).
is thought to occur for several reasons. Apart from re-establishing the immune repertoire,
it is believed to be a mechanism of inducing penpheral tolerance to self and foreign
antigens (17-23) and, more importantly. as a mechanism of eliminating potentially
autoreactive lymphocytes from the periphey (24.25).
1.1.3-Activation-Induced Apoptosis in vitro
Several in vitro systems have been established to mimic the in vivo process of
AIA. Of course, these systems do not completely represent the process of penpheral
deletion but, provide an opportunity to get a bener understanding of the molecular
mechanisrns involved in this phenomenon. The fact that cross-linking the TCRICD3
complex of the T cells using anti-TCRICD3 antibodies causes apoptosis in T cells was
first characterised by Ucker et al. (26). Odaka et al. (27), and Shi et al. (28) using murine
T ce11 hybridomas. Since then, numerous studies have s h o w that activated non-
transformed T cells, both CD4' and C D ~ ' T cells, also undergo apoptosis following
cross-linking of the TCRKD3 complex using the appropriate, imrnobilised antibodies
(29-36). The majority (60-80%) of activated SP T ceils undergo apoptosis within the first
24 hours after cross-linking the TCR.
1.2-Pathways and Molecules Involved in the Regulation of Apoptosis
Many advances have been made over the last few years in understanding some of
the major regulators of apoptosis in activated T cells. It is generally well accepted in the
field of apoptosis that the ultimate outcome of a ce11 will depend on the balance between
the death preventing and death-inducing signals received by the cell. There are three
major families of molecules involved in the regulation of apoptosis. Some of the kry
molecules are discussed below.
1.2.1-TNF-Receptor Family and their Ligands
The Turnor Necrosis Factor Receptor (TNF-R) farnily consists of a number of
t g e I integral membrane glycoproteins that are charactensed by the presence of multiple
cysteine-rich repeats of approximately 40 amino acids in the extracellular amino-terminal
(Cys) domain. There is about 25% homology in the Cys domains of the different farnily
members. Furthexmore, these receptors have their respective ligands that are collectively
known as the TNF family. The M F farnily niembers are al1 type II transmembrane
proteins which display significant sequence hornology with TNF and Lymphotoxin (LT)
a. This sequence hornology is a stretch of about 150 amino acids confined to the carboxy-
terminal (receptor-binding) reg ion of the extracellular domains. Mem bers of the TNF-R
Family include two TNF receptors (TNF-RI (55kDa) and TNF-R2 (75kDa)). nerve
growth factor receptor. CD40. CD27. CDN. 0x40. 4-1BB. and Fas, whereas the TNF
5
Family includes TNF, LT-a and -P. CD40 Ligand (CMOL), CD27L, CD30L. OX40L. 4-
IBBL, and Fas-L (reviewed in (37)). Among these families. Fas/Fas-L and TNF/MF-R
play a critical role in inducing apoptosis of T lymphocytes.
Fas. APO-1 or CD95, is a 36kDa ce11 surface protein that is prirnarily expressed
on activated T cells. B cells, monocytes and neutrophils (38). It was first identified in
1989 by using mouse-derived monoclonal antibodies that induced death in human tunior
ce11 lines (39.40). It was subsequently cloned in humans in 199 1 by Itoh et cd and in micr
by Watanabe-Fukunaga rr al in 1997 (38.41). The structure of Fas is similar to other
members of the TNF-R farnily, which suggested that it was probably a receptor for an
unknown cytokine. This cytokine was initially described in 1993 by Rouvier er ai when a
subline fiom a cytotoxic T ce11 hybndoma (PC60) between a mouse Cytotoxic T
Lymphocyte (CTL) line and a rat lymphoma could kili target cells expressing Fas but.
not target cells which did not express Fas (42). The Fas-Ligand (Fas-L) was purified
from the CTL lymphoma subline and subsequently cloned. first in rats, then in mice and
humans (43-45). It exhibited a 27-28% identity with TNF and was found to be expressed
only on activated. as opposed to resting, T cells (46). Furthemore. the ability of
fibroblast-like COS cells that express recombinant Fas-L on the ce11 surface to induce
apoptosis in Fas-expressing targets suggested that Fas-L was indeed a death factor whose
receptor was Fas (43).
The importance of FasFas-L in the elimination of peripheral lymphocytes was
first observed in Ipr ( lymphoproliferation) and gZd (generalised lymphoproliferative
disease) mice. These mice exhibit spontaneous autosomal recessive mutations on
chromosomes 19 and 1' respectively, corresponding to Fas and Fas-L. respectively
(45,47,48). They develop severe forms of lyrnphadenopathy and splenomegaly. and
produce igG and IgM antibodies. particularly anti-DNA antibody and rheumatoid factor
autoantibody. They are also characterised by an unusual expansion of CD4CD8-B220' T
cells that promote autoimrnunity (-49.50). The main observation that came from these
mice was that the T cells were resistant to activation-induced apoptosis. Russell et ai. and
Bossu et al. have shown that activated. mature T cells from Ipr mice continue to
proliferate following antigenic stimulation in vitro. Russell et al. have also shown the
same phenornenon with T cells fiom gld mice in vitro(51-53). Similady. when
staphylococcal enterotoxin B (SEB) was injected into Zpr mice, the VP TCR espressing T
cells that respond to SEB failed to undergo apoptosis following clona1 expansion (54).
These data together strongly suggest a role for Fas and Fas-L in the deletion of activated
T cells. This has been confirmed by a nurnber of groups using various rnodels studying
activation-induced apoptosis in peripheral cD4' and CD8' T cells and hybridomas (55-
60).
Following these observations, a vast arnount of work has been done to
characterise the mechanisms involved in Fas-mediated death in lymphocytes. As
previously mentioned. Fas and Fas-L are predominantly expressed in activated
lymphocytes. Therefore. when the activated lymphocytes are re-stimulated through their
TCR. the increased expression of Fas-L delivers a death signal to activated T-cells
7
through Fas. This death signal can be delivered in an autocrine or paracrine fashion, Le..
cells expressing Fas-L can bind to Fas expressed on themselves or neighbouring cells
(61). The binding of the trimeric Fas-L cross-links the Fas receptor and induces
trimerization of the receptor. which. in turn transduces an apoptotic signal to the ce113
intemal apoptotic machinery (62). Mutational analyses have shown that the cytoplasmic
domain of the Fas receptor contains a region of about 70 arnino acids that is necessary
and suficient for the transduction of the apoptotic signal. This domain was first identified
by Itoh et al. in 1993 and was called the "death domain" (63). Several intracellular
molecules c m bind to the death domain
FADD (Fas-Associating protein with a
residue protein with a death domain of it
of the Fas cytoplasmic region. One of these is
Death Domain). or MORT1. which is a 208-
s own at the carboxyl terminus (64.65). FADD
binds to Fas via the interactions between the death domains. whereas the arnino terminal
of FADD contains a Death-Effector-Region that transduces signals further downstream.
Similady, many other proteins corne together at the intracellular region of the Fas
receptor to form what is known as a Death-Inducing Signal1
The transduction of this apoptotic signal through the cell's
eventually leads to ce11 death with a11 the morphological ha111
ing Cornplex (DISC) (66).
interna1 signailing cascade
narks of apoptosis- loss of
plasma membrane integrity. cytoplasmic condensation and extensive fragmentation of
chrornosomal DNA into nucleosome units (61). Interestingly, it was found that even cells
that do not have a nucleus could undergo apoptosis upon Fas activation, indicating that al1
the components necessary for the apoptotic signal transduction are present in the
cytoplasm of cells and that Fas activation only triggers this machinery (62).
1.2.1.2-TNF/TNF-Recep tor (TNF-R)
Historically, TNF has been seen as a pleiotropic cytokine, associated with a
variety of biological activities ranging from imrnunoregulation to cytotoxic mediation of
the cellular immune response. It is also involved in antiviral defence but, is most
cornrnonly known as a potent inflarnmatory mediator. Activated macrophages are the
highest producer of this cytokine, but it is also produced by activated T cells. B cells and
Natural Killer (NK) cells (reviewed in (67)). TNF rxerts its effects by binding to two
distinct, high afinity ce11 surface receptors. These two receptors have distinct molecular
masses of S k D a (TNF-RI) and 75kDa (TNF-R2) and were first identified on myeloid
and epithelial cells by cross-linking monoclonal antibodies against the receptors (68.69).
Human isoforrns of the MF-Rs were first cloned in 1990 (70-72), followed by the
murine sequences in 1991 (73.74). The two murine receptors have lirnited sequence
homology (about 20%) in their extracellular regions but. no apparent similarity in their
cytoplasmic portions (73). This suggests that these two receptors, although having a high
affinity for TNF, may mediate different signalling events and produced different cellular
responses (73,75). No intrinsic catalytic activity has been found in the intracellular
domain of the RIF-RI but, deletional mutagenesis of the intracellular region of TNF-RI
has identified an 80 arnino acid domain near the carboxy terminus responsible for
signalling cytotoxicity (76). This region is homologous to the death domain of the Fas
receptor suggesting that TNF-R1 also plays a role in transducing an apoptotic signal. The
TNF-R2 does not display this cytoplasmic death domain but it c m also induce apoptosis
(see below). Similar to Fas, the death domain of TNF-R1 binds to other molecules within
the cytoplasm that also contain a death domain (77.78). One of these molecules is
TRADD. or TNF-RI Associated Death Domain protein (79), which. in turn can bind to
FADD (80,81). This interaction witi FADD suggests the possibility of receptor "cross-
talk" between Fas and TNF-RI further down the apoptotic signal pathway once the signal
has been transduced from these surface receptors (82).
The involvement of TNF and its receptors in penpheral deletion of T lymphocytes
has recently been established. In 1995. Zheng et al. demonstrated that apoptosis was
reduced in mature T cells from TNF-RI deficient mice following TCR stimulation and
the addition of TNF-R-Fc blocking agents. Furthemure, only a Fas-Fc blocking agent
reduced apoptosis in T cells from TNF-R2 deficient and TNF-RIITNF-R2 deficient mice
following TCR stimulation (83). This study also suggested that mature CD8' T cells. as
opposed to CD4' T cells, are eliminated primarily through the TNFIINF-R pathway.
Similarly. Lin et al. have recently s h o w that direct cross-linking of TNF-EU with anti-
TNF-R2 antibodies in vitro induces apoptosis in Con A activated. murine T cells and this
process could be inhibited by CD28 CO-stimulation (84). In vivo studies have also
revealed the importance of MF-Rs in penpheral deletion of activated T cells.
McDevitt's group has shown using Fas deficient (i'pr) mice that peripheral deletion of
activated T cells is still possible and that the injection of anti-TNF antibodies can prevent
this deletion. Furthemore, both pathways need to be suppressed for peripheral deletion to
be inhibited (60). Using TNF-~1"' transgenic mice, Speiser et al. have also shown the
persistence of activated C D ~ ' T cells in the penphery of these mice compared to TNF-
R 1 "' controls where profound lymphocyte deletion was observed (85). Together these in
vivo and in vitro results suggest the importance of both TNF-Rs in mature T ce11
apoptosis.
1.2.2.1-Bc1-2 and B ~ 1 - x ~ : Apoptosis repressing moIecules
Two of the best known intnnsic survival factors expressed in lymphocytes are
Bcl-2 and Bcl-xL. Bcl-2 was originally identified and cloned frorn the breakpoint of a
t(14: 18) translocation present in many human B ceIl lymphomas (86-88). This
translocation fuses the Bcl-2 locus to the immunoglobulin heavy chah gene. resulting in
the overexpression of normal Bcl-2 protein in B cells. The importance of Bcl-2 as a
repressor of programmed ce11 death was established when it was f o n d to be homologous
to the ced-9 death repressor gene found in the nematode Caenorhabtidis eleguns (C:
elegans) (89.90). It is located predominantly in the outer rnitochondrial membrane. the
endoplasmic membrane and the nuclear membrane and has a relative molecular mass of
25-26kDa (91,92). Furthemore, Bcl-2 has been shown to inhibit various forms of
apoptosis, such as ce11 death seen in cultured cells deprived of growth factors (92-94) and
in thymocytes induced by glucocorticoids or y-irradiation (95,96). Western blot analyses
have revealed a high expression of Bcl-2 in resting T cells and have also shown that
activation had no ef5ect on the level of expression (97). This suggested that Bcl-2 plays an
important role in maintaining the survival of resting cells. This was experimentally
demonstrated when resting lymphocytes from Bcl-2 deficient mice were more susceptible
to ceil death than their normal counterparts (98) and that animals deficient in Bcl-2
becarne lymphopenic within several weeks afier birth (99). Due to its mitochondrial
localisation, it has been suggested that Bcl-2 may regulate apoptosis by interfering with
the release of cytochrome c fiom the mitochondria. Cytochrome c is necessary for the
initiation of apoptosis and is elevated in the cytosol during apoptosis. Recently. two
groups working independently have shown that Bcl-3 does in fact block cytochrome c L.
release and prevents the initiation of apoptosis (9 1.100).
Bcl-xL was identified in 1993 by Boise et al. as an altematively spliced prorein
product from the Bcl-x gene (101). Like Bcl-2, BcI-xL is a dominant repressor of
apoptosis (101,103) and is localised in the outer mitochondrial membrane (1 03). it is
3OkDa in size. shares 44% homology with Bcl-2 and is only present in activated T cells,
as opposed to resting T cells (97,101). Upregulation of Bcl-xL, particularly by CD28
costimulation. has been s h o w by several groups to enhance T ce11 survival and
preventing apoptosis ( 10 1.104- 106). The mechanism by which B ~ 1 - x ~ exerts its protective
effect is still not known.
1.2.2.2-Bad, Bak, Bax and Bcl-xs: Apoptosis inducing molecules
Several members of the Bcl-2 family have also been shown to have pro-apoptotic
activities. Amongst these are Bad. Bak. Bax and Bcl-xs that can form heterodimers with
Bcl-3 and Bcl-xL. Various studies have indicated tliat the decision to undergo apoptosis
12
depends on the relative expression of the death-promoting molecules of the Bcl-2
family( 10 1.107- 1 1 1 ). For example, Oltvai et al. have s h o w that Bcl-2 heterodimerizes
with Bax in vivo. Bax is a 2 1 kDa protein that has extensive sequence homology with Bcl-
2. If Bau is overexpressed in an Interleukin (IL)-3 dependent cell line. it will counter the
effect of Bcl-2 and promote apoptosis (108). Similady. Chittenden et al. have shown that
overexpression of Bak induces rapid and extensive apoptosis in serum-deprived
fibroblasts (1 10). Bak and Bax have been s h o w to heterodimerize with B ~ 1 - x ~ ( 1 12).
Bcl-xs was also identified by Boise el al. in 1993 as the srnaller protein product of the
altematively spliced Bcl-x gene (101). An IL-3 dependent ce11 line transfected with BcI-
x~ underwent apoptosis upon IL-3 withdrawal. despite the presence of Bcl-2. This
suggested that. unlike the larger Bcl-xL molecule. Bcl-xs was a dominant inducer of
apoptosis. Together these results suggest that the overall ability of the ceils to survive or
undergo apoptosis depends on the relative expression of these death preventing and
death-inducing molecules.
1.23-Interleu kin- 1 p Converting Enzyme (ICE) Family of Proteases
In the nematode C. elegans, 131 cells undergo apoptosis during somatic
development. Horvitz et ai. identified 3 genes that were involved in the death of al1 13 1
cells. These were identified as ced-3. ced-4 and ced-9 ( 1 13). Ced-9 has already been
described as a ce11 survival and gain of function gene homologous to mammalian Bcl-2.
Ced-4 was required for ce11 death and has no known mammalian counterpart at this time.
Ced-3, on the other hand, was dso required for ce11 death and cloning of this gene
showed that its product is a homologue of mammalian ICE (1 14). ICE was originally
identified as an enzyme that converts the IL-1P precursor to the mature form, which is an
inflarnrnatory cytokine. However. ICE has turned out to be a prototype of a cysteine
family of proteases that have characteristic conserved sequences for substrate binding and
catalysis (62). To date. 11 molecules comprise the [CE family but, new molecules are
being identified at a rapid rate. Recent studies have provided compelling evidence that the
ICE family of proteases are involved in Fas-mediated and TNF-R-mediated apoptosis.
For example. by using specific inhibitors for [CE-like proteases, like the product of c r d .
a cytokine-response modifier gene encoded by cowpox virus, Fas and TNF-R-triggered
death was substantially suppressed (1 15- 1 18) These initial results suggested the role of
ICE-like proteases in apoptosis and since then. a flurry of research has identified the
involvement of a number of key ICE family molecules in Fas and TNF-RI-rnediated
apoptosis ( 1 19.120). Among them. FADD-Like ICE 1 and 2 (FLICE 1 and 2) are
recruited to the DISC and bind to FADD (12L.122). Similarly. crnvl and other inhibitors
were also able to inhibit the activity of the more downstream ICE-like protease CPP32
and its family members (123-125). The CPP32 family of enzymes are involved in
cleaving the nuclear protein poly(ADP-ribose) polymerase (PARP) during the induction
of apoptosis. (125-128). Upon inhibition of this family, Fas-mediated and MF-R1
mediated apoptosis was also suppressed, suggesting that these ICE-like proteases could
be involved further downstream in the Fas and TNF-R apoptotic signalling cascade.
These results together show the complexity of the apoptotic signalling cascade and also
14
provide M e r evidence for the possibility that Fas and TNF-RI-rnediated apoptosis
might eventually converge on a similar death signalling pathway (82.1 19).
Figure 1 is a schematic of some of the molecules discussed and examined in this
study and their relationship to one another. Many pathways still have to be detined but the
simple tiamework between the three major families is illustrated.
Figure 1: Schematic diagram of apoptotic pathway
The three major families involved in apoptosis and their relationship to one another are
illustrated. The top of the page represents the ce11 surface.
Figure 1
DEATH SIGNAL
n T N F n TNF n Fas-L + Fas + TNF-R1 TNF-R.2
Plasma Membrane
FLICE i d 2~.
ICE (-like) Family
CPP32NAMA (-like) Proteases
Mitochondria
Cytoplasm
4-' Nucleus
APOPTOSIS
1.24-Cytokines in Apoptosis
Murine CD4' and CD8' T cells c m be sub-divided into two main groups
depending on their cytokine profile. For C D 4 T cells. they are divided into T-Helper 1
and T-Helper 2 cells (TH1 and TH2) and CDgz T cells are divided into T-Cytotoxic 1 and
T-Cytotoxic 2 cells (Tc] and Tc2). The Type I cells from both groups predominantly
produce Interleukin (IL)-2. Interferon-y ( I M - y ) and lymphotoxin (LT). The Type 7 cells
producr IL-4. IL-5. IL-5. IL-9, I L 4 0 and IL-13. (129). -4s well, cells producing high
levels of TGF-P have been tenned TH3 and Tc3 (130). Whether any of these cytokines
play a rote in apoptosis, particularly activation-induced. remains controversial.
Lenardo and Russell's group have independently shown that IL-2 plays a negative
role in the regulation of mature T cells ( 3 1.13 1 ). Lenardo has shown that CD4- and CD8'
T cells activated in the presence of IL-2 undergo apoptosis after antigen-receptor
stimulation ( 3 1). Similarly Russell's group demonstrated that conversion of naïve. mature
CD4' T cells from an AIA-resistant to a sensitive phenotype required IL-2 (13 1). This
data was recently cornplemented by Parijs et al. and Kneitz et al. who demonstrated that
IL-2 Receptor (IL-2R) deficient T cells and IL-2 deficient mice were resistant to Fas-
mediated AIA. respectively (137,133). IFN-y has also been shown to play a negative role
in T ce11 regulation. Liu and Janeway first drmonstrated in 1990 that anti-IFN-y
antibodies were able to prevent anti-CD3 stimulated death in TH1 clones (29). Similady.
Groux et al. demonstrated that anti-CD3 mediated death of PhytoHaemAgglutinin
(PHA)-stimulated T lyrnphoblasts correlated with IFN-y gene expression and was prevent
by anti-IFN-y antibodies (34). The results for IL-2 and IFN-y together suggested that TkI1
18
type cells were susceptible to activation-induced apoptosis. In fact. two study has s h o w
that TH1 cells are more likely to undergo AM compared to TH2 cells due to a higher
expression of Fas-L (134,135). Because activated T cells express Fas and because IL-?
seems to upregulate Fas-L expression (46). it is possible that TH 1 cells are more sensitive
to apoptosis because they are the cells that produce IL-2. TH^ cells also express Fas on
their surface. but since they are not producers of IL-2. they rnay not upregulate Fas-L
expression. Consequently. they do not receive the death signal from Fas-L and. therefore.
are less susceptible to AIA.
On the other hand. it is possible that the cytokines produced by TH2 rnay play a
role in protecting these cells From apoptosis. One such candidate is IL-4. The majority of
work involving IL-4 and apoptosis h a . been done on B cells. Dancescu et al. have shown
that chronic lymphocytic leukaemic B cells are protected by IL-4 from spontaneous and
hydrocortisone-induced apoptosis, possibly by an upregulation of Bcl-2 (1 36). Similarly.
Rothstein's group has demonstrated that activated B cells can be protected from Fas-
mediated apoptosis by IL-4 ( 1 37). In T cells. IL-4 has been shown to protect Tt{ 1 and Tti2
cells from glucocorticoid-mediated apoptosis (138) but, its role in N A remains unclear.
TGF-P has also been shown to play a protective role against apoptosis. Swain's group has
s h o w that TGF-P protects T112 cells from antigen-induced AIA (1 39). Similarly.
Cerwenka et ai. have recently demonstrated that the presence of TGF-P during PHA-
activation of T cells prevents the T cells fiom undergoing anti-CD3 antibody or Fas-
mediated apoptosis (140). The role of another important cytokine, M F - a , has already
been discussed above. Together these results suggest that some cytokines might be
involved in AIA. but the simple presence or absence of such factors is insufficient to
regulate AIA.
The majority of work done on apoptosis has corne fiom CD4' or CD8- T cells.
There are. however. some TCR- T cells that express neither CO-receptor and are known as
DN T cells. These cells have been largely overlooked in the field of apoptosis. despite
being assigned some crucial functional characteristics. The next two sections deal with
these DN T cells in more detail.
1.3-Dou ble Neeative Cells
The majority of ab-T celis in the periphery of normal mice express the CD4 or
CD8 molecule and are restricted to either the Major Histocompatibility Class (MHC) II or
class I molecule. respectively (141). About 1 4 % of peripheral T cells express neither
CD4 or CD8 on their surface, Le.. they are ap-TCR- and CD4, CD8- (Double Negative
ap' (DNaPT)) (1 42-144). The penpheral DNaP' T cells differ €rom immature DNaP' T
cells found in the thymus. which develop into CD4' or C D ~ ' T cells by positive selection.
The origin of these peripheral D ~ a p ' T cells remains unclear. von Boehmer's group has
shown using transgenic mice that the majority of these cells map undergo negative
selection in the thymus but. some escape negative selection and end up in the periphery
(145.146). On the other hand. Cnspe's group and Fowlkes' group have shown thai these
ce11 do not undergo any selection process in the thymus ( 147.148). Furthemore. Strober's
group has shown that D N ~ P ' T cells c m also anse from the bone marrow via an
ertrathyrnic pathway (149). Regardless of ongin, these penpheral D N ~ P ' T cells sxhibit
a functional TCR and are capable of responding to antigens (1 50- 152).
The percentage of D N ~ P ' T cells is usually expanded in transgenic mice. (134).
For exarnple, von Boehmer's group has s h o w mice with a transgenic TCR For the H-Y
antigen express about 20% of DNaP' T cells in the spleen and up to 70% DNaPt T
cells in the lymph node(146). Sirnilarly. the same group has shown an increased number
of DNuBt T cells in Cochrome C transgenic mice (153). The increased nurnber of
DNaP' T cells in these rnice rnight arise due to an absence of positively selecting MHC
elements or because they are resistant to clonal deletion in the thymus ( 148.150).
The naturally occurrïng DNaPt T cells that exist in normal and transgenic mice
are different from the DNaP' T cells that accumulate as a result of autoimmune disease
in gld and lpr mice with mutations in Fas-L or Fas, respectively (49,154). The DNaP- T
cells found in these mice appear to be single positive cells which have down-regulated
their CD4 and CD8 coreceptors ( 155). The existence of this type of DNaB' T cells has
aiso been shown in some in vitro studies. Erard et al. have shown that the activation of
mature mouse CD8' T cells in the presence of I L 4 switched these cells into DNaP' T
crlls that were non-cytolytic and produced TH2 cytokines ( 1 56,157). This phenornenon
has also been observed in patients infected with HIV- 1. making the host more susceptible
to the infectious agent ( 1 5 7.1 5 8).
The function of DNaP- T cells remains unclear. However, recent evidence has
suggested that these cells may play an important role in regulating immune responses.
First, Strober's group and Sykes' group have s h o w that these cells play an important role
in preventing grafi versus host disease (GVHD) after allogeneic bone marrow
transplantation (143,159-161). The transplantation of single positive CD4 or CD8 T cells
alone induced lethal GVHD. However, the presence of D N ~ P ' T cells in the donor
marrow helped downregulate the alloreactive responses of donor CD4' and CD8' T cells
and prevented GVHD. These DNaP' T cells. found in the bone marrow and spleen. have
been called "natural suppressor" cells ( 1 59). Secondly. Strober's group has also shown
that DNuP* T cells regulate autoreactive responses of single positive T cells in transgenic
mice which othenvise would result in a "lupus-like" autoimmune glomenilonephntis
(162). Clearly. these DNaP- T cells are diffcrent from the DNaP' T ceils found in lpr
and gld rnice that seem to promote autoimrnunity. Lastly. several groups have also shown
that D N ~ B ' T cells may be a potential source of IL-4 during a pnmary response and could
help initiate a THZ response (163,164). These results suggest that DNaP' T cells do have
the potential to be immunoregulatory cells and could promote tolerance induction.
Although DNaP' T cells have been shown to play a role in regulating immune responses.
no study has been done so far to elucidate the regulation of apoptosis in DNuP' T cells.
1.4-Our Previous Work
1 .l. 1-Description of 2C Transgenic Mouse System
The 2C transgenic mouse system has been used in our lab to study the
mechanisms leading to the induction and maintenance of tolerance. The 2C transgenic
mice were originally developed in 1988 by Sha el ( i f . using the a p antigen receptor from
the cytotoxic T lymphocyte clone 2C (165). The ZC clone expresses a TCR reactive
against the L* MHC class 1 antigen. This clonospic TCR of the ZC clone c m be
recognised by the monoclonal antibody (rnAb), 182 (166). 1 B2 recognises both the a and
B chahs of the 2C TCR. The 2C TCR can also be recognised by the F23 . 1 mAb. which
recognises al1 three antigenic determinants of the Vp8 family (167). In the 7C transgenic
mice, the clonotype-positive (1~2 ' ) TCR is expressed on 20-95% of peripheral T cells
and contains one copy of the a transgene and eight copies of the P transgene (165). The
vast majority of these 1 82' T cells are CD4-CDSy and undergo positive selection to K~
(H-zb) molecules (168).
We have used these 2C transgenic mice and backcrossed them ont0 C57BL/6 (86)
mice for 8-10 generations to obtain the transgenes on the B6 (H-2bh) background. The
resultant mice were bred with H-2-dm2 (dm2) mice (a BALBIc L~ IOSS mutant) to obtain
2CFi mice that are H-2", L ~ - , 1 ~ 2 + . These mice were used as recipients. The donor mice
were (BALBk x B6)Fi mice that are H-zb", L ~ ' (169). The advantages of using this
system are two-fold. First, there is only one mismatched antigen, so we know precisely
which immune reaction is taking place. Secondly, the fate of the donor-reactive cells can
be foilowed using the 1B2 mAb. Therefore. this system has allowed us to study the
mechanisms of tolerance induction.
1 A.2-Donor Specific Transfusion (DST)
Recently, our lab has discovered that a single pre-transplant infusion of viable
splenocytes from an L ~ ' donor is able to induce permanent donor-specific skin allograft
survival in 2CFi mice in the absence of any immunosuppression (submitted to BLOOD).
Furthemore. DST led to a deletion of the rnajority (%O%) of donor-reactive ( 1 BTCD8-)
cells in the periphery of the recipient. This deletion occurred by AIA. similar to previous
results obtained by adoptive transfer of viable 1 B2' T cells into SCID mice expressing L~
antigen ( 169). In contrast, no AIA was observed in 1 BTCD8-CD4- cells and 3040% of
the remaining population was I B ~ ' c D ~ - c D ~ - , suggesting that these D N ~ P ' T cells rnay
be resistant to AIA. To further understand the role of DNaP' T cells and how apoptosis is
regulated in these cells, our lab generated D N ~ P ' T cell clones from the remaining
population of the tolerant mice. Two clones. named L2 and L 12. were successfully grown
and maintained from an initial generation of 28 clones and were I B ~ ' C D ~ ' C D ~ - . We
found that these clones were able to inhibit the proliferation of naïve T cells in an antigen
speci fic and dose-dependent manner (L. Yang- unpublished observation), and were
resistant to TCR cross-linking-induced apoptosis. Together, these results suggest that the
remaining DNaP' T cells have a suppressor function and could play a rote in the
induction and maintenance of peripheral tolerance.
1.5-Hvpothesis and the Aim of This Studv
Recent work done by us and others has demonstratcd that DNuP- T cells have a
regulatory function and may play a role in inducing tolerance . Furthermorc. we and
others have also s h o w that these cells. like their CD4' and CD8' counterparts. are
capable of responding to antigen. The mechanisms involved in the deletion of activated
CD4' and CD8' T cells has and is being studied extensively. However. no studies have
investigated the regulation of apoptosis in peripheral D N ~ P ' T cells. Because these cells
possess the capability to be activated. they must also possess a mechanism(s) by which
they are deleted. similar to CD4- and CD8' T cells.
We hypothesise that this DNaB- T ce11 clone utilises pathways similar to those in
mature CD4' and CD8' T cells to regulate apoptosis and that the overall fünction of these
pathways will decide the ultimate fate of these cells. The purpose of this study is to
examine some of the major molecules and pathways involved in apoptosis in peripheral
D N ~ P * T cells, using a T ce11 clone derived fiom skin grafi tolerant mice.
CHAPTER 2-MATERIALS AND METHODS
2.1-Generation and Maintenance of L ~ + Specific T Ce11 Clones
28 T-ce11 clones from 2C mice that accepted skin allograft permanently were
generated by Our Research Fellow. Dr. Liming Yang. Brietly. the spleen cells were
collected from the tolerant mice and cloned using standard cloning and subcloning
procedures. To maintain the clones. 5x10'" clone cells were cultured in a 24-well plate
containing 5x 10' irradiated L ~ ' celk in a-Minimum Essential Media supplemented with
10% Foetal Calf S e m and 40U/rnl IL-2 and IL-4. The cells were incubated at 37°C with
5% CO2. From the initial clones generated, 6 were grown successfully and two (L2 and
L 12) were chosen for further study. T ce11 clones were stimulated in the above manner
every 3-5 days. Viable cells after activation were collected by Ficoll-Hypaque (Pharmacia
Biotech. Uppsala. Sweden) gradient centrifugation.
2.2-MLR Culture of 2CFi Splenocytes
Splenocytes from naive 2CFI were collected and used as responder cells in an
Mixed Lymphocyte Reaction (MLR). 7.5 x 10' cells/ml were CO-cultured with irradiated
(20Gy) sex-matched L ~ ' splenocytes (7.5 x 10' cells/ml) from BYJFi in a-MEM
supplemented with 10% FCS and 30U/ml rIL-2 and rIL-4 as a source of growth factor in
24-weII plates. After a 3.5-day incubation at 37°C and 5% COz, the activated 2CFI
lymphocytes were collected using Lympholyte M (Cedarlane, ON, CANADA) for further
analysis.
2 . 3 4 1 0 ~ Cytometric Analysis
LI2 cells were stained with FITC-labelled IB2 mAb (which recognises the
transgenic TCR: hybridoma kindly provided by Dr. Herman Eisen. MIT) and PE-
conjugated anti-CD8 rnAb (Pharmingen. San Diego. CA). anti-CD4 mAb (Cedarlane.
ON. Canada) and FITC-conjugated anti-CD3 rnAb (Pharmingen. San Diego. CA) to
examine the surface phenotype. BALBk lymph node cells were used as positive controls.
PE-conjugated anti-Fas Ab (Pharmingen, San Diego. CA) was used to examine surface
Fas receptor expression. Hamster anti-mouse anti-TNF-RI (T55-593.4) and anti-TNF-R2
(T75-54.7.14) Abs (kindly provided by Dr. Josef Penninger. AMGEN Institute. ON.
Canada and described in ( 1 70)) followed by anti-hamster FITC-conjugated Ab were used
to examine surface expression of TNF-R. The murine L929 fibroblast ce11 line was used
as a positive control for M F - R staining (kindly provided by Dr. Thomas Issekutz.
University of Toronto, ON, Canada). Data were acquired and analysed on an EPICS@ XL-
MCL flow cytometry machine (COULTER CORPORATION, Miami. Florida).
2.4-Induction of Apoptosis by Cross-Linking of T Ce11 Receptor
The 1 B2 mAb was titrated at 10, 25, 50, 65, 75 and 100pg/ml to find the optimal
concentration for TCR cross-linking-induced death. 65pgIml 1 B2 rnAb was chosen after
it was found that concentrations above 65pe/ml did not induce excess death in the LI2
cells and concentrations below 65pg/ml did not induce maximal death. The lB2 was
diluted in Phosphate Buf3ered Saline (PBS), plated in a 24-well plate and left in I °C
ovemight. Non-specific binding sites were blocked with 1 O%FCS/PBS. Following
activation of the clone and naïve 2C lymphocytes. 1.25 x 10' cells were plated in each
well that was either pre-coated with 1B2 mAb (expenmental) or without 1B2 mAb
(control). Initially. anti-CD4 Ab was used as an isotype control and found to be similar to
media alone. Ce11 viability and death was measured using Eosin exclusion at 2 1. 44. 72
and 96 hours afier cross-linking.
2.5-Detection of Apoptosis by In situ Nick Translation
Apoptosis was also detected at the single ce11 level using the combined techniques
of ceIl surface marker staining and in situ nick translation (171). Cells were collected at
21 and 44 hours and stained for 1B2 and CD8 as described above. Cells were then gently
permeabilised with 0.1 % Triton X- 100 and incubated with Phycoerythrin-conjugated
dATP and DNA Polymerase 1. New DNA is synthesised at the site of DNA breakage and
fluorescent DNA is incorporated at the fragmented sites. Using three-colour flow
cytometry. 1 B2-CD8' T cells that were undergoing apoptosis c m be detected at the single
ce11 level.
2.6-Detection of Fas/Fas-L mRNA levels by RT-PCR
Total RNA was extracted fiom LI2 clone ( 5 x 106 cells) with Tri201 reagent
(G1BCO B U ) . cDNA was prepared from RNA with O.jmg/ml pd(N)6 Random Hexamer
Primer (Pharmacia Biotech, Uppsala, Sweden) and 300 units of murine MLV reverse
transcriptase (GIBCO BRL); 2pi of the cDNA mixture was used in a PCR reaction with
10 pmol of forward and reverse primers as s h o w below and 2.5U of Taq DNA
30
polymerase (GIBCO B U ) . The sequences of the specific sense and anti-sense
oligonucleotide primer pairs were as follows:
Fas ( 1 69): Sense 4'-ATCCGAGCTCTGAGGAGGCGGGTTCATGAAAC-3'
Anti-sense = Y-GGAGGTTCTAGATTCAGGGTCATCCTG-3 '
GAPDH (1 72): Sense = 5'-TGATGACATCAAGAAGGTGGTGAA-3'
Anti-sense = 5'-TCCTTGGAGGCCATGTAGGCCAT-3'
Samples were amplified through 30 (Fas and Fa-L) or 35 (GAPDH) cycles at an
annealing temperature of 59OC in a PCR Thermal Cycler (MJ Research. Watenown.
Mass). The amplified products were separated on a 1.5% agarose gel stained with
Ethidium Bromide. GAPDH was used as an intemal control for RNA integrity.
Supematant was collected from cells three days after activation and 96 hours after
cross-linking and no cross-linking. TNF-a cytokine Ievel was assayed by inhibition of
grow~li of the WEHI 1643 (ATCC) ce11 line as measured by Tritiated Thymidine ( 3 ~ ~ d ~ )
incorporation at 10 hours. The sensitivity of detection was 30pg/ml cytokine as
determined by standardisation using recombinant matenal. The assay was kindly
perfonned by Dr. Reginald Gorczynski. University of Toronto. ON. Canada.
2.8-Western Biot Analysis
L12 clone cells were collected at 21. 44 and 96 hours after treatment with or
without II32 mAb, washed with PBS. pelleted by centrifugation, then lysed using RIPA
buffer (1% Nonidet P-40, 15OrnM NaCl, 50mM Tris-HCl pH 8.0. 5OmM NaF. 2mM
EDTA, 1mM sodium orthovanadate and 0.05% NaN3) supplemented with 0.1%
aprotinin. 0.1 % leupeptin, and 1 mM phenylmethylsulfonyl fluoride (PMSF). Samples
were incubated on ice for 15 min. and then centrifuged at 13.000 g to remove cellular
debris. Protein concentration of the supernatant was determined using the colourimetric
bicinchoninic acid (BCA; BioRad) assay. 10pg of total protein for each sample was
denatured in SDS sample loading buffer and separated by 12% SDS-PAGE. Proteins
were transferred to nitroceliulose membranes and blocked with 5% milk and 0.5%
Tween-20 in Tris Buffered Saline (T-TBS). After blocking, blots were incubated with
anti-bcl-x (1:5000), anti-bcl-2 (1:2000), and anti-p-actin (I:5000) Abs in T-TBS. Blots
were washed in T-TBS and subsequently incubated with horseradish peroxidase-
conjugated anti-rabbit ( 1 : 10000) (Sigma) and anti-mouse (1 : 10000) (BioRad) Abs.
Western blots were developed using the enhanced cherniluminescence (ECL) system
(Amers ham).
2.9-Statistical Analysis
The Student's T-test was used for statistical analysis throughout the study.
CHAPTER 3-RESULTS
3.1-Surface Staining of LI2 Cells
In order to know the phenotype of the L 12 clone. L 12 cells were collected three
days after stimulation and stained with fluorescent labeiled mAbs that recognise the cell
surface molecules CD4, CD8 and the transgenic TCR. BALB/c lymph node cells were
used as positive controls. Figure 1 shows the FACS@ analysis of L 12 cells. L12 is 1 B T
(ap-KR') and CD4-. CD8- (DN) ap'. The staining results of BALBlc lymph node cells
show clear CD4 and CD8 staining. Similar results to L 12 were obtained for the L2 clone
but. only L 12 was used for the present study.
3.2-LI2 Clone is Resistant to TCR Cross-Linking-Induced Apoptosis
Cross-linking the TCR of activated T-cells using monoclonal antibody, either
anti-TCR or anti-CD3. in vitro causes the ce11 to die by a process known as TCR cross-
linking-induced apoptosis (3 1,36,173,174). The response of the L 12 clone to TCR cross-
linking-induced apoptosis was examined using the I B2 mAb which specifically
recognises the transgenic TCR expressed on the L I 2 cells (166). The activated L I 2
( 1 B2'CD8-CD4-) and primary activated 2CFi (1 B~ 'cD~ ' cD~- ) cells were plated in 24-
well tissue culture plates that were pre-coated with 1B2 mAb. .At various time points
(21.44.72 and 96 hours) afier cross-linking, the cells were harvested and ce11 viability was
measured by Eosin exclusion. Figure 2 shows that the primary activated 2CFi cells
exhibited dramatic ceil death (about 60%) within the first 2 1 hours after cross-linking. By
96 hours. only 25% of the cells were viable. in contrat to the 2CF\ cells. the L I 2 clone
was highly resistant to cross-linking-induced ce11 death. No more than 30% of ce11 death
was observed at any time after cross-linking (figure 2). Similar results were found for
another 1 B2'CD8TD4' clone. L2 (data not shown). These data suggest that the DNaP' T
ce11 clones generated fiom mice that perrnanently accepted an allo-skin grafi afer DST
are resistant to cross-linking induced ce11 death.
3.3-Cross-linking of TCR by 1B2 mAb Induces Ce11 Death by Apoptosis
To confirm that ce11 death was due to apoptosis. the in situ nick translation
technique was used. Figure 3 shows that about 65% of the 1~2'CD8' T cells (right side)
were undergoing apoptosis after 21 hours of cross-linking compared to the cells treated
with control anti-CD4 antibody (lefi side). Simiiarly. no apoptosis was drtected in the
control cells after 44 hours, whereas about 35% of expenrnental cells were still
undergoing apoptosis (data not shown). These results confirmed that ce11 death in the
1 B2-treated cells was due to apoptosis.
Figure 2: Surface Marker Expression of LI2 Clone
L I 2 cells were collected after a 3-day MLR with irradiated L ~ ' cells (Ieft) and naïve
lymph node cells fiom BALBIc mice (right) were collected and stained for TCR (either
lB2 or CD3), CD8. and CD4. The data were collected and analysed using flow
cytometry.
Figure 2
1B2lCD3 log fluorescence
CD4 log fluorescence
CDS log fluorescence
Figure 3: Resistance of D N ~ P ' T ce11 clone L12 to TCR-cross-linking-induced
apoptosis
L12 cells (open bar) and naïve 1 B ~ ~ C D ~ ' lymphocytes (closed bar) were stimulated by
L" cells in an MLR. Three days later. viable cells were collected and plated in wells pre-
coated with or without 1B2. The cells were collected at various time points and ce11
viability was measured using Eosin exclusion. The data is shown as viability as a percent
of control (i.e., nurnber of viable cells in 1B2-treated wells as a percent of number of
viable cells seen in non-1B2-treated wells). Data for L 12 clone is the mean of nine
independent esperiments and data for 1 B3'CDB' cells is the mean of three independent
ex periments.
Figure 1: TCR Cross-Linking Induced Ce11 Death is due to Apoptosis
Activated 1 B2+C08' cells were collected 21 hours after TCR cross-linking and stained
for the surface markers lB2 and CD8. Following gentle penneabilisation. cells were
treated with Phycoerythnn-conjugated dATP in the presence of DNA polyrnerasc 1 to
allow DNA synthesis at 'nick' sites. Using three-colour flow cytometry. nick staining was
detected in cells that were cross-linked with 1B2 mAb (right panel), as opposed to cells
treated with anti-CD4 mAb (left panel). Region K ( I B ~ ' c D ~ ' cells) was separately
analysed for the presence of DNA nicks where percentages of apoptotic cells are
indicated. Similar results were obtained at 44 hours (data not show) .
Figure 4
Control Experimental
CD8 Log Fluorescence
Nick Log Fluorescence
3.4-Expression of Fas and Fas-L on L l2 cells
The Fas/Fas-Ligand (Fas-L) interaction is known to be important for the deletion
of activated lymphocytes (55,56.59.175.176). The deficiency of Fas or Fas-L seen in Ipr
and gld mice results in an accumulation of T-cells in the periphery that are autoreactive
and resistant to Activation-Induced Apoptosis (AIA) (45.47). Because our clone was
highly resistaqt to cross-linking-induced apoptosis. we wanted to see if this resistance was
due to a lack of these two molecuies. Using RT-PCR analysis. we found that the level of
F a - L mRNA was similar for L 12 cells and activated 1 B2TD8' cells. but the tevel of Fas
rnRNA was lower in L12 cells compared to the activated IB2'CDS' cells that are
sensitive to TCR cross-linking-induced apoptosis (figure 4a). in addition. we exarnined
the surface expression of the Fas molecule using FACS@ analysis and found a low
expression of Fas on 3-da- srimulated L12 ceils cornpared to activated lB2'CDS' cells
(figure 4b). These results suggest that resistance to cross-linking-induced apoptosis by the
L12 clone was not due to a lack of Fas-L but. may be due to a lowered surface expression
of Fas.
3.5-Induction of Apoptosis by anti-Fas Antibody
Despite the presence of Fas and Fas-L, two possibilities might explain why the
L I 2 cells were highly resistant to apoptosis with regards to these two molecules. One
could be that Fas-L was unable to bind to the Fas receptor to induce a death signal. The
other could be that there was a defect in the Fas receptor and even if Fas-L was binding,
no signal was being transduced. It has been shown that agonist anti-Fas antibodies can
42
induce the death signal via the Fas receptor ( 1 77- 180). Therefore. we added soluble anti-
Fas antibody. that would act like Fas-L. during cross-linking to examine whether the
FaslFas-L pathway was functional in L12 cells. Figure 5a shows that at the eariy time
points until 72 hours, the addition of anti-Fas antibody significantly increased the number
of dead cells seen after cross-linking compared to the control that was not treated with
anti-Fas antibody. A significant decrease in cell viability (about 20%) was also observed
after the addition of anti-Fas antibody (figure 5b). By 96 hours. there was neither a
difference in the number of dead cells nor percent viability between the anti-Fas treated
and untreated cells. Interestingly. the percent viability did not fa11 below 60% afler 44
hours. indicating that this antibody was unable to increase the death rate afier this time
point. Taken together. these results suggest that the FaslFas-L pathway was only partially
responsible for cross-linking-induced apoptosis in these cells.
Figure 5: Expression of Fas and Fas-L by LI2 cells
a- Fas and Fas-L mRNA expression was detected by RT-PCR. Total RNA was extracted
as descnbed previously. GAPDH was used as a positive intemal control for RNA
integrity. Data shown here are from one expenment representative of three
independent experiments.
b- FACS Analysis of Fas surface receptor. Primary activated 1B2'CD8'CD4- cells and
1 B2-CDS-CD4- L 12 cel!s were collected 96 hours after cross-linking and stained with
PE-conjugated anti-Fas antibody. Surface expression was compared to unstained
controls. Data represent staining of at least 3 independent experiments.
Figure 5
Fas
Unstained
.1 1 i 9
Fas log fluorescence
Figure 6: Addition of agonist anti-Fas Ab increases ce11 death and decreases ce11
viability in cross-linked cells
L 12 cells were cross-linked with 1 B2 mAb in the presence (m) or absence (e ) of 1 pg/ml
soluble anti-Fas antibody. Cells were collected at the indicated tirne points and ce11 death
(top panel) and viability (bottom panel) was measured using Eosin exclusion. Viability is
s h o w as a percent of viable cells in 1B7-treated wells over the number of viable cells in
non4 BZ-treated wells. ". p < 0.05; *", p < 0.01 versus I B2 only.
Figure 6
O ! 1 I 1 I I I I O 21 44 72 96
Time After Cross-Linking (hrs)
3.6-Resistance to Apoptosis is Sot Due to a Lack of TNF-a
Many recent studies have shown the TNFITNF-Receptor (TNF-R) pathway to
induce apoptosis in activatrd T-cells (60.83-85). To study if resistance to apoptosis of
L 12 cells was due to a defect in the MFKNF-R pathway, we first examined if L 12 cells
cm produce MF-u after cross-linking. Figure 6 shows that activated L 12 cells (time O)
produce a low level of TNF-a. TNF-u levels rise in both cross-linked and non-cross-
linked cells 21 hours afier cross-linking. However. the level of TNF-a in non-cross-
linked cells falls dramatically by 41 hours but. remains significantly higher in cross-
linked cells. By 96 hours after cross-linking, the level of TNF-a in non-cross-linked cells
retums to basal level. whereas the level of TNF-a production remains significantly higher
in cross-linked cells. Despite this high level of TNF-a production. it was possible that
these cells were resistant to cross-linking induced apoptosis because they were not
producing enough TNF-CY to induce a death signal. Therefore, exogenous TNF-a was
added during cross-linking to see if this would increase ce11 death. The addition of
exogenous TNF-a did not increase the number of dead cells at any time point compared
to the control cells that were not treated with TNF-a (figure 7). These results indicate that
it was not due to an insufficient quantity of TNF-a produced by the L 12 cells that was
preventing them from undergoing apoptosis.
3.7-L12 cells express both TNF-RI and TNF-R2
To test the possibility if L12 cells were resistant to cross-linking-induced
apoptosis because they lacked the MF-RI a d o r TNF-W. the expression of both
receptors was esarnined by flow cytometry. The murine L929 fibroblast ceIl line which
constitutively expresses both forms of the TNF-R (170) was used as a positive control for
both receptors. Using antibodies against the TNF-RI and TNF-R2 isofoms of the TT*IF-R
respectively. we found that the Level of TNF-RI expression on L 12 ceils $vas similar to
that seen in L929 cells (figure 8 - top) but. the expression of TNF-EU was lower than that
seen on L929 (figure 8 - bottom). These results suggest that the L 12 cells may be resistant
to cross-linking-induced apoptosis due to a lower expression of TNF-Rs.
3.8-Both TNF-RI and TNF-R2 rnay play a role in cross-Iinking-induced apoptosis
Because TNF-a and its receptors were expressed by the LI2 cells. we wanted to
see which. if any, TNF-R might be playing a role in cross-linking-induced apoptosis.
Soluble anti-TNF-R1 and anti-RIF422 mAbs that block the RIF-a binding sites on the
MF-Rs were added either alone or together during cross-linking to block their respective
TNF-P.S. Twenty-one houn d e r cross-linking, the addition of anti-RIF-RI rnAb shows
a significant decrease in ce11 death (table 1 ) and increase in ce11 viability (table 2). By 44
hours. anti-TNF-R2 plays a more significant role in preventing ce11 death and increasing
viability compared to anti-TNF-RI. Afier 72 hours, however, both anti-TNF-RI and anti-
TNF-R2 Abs alone significantly decrease ce11 death and increase ce11 viability. No
significant difference was seen after 96 hours of cross-linking with either antibody. These
results suggest that either both recepton are functional and only play a minor role in
cross-linking-induced apoptosis in these cells. or there is a defect in the TNF-Rs on L 12
cells.
Figure 7: Cross-linked Ll2 cells produce higher levels of TNF-a
Supernatant was collected from 3-day stirnulated L12 cells and from wells containing
cells treated with or without IBZ for 21. 44 and 96 hours. TNF-a cytokine levels were
measured by the inhibition of grow-th of the WEHI 1643 ce11 Iine using the ' H T ~ R
incorporation assay. Concentration of TNF-a is expressed as @ml. Data represent one
expenment of two performed.
Figure 7
21 44
Time (hm)
Figure 8: Addition of exogenous TNF-a did not increase ce11 death
L 12 cells were cross-Iinked in the presence (m) or absence (*) of 200pg/ml TNF-a. Ce11
death was measured at the indicated time points using Eosin exclusion and expressed as
number of dead cells/well. Data is an average of three independent expenments.
Figure 8
O 21 44 72 96
Tirne After Cross-Linking (hrs)
Figure 9: Expression of TNF-Receptors on L12 Clone
L 12 crlls (left) were collected at 96 hours afier cross-linking and stained with anti-TNF-
RI (top) and anti-TNF-R2 (bottom) Abs followed by goat anti-Hamster-FITC (dotted
lines). The murine L929 fibroblast ce11 line (right) was used as a positive control for
TNF-RI and TNF-R2 expression. Cells stained with goat anti-hamster-FITC alone (solid
lines) were used as negative controls. Data was analysed by FACS" flow cytomctry and
percent of cells expressing TNF-Rs is indicated. Data is representative of at least two
independent stainings.
Figure 9
FITC
FITC I L
TNF-R1 log fluorescence
.. j..
4 - (II
i œ
TNF-R2 log fluorescence
Table 1: Addition of anti-TNF-R1, anti-TNF-R2 or both Abs together decreases ce11 death
L12 cells were cross-linkrd in the presence or absence of 500ng/well anti-TNF-R1.
500ng/well anti-TNF-EU or both Abs together to block the TNF-RI and TNF-EU
isoforms of the TNF-Receptors. Cells were collected at 21. 14 and 72 hours afier cross-
linking and ce11 death was measured by Eosin exclusion. Data is expressed as the number
of dead cells as a percent of total number of cells I SEM and represents results from t h e
independent expenments. *' p < 0.05 versus control without antibodies.
Table 2: Addition of anti-TNF-RI, anti-TNF-R2 or both Abs togetber increases
ce11 via bility
LI2 cells were treated similar as above afid collected at the same tirne points. Ce11
viability was measured by Eosin exclusion and is expressed as the number of viable cells
as a percent of the total number of cells + SEM. Data represents results from three
independent experiments. *. p < 0.05 versus control without antibodies.
Both TNF-R1 and TNF-R2 Play a Role in L12 Apoptosis
Table 1- Effect of TNF-R Abs on Ce11 Death
Percentage of Dead Cells (%) Treatment Antibody 21 hours 44 hours 72 hours
Control 39*4 1912 36k4
Table 2- Effect of TNF-R Abs on Ce11 Viability - -- -- - -- -. - - . -
Percentage of Viable Cells (%) Treatment Antibody 21 hours 44 hours 72 hours
3.9-Upregulation of Bcl-2 and Bel-xL but, not Bel-%, in 1B2-treated cells
The fate of a ce11 to undergo apoptosis or not is not solely dependent on the death
pathways. It is also influenced by the intrinsic or extrinsic survival signals received by the
cell. One set of intrinsic signals cornes from the Bcl-7 farnily of gene products that play a
dominant role in regulating apoptosis (92.10 1.107.108). Among them. Bcl-2 and Bcl-.uL-
are potent repressors of lymphocyte apoptosis ( 1 02.1 8 1 ). Because these L 1 2 cells were
highly resistant to cross-linking-induced apoptosis. we wanted to know if there was an
increase in Bcl-2 and Bc1-x~ expression in the LI2 cells that was protecting them from
ce!l death. Using Western blot analyses. it was found that Bcl-2 was not constitutively
expressed in L 12 cells compared to what is seen in resting and activated CD4' or CD8' T
cells (97). The level of Bcl-2 was substantially increased at a11 time points after cross-
linking, cornparrd to the non-cross-linked cells (figure 9a). Cells that were not cross-
linked actually down-regulated their Bcl-2 expression. Figure 9b shows that the level of
Bcl-xL is higher in the 1 B7-treated cells at the earlier time points (21 and 44 hours afier
cross-linking) compared to the non-cross-linked cells. By 96 hours, however. the level of
Bcl-xL in the cross-linked cells is comparable to the non-cross-linked cells. These results
together suggest that L12 cells that are not cross-linked might use Bcl-xL for survival but.
if the cells are cross-linked. then the upregulation of both Bcl-2 and Bc1-x~ rnay be
contributing to the high resistance of these cells to cross-linking-induced apoptosis. In
contrast, the level of Bcl-xs, which is the protein product of a smaller. altematively
spliced mRNA of the Bcl-x gene and potent apoptosis inducer (1 Ol), was not changed at
any time point, regardless of cross-linking or not (figure 9c). This suggested that either
Bcl-xs was playing no part in cross-linking-induced apoptosis. or the ratio of Bcl-xs to
Bcl-3 and Bcl-xL favoured the death-preventing molecules. Taken together. these results
suggest that Bcl-2 and B ~ 1 - x ~ may be involved in a dominant fashion to prevent these
cells from undergoing cross-linking-induced apoptosis and that they could be exerting
their effect by over-riding the death signais being induced by the FadFas-L and
TNF/TNF-R pathways.
Figure 10: Expression of Bcl-2, Bcl-xL and Bcl-xs in L12 cells
L 13 cells were collected before (time 0) and 2 1. 44 and 96 hours afier 1 B2 or no 1 B?
treatment and lysed with NP-40. The expression of Bcl-2, Bcl-xL, BcI-xs and p-actin as
an intemal control for protein integrity was examined using standard Western blot
analysis. Data is representative of two independent expenments.
Figure 10
3.10-IL-4 protects against cross-linking induced apoptosis
Numerous studies have s h o w IL-4 to protect against a variety of apoptotic
stimuli. particularly in B celis ( 132.137.138.1 81). Its role in cross-linking-induced T ce11
death is not known. Because the LI2 clone requires the presence of exogsnous IL-4 to
grow and proliferate. and our previous results indicated that apoptosis-resistant T-cells
expressed a high level of IL-4 rnRNA (169). these results suggested that IL-4 might play
a protective role in cross-linking-induced apoptosis. To test this possibility. L 12 cells
were stimulated and grown in the absence of IL-4 for 6 days (two stimulation periods).
The cells were harvested and plated in wells pre-coated with or without lB2. in the
presence or absence of IL-4. L12 cells activated and cross-linked in the presence of IL-4
were used as controls. Figure I Oa shows that as early as 2 1 hours. there was a significant
difference in the number of dead cells seen in the cells activated and cross-linked without
IL-4 (-IL-4.-IL-4) compared to cells activated without IL-4 but. cross-linkrd in the
presence of IL-4 (-IL-4.+IL-4). By 96 hours. there was much greater cell death in the cells
stimulated and cross-linked without IL-4 compared to the cells given IL-4 only during
cross-linking. The ce11 death in cells given IL-4 only during cross-linking was also
significantly higher but. it was not much higher than the ce11 death seen in the control
cells that were activated and cross-linked with IL-4 (+IL-4.+IL-4). Together. these results
suggest that IL-4 plays a protective role against cross-linking-induced apoptosis and it is
only necessary during cross-linking.
Figure II: Absence of IL-4 increases ce11 death
LI2 cells were activated in the absence of IL-4. and then cross-linked in the presence
(-IL-4.+IL-4 : (A)) or absence of IL-4 (-IL-+IL-4 : (W)). Cells were collected at the
indicated time points and ce11 death was measured using Eosin exclusion. Dead cells are
shown as a difference between the number of dead ceIls in the 1 B2-treated wells and non-
IBZ-treated wells. Viable cells are shown as nurnber of viable cells/welI. L I 2 cells
activated and cross-linked in the presence of IL-4 were used as a control (*). Data is an
average of three independent expenments. *, p < 0.05; **, p < 0.0 1 versus control.
Figure 11
9
-t (+IL4,+1L4) ** U L
+ (-1 L 4 , -1 L 4 ) .I œ + (-IL-4,+IL-4)
m œ
- - - œ
m œ
Time After Cross-Linking
CHAPTER 4-GENERAL DISCUSSION
The present study investigated the regulation of apoptosis in penpheral DNaP' T
cells, using the T-ce11 clone. L12. The LI2 clone was generated fiom the penpheral T-ce11
population of 2C a d - l d TCR transgenic mice that permanently accepted an allogeneic
skin grafi afier Donor Specific Transfusion (DST) (submitted to Blood). The clone was
CD4'. CD8'. 1B2- (TCRT), a~' and highly resistant to TCR cross-linking-induced
apoptosis.
We first exarnined if these cells were resistant to cross-linking-induced apoptosis
due to a Iack of two of the major death pathways. FasFas-L and TNF/TNF-R. We found
that both Fas-L and TNF-a were expressed at high leveis but, Fas and the TNF-E22 were
expressed at lower levels compared to activated cells that are sensitive to apoptosis as
shown by us and others. The addition of anti-Fas antibody only decreased ce11 viability by
about 10% in our system. whereas other groups have shown a greater effect by anti-Fas
antibody. For example. both Thompson's and Krarnmer's group have shown that the
addition of soluble anti-Fas antibody to activated T cells caused about 80% apoptosis in
the cells (1 79,180). Similarly, the addition of exogenous TNF-a did not show an increase
in ce11 death of the L I 2 cells. Sarin et al. and Vandenabeele et al. have shown that TNF-a
caused about 60-70% apoptosis in activated T lymphocytes and hybridomas (1 83,184).
These results suggest that these pathways were not exerting a very dominant negative
effect on these cells. Several possibilities could account for this behaviour in the L I 2
cells.
First. it is possible that the level of expression of the surface death receptors was
not high enough to induce an effective death signal. Our results show that the expression
of both Fas and TNF-R. especially TNF-R2. on activated L 12 cells is lower than that seen
on activated 1 ~ 2 ' ~ D 8 ' T cells for Fas and on activated SP CD4' and CD8' T cells, as
s h o w by other groups (84,185). Both MF-RI and TNF-R2 are increased by about 5- 10-
fold in activated cD4' and C D ~ ' T cells compared to naïve SP cells. L 12 cells only
showed a maximum of 2-3-fold increase in both receptors by 96 hours after cross-linking.
So, even though there was a comparable leve! of Fas-L expression on L12 ceils as
cells and there was sufficient TNF-u being produced by the L I 2 cells. it is possible that
the expression of death receptors on the surface of the L12 cells was not sufficient to be
effective.
A second possibility could lie in the signalling pathway further downstream of the
surface molecules. Nurnerous studies have indicated the role of ICE farnily mernbers in
Fa-mediated apoptosis (1 1 5,116,120,123) and there is considerable evidence suggesting
that the TNF-R pathway may also involve some ICE family members ((1 20.186), see ref.
(82) for review). It is possible that the Fasfias-L and TNFlTNF-R pathways in these
DNaP- T cells are not playing a dominant role due to a defect in molecules that converge
on the ICE pathway. Peter et al. have shown that peripheral T cells cultured for one day
are resistant to apoptosis due to a lack of recmitment of FLICE to the CD95 (Fas/APO-1)
death-inducing signalling cornplex (DISC) jl80). This step is thought to be the initial step
in the Fas-mediated pathway and the one that would be most necessary for Fas-mediated
death to be achieved (1 2 1,186). Boldin et al have also been s h o w FLICE to play a role in
RIF-R mediated ce11 death (186). Therefore, it is possible there is a lack of FLICE in the
67
DNaPt T cells, or other ICE family rnembers. e.g. CPP32 (124), that are involved in Fas
and TNF-R rnediated death. However, because we found both the FadFas-L and
TNFiTNF-R pathways were at least partially functional. it is unlikely there is a lack of the
ICE farnily of molecules but. rather. an ineffective formation of the DISC.
Third. it has been shown by several groups that Bci-2 and Bcl-xL can prevent Fas-
and TNF-R-mediated death. For example, Jurkat cells transfected with &LxL become
resistant to anti-Fas Ab-induced apoptosis (1 79). More recently, it was shown that Bcl-x~
exerts its protective effect by preventing the loss of mitochondrial membrane potential
that occurs as a consequence of ICE protease activation by Fas and TNF-R (187).
Similady, Krammer's group has shown that the resistance to Fas-mediated apoptosis is
correlated with an increase in Bcl-xL (1 80). Itoh cl al. have also shown using transfection
studies that Bcl-2 can protect an IL-3 dependent ce11 line and a murine lymphoma from
Fas- and TNF-R-mediated apoptosis (188). The protective effecr of Bcl-2 and B ~ 1 - x ~
against TNF-R mediated apoptosis has also been shown by Vandenabeele et al. and Lin et
al. using transfection studies in hybridomas and activated T cells, respectively (84.184).
Furthemore, extensive studies by several groups have indicated that Bcl-2 and Bcl-xL c m
prevent the ICE cascade fiom being activated and that these two molecules act upstream
of the ICE-like proteases (124,189-191). In o u . studies, we found that Bcl-2 and Bc1-x~
proteins were highly upregulated in the cross-linked cells cornpared to the non-cross-
linked cells and there was no increase in the death-inducing molecule, Bcl-xs. Taking into
consideration the work done by others and assuming that Fas- and TNF-R-mediated
pathways utilise the ICE pathway hlly to transduce the death signal, it is very possible
that Bcl-2 and Bcl-xL together blocked the death signal and prevented the [CE pathway
68
from being activated in these D N ~ P ' T cells. This would explain the high resistance of
this clone to apoptosis but. it still does not fully explain the additional death seen by the
addition of anti-Fas antibody or the increased viability seen by the addition of anti-TNF-R
antibodies.
The answer to this question could lie in some studies that have shown that Fas
mediated apoptosis is not blocked by Bcl-2 and Bcl-xL. Parijs et al. have shown that
activated T cells are not protected by Bcl-xL during Fas-mediated apoptosis (192) and
Zacharchuk's group has shown that transient overexpression of Bcl-2 in murine T blasts
did not prevent Fas-mediated death (1 93). Taken together. these results strongly suggest
that Fas-mediated apoptosis may occur via two distinct pathways. one that utilises the
ICE(-like) protease family and is blocked by Bcl-2 and Bcl-x~, and the other which is not
affected Dy Bcl-2 and Bcl-xL and bypasses the majority of the ICE proteases.
Why would a ce11 possess the capability of triggenng two different pathways via
the same stimulus? The significance of this becomes apparent if the results are
extrapolated to the in vivo physiological level. If a ce11 becomes autoreactive and
potentially dangerous to the host, it would be advantageous for the host if the ce11 utilises
the shorter pathway that is not inhibited by Bcl-2 and Bcl-xL to get deleted. On the other
hand, if the cells are beneficial to the host. like D N ~ P + T cells that have a suppressor
function, then it would be advantageous for the host if the ce11 upregulates Bcl-2 and Bcl-
x~ to block the more downstream ICE-like proteases that are activated by Fas and TNF-R,
e.g. ICE and CPP32. Subsequently, this would prevent the ce11 fiom undergoing apoptosis
and allow the ce11 to survive for longer periods of time in vivo to help promote and
maintain tolerance. However. it rnust be kept in rnind that most of the in vitro work that
69
has shown Bcl-2 and Bc1-x~ to prevent Fas- and TNF-R-mediated apoptosis has corne
fiom snidies where these molecules were overexpressed. In a normal physiological state.
it is unlikely that the levels of Bcl-2 and B ~ 1 - x ~ ever reach the high levels seen in these
artificial systems to block the effect of the death receptors. In this case, the DNUP* T cells
might 'adapta in other ways to become more resistant. For example. they might
downregulate Fas and MF-Rs on their surface so that the death signai is not 'strong'
enough to induce apoptosis and/or is 'weak' enough to be blocked by Bcl-2 and Bcl-XL.
This study also examined the effect of IL-4 on cross-linking induced apoptosis.
The role of cytokines in general in the regulation of apoptosis in D N ~ P ' T cells is largely
unknown but. we particularly chose to study the role of IL-4 for a couple of reasons. First.
our previous data suggested that apoptosis-resistant 1 B2-CD8' T-cells expressed IL-4
mRNA but, no IL-? mRNA (169). Second. the s e m of skin graft tolerant mice
expressed high levels of IL-4 (L. Yang, submitted to Blood). In our system. we found that
the addition of IL-4 during cross-linking protected L12 cells from apoptosis. It is still
unclear, however, how IL-4 plays its protective role. Studies from B-ceils indicate that IL-
4 can protect from Fas-mediated death and can also upregulate Bcl-2 (1 36,13 7). Studies
from T cells have shown that TH2 cells are preferentially protected from apoptosis using
the FadFas-L pathway as opposed to TH1 cells (134.135). It is possible that IL-4 could
prevent the upregulation of Fas-L as mentioned previously, or it could help upregulate the
expression of the deadi-repressing molecules Bcl-2 and Bcl-xL. In our system, we found
that Fas-L mRNA was highly expressed so it is unlikely IL-4 is playing a role in
regulating Fas-L expression in D N ~ P ' T cells. Whether IL-4 affects the expression of
Bcl-2 and Bc1-x~ in our system is not known at this time.
70
In conclusion, a nurnber of factors may be involved in regulating apoptosis in
DNaP' T celis. including the downregulation of ce11 surface expression of death
signalling molecules. such as Fas and TNF-R, and the upregulation of apoptosis-
repressing molecules, such as Bcl-2 and Bcl-xL. Together, these factors would prevent the
death signal fkom being activated m e r downstrearn corn the surface death receptors
and would allow the cells to survive for longer periods of tirne.
Although this study provided us with an introduction to the regulation of
apoptosis in peripherai DN@ T cells, there are still a few limitations that prevented the
study fiom being completely thorough.
One of the biggest limitations of this study was that we were only able to test one
T ce11 clone. It would be ideal to study at least 5-10 more D N ~ P ' T ce11 clones. so that the
results obtained would be more representative of DNaP' T cells. Furthemore. we should
study DNaB' T cells isolated directly from the periphery of mice because we do not know
if the clone is representative of penpheral DNaP' T cells. In addition, al1 the T ce11 clones
we generated were resistant to cross-linking-induced apoptosis. It would have been ideal
if we had obtained a D N ~ P ' T ce11 clone that was sensitive to cross-linking-induced
apoptosis and used it as a control. This would have been a much better control than the
primary activated IBZ'CDS' T cells used in this study.
Another limitation of this study is that we only examined certain mernbers of the
Bcl-2 farnily. As mentioned in the introduction, three other molecules. narnely Bad. Bak
and Bay. also have a death-inducing function. It is possibie that these death-inducing
molecules were not present or that their expression levels also did not change. Therefore,
71
the protein expression Ievel of Bad. Bak and Bax should be checked to see whether these
DNaP' T cells are resistant to apoptosis due to a lack or lower expression of these
molecules.
Apart fiom the TNF-R and Bcl-2 families. one of the most intensely studied areas
in apoptosis is the ICE farnily of cysteine proteases. It has particularly been linked to Fas-
and TnF-mediated death. In our system. the Fas and TNF death pathways were not a
dominant negative factor. This could be because several members of the ICE fmi ly were
absent or because they were prevented from getting activated. It would be interesting to
see if there was a defect in the ICE family in DNaP' T cells. The protein expression
levels of several key ICE family members. e.g. FLICE 1 and 2. ICE and CPP3Z. should be
exarnined as well as their activation levels. Even if the ICE(-like) proteins are present in
their inactive form, they might be prevented from getting activated by the action of
molecules like Bcl-2 and Bcl-xL that act upstrearn of molecules like ICE and CPP32.
Our study also suggests that IL-4 may prevent TCR cross-linking-induced
apoptosis. However, the molecular mechanism underlying this phenomenon is not
known. One possibility would be that IL-4 plays a role in upregulating the expression of
Bcl-2 and B ~ 1 - x ~ proteins. We found that L12 cells stimulated and cross-linked in the
presence of IL-4 upregulated Bcl-2 and Bcl-xL and were resistant to apoptosis. We also
know that cells stimulated and cross-Iinked in the absence of IL-4 were more susceptible
to cross-linking-induced apoptosis. However, we do not know if these cells were more
susceptible to apoptosis due to a decrease in Bcl-2 and B ~ 1 - x ~ protein level. It would be
worth examining the expression levels of these proteins in the absence of IL-4 to see if
this cytokine does play a role in regulating these potent death-repressing molecules. It
72
would also be worth examining the effect of IL-4 on the surface expression levels of Fas
and TNF-Rs. Because we found a lower expression of Fas and TNF-Rs. particularly TNF-
EU. it is possible that IL-4 is down-regulating these receptors to prevent these cells From
being susceptible to Fas-L and TNF-a.
There are a number of other cytokines that were not touched in this study and that
have been shown to play a role in regulating apoptosis. For exarnple, IL-2 and I M - y have
been shown to play a negative role and TGF-P has been shown to play a protective role. It
would be worth checking whether these cytokines play any role in regulating apoptosis in
DN@' T cells, similar to the way IL-4 might. First. we should see if these cells produce
any of these cytokines and second, see if the addition or neutralisation of these cytokines
with antibodies has any effect on apoptosis.
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