dissecting the roles of distinct dendritic cell subsets … · the mattresses” for me. i will...
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DISSECTING THE ROLES OF DISTINCT DENDRITIC CELL SUBSETS IN
THE IMMUNE RESPONSE TO THE INTRACELLULAR BACTERIAL
PATHOGEN, LISTERIA MONOCYTOGENES
By
LATOYA MICHELLE MITCHELL
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES
In Partial Fulfillment of the Requirements
For the Degree of
DOCTOR OF PHILOSOPHY
In the Department of Microbiology and Immunology
December 2009
Winston-Salem, North Carolina
Approved by:
Dr. Elizabeth Hiltbold Schwartz, Ph.D., Advisor
Examining Committee:
Dr. Douglas S. Lyles, Ph.D., Chairman
Dr. Martha Alexander-Miller, Ph.D.
Dr. Purnima Dubey, Ph.D.
Dr. Griffith Parks, Ph.D.
Dr. William E. Swords, Ph.D.
ii
ACKNOWLEDGMENTS
After five long years I have accomplished much more than I ever thought I would
when I started. I have learned so much and have grown even more independent as a
person and a scientist during the last five years. This manuscript is dedicated to those
who paved the way for me to achieve all that I have. First to my parents, Lloyd and
Hallistine Mitchell, who have supported me in whatever I have done, THANK YOU.
You have loved me unconditionally, sacrificed to keep me in great schools, put up with
my moods when I was stressed, prayed for me, and gave some well-needed advice when I
needed direction but may not have known it (or didn’t want it!). Mom, you gave me my
love of books, a shoulder to lean on, and have shown me what hard work, hospitality,
prayer, and selflessness can achieve. Dad, you were my go-to person when I needed a
laugh during a stressful day, instructions on how to fix something I had broken, and a
source of wisdom when I was in a situation I didn’t know how to handle. You two have
always been my biggest fans, and for that I am eternally grateful. I love you both!
To my relatives and loved ones – my brother Derrick, my niece A’Deijsha,
Grandma Ethel, Aunt Bev, my cousin Kathryn and numerous other aunts, uncles, and
cousins – thank you for your prayers, phone calls, emails, text messages, Facebook
messages, cards, and gifts in the mail, especially as the finish grew nearer and my stress
levels were mounting high. To “The Fam” – Tiffany, Reeko, and Mike – what would my
last two years of grad school have been, if not for you? A huge thank you for Football
Sundays, Soul Food, chats, Old School Saturdays, birthdays, Friends Football Weekends,
and Candy Bowls I and II. I look forward to all that the future holds.
iii
To Furman University and all of the faculty and staff that supported and
encouraged me, trained me, and challenged me to go to the next level. Furman has
become my home away from home and will always hold a special place in my heart. I
look forward to giving back to the Furman community in the years to come. GO
PALADINS!!
Thank you to my WFU friends over the years who kept me sane with cookouts,
tailgates, game nights, happy hours, group runs, wine tastings, and movie outings–
Cheraton Love, Dr. Candace Wilson, Dr. Elizabeth “Joy” Akins, Nicole Beauchamp and
Chris Dowd, Dr. Keith Keene, Dr. Josh Lewis, Dr. Jerry and Alicia Saunders, Dr. Monica
Hall-Porter, Dr. Yuval Silberman and Dr. Amy Arnold, Stacy Reeves, Dr. Rhea Busick,
Beth Holbrook, Amanda Wibley Brown, Caitlin Mattos Briggs, Dr. Keith Keene, and Dr.
Monica Hall- Porter. Thank you to my classmates who helped me get through the years
of classes, tests, AT, complaining, and seminars and who I’m sure will go on to do
GREAT things with their degrees – Dr. Ashley East, Dr. Ryan Michalek, Elizabeth
Waligora, Anne Stanner Cook, Dr. Neelima Sukumar, and Dr. Gena Jester Nichols – We
were (and will forever remain) this department’s “Magnificent Seven.”
Thank you to my lab mates, past and present– Dr. Curtis Henry, Kristina Brzoza-
Lewis, Dr. Marlena Westcott, Anne Cook, and Gunjan Arya – for your support,
experimental help and know-how, paper editing skills, general happiness, singing ability,
and love of gummy bears in the lab when I needed it the most. Thank you especially to
Curtis for your well-timed advice and also to Kristi and her husband, Dr. Eric Lewis, for
allowing me to crash at their place when I was homeless my final month.
iv
Thank you to the Sorors of the Winston-Salem Alumnae Chapter of Delta Sigma
Theta Sorority, Inc. for your support in the last five years. All of you have special places
in my heart and I carry with me fond memories of the times in the Coliseum Concession
Booth #103, sorority meetings, and May Week and Founders Day celebrations. You
have shown true Delta love and spirit in making sure I was doing well while I was in
school.
To my church family at Emmanuel Baptist Church, Pastor Mendez, and Mrs.
Elizabeth Fields, thank you for giving me guidance, perspective, solace, and a place to
worship and call home over the last 5 years. Prayer availeth much and joy comes in the
morning. I am truly going to miss communing with you as I move on.
To my mentor who supported my research and allowed me to become the more
independent researcher that I am today, thank you Dr. Elizabeth Hiltbold Schwartz aka
“Dr. H.” You always encouraged me to keep my focus on what was important, pushed
me to take a really good look at my data before moving on to the next experiment, and
guided what route I took next. Thank you for the pep talks and laughs as well.
Thank you to Dr. Griffith Parks for recruiting me to the department back in
2003/2004 as well as the office chats we have had as my project has progressed. Our
little talks really helped and showed that you cared about my well-being as a graduate
student and also taught me that “it’s not the circumstances that are the problem; it’s how
you react!” Thank you to Dr. Jason Grayson for the conversations we had during
numerous mouse infections and even more importantly the fresh insight into my project.
I also appreciated the career advice when I really needed perspective. To Dr. Ed Swords,
v
thank you for your constant encouragement that I could go farther than I imagined.
Thank you for also having my back when it counted the most and being willing to “Go to
the Mattresses” for me. I will forever remember and appreciate what you have done for
me. Thank you also to the members of the Immunology Group (in particular Dr. John
Bates and the Alexander-Miller lab) for their input in my research during group meetings
as well as reagents, advice, and protocols when I needed them… many times last minute!
Thank you to Tina, Mary, Mandy, Joyce, and Nancy in the Micro/ Immuno office for
smiling, saying hi every morning, and the encouragement during my marathon training.
And last but definitely not least, I would not have been able to do this without
knowing that my heavenly Father was there for me the entire time and no matter what I
was going through, dealing with, or stressing about, I had Him to go to, anytime,
anyplace. His love has kept me through the ups and downs, joys and pains and more joys
of the last five years. I truly “…never would have made it without You.”
vi
Do not let anyone look down on you because you are young,
but be an example for other believers
in your speech, behavior, love, faithfulness, and purity.
~I Timothy 4:12
God grant me the serenity
to accept the things I cannot change;
courage to change the things I can;
and wisdom to know the difference.
~Reinhold Niebuhr
“Yes We Can.”
~President Barack Obama
vii
TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ………………………………………………………...….. ii
LIST OF ILLUSTRATIONS ……………………………………………………………... ix
LIST OF ABBREVIATIONS USED ……………………………………………………… xii
ABSTRACT …………………………………………………………………………... xvii
CHAPTER I. INTRODUCTION ……………………………………………………… 1
DENDRITIC CELLS …………………………………………………... 1
DENDRITIC CELLS SUBSETS IN THE SPLEEN…………………………. 2
IN VITRO DERIVED BONE MARROW DENDRITIC CELLS …………….... 9
LISTERIA MONOCYTOGENES (LM) …………………………………….. 11
T CELL RESPONSES TO LM ………………………………………….. 16
THE ROLE OF PATTERN RECOGNITION RECEPTORS IN
SENSING LM …………………………………………………………. 17
THE SPLEEN AND DC IMMUNITY TO LM …………………………....... 22
SUMMARY TO INTRODUCTION ………………………………….......... 27
CHAPTER II. MATERIALS AND METHODS ………………………………………..... 28
CHAPTER III. IMMUNE RESPONSES OF IN VITRO- DERIVED DENDRITIC CELL
SUBSETS TO AN INTRACELLULAR BACTERIAL PATHOGEN,
LISTERIA MONOCYTOGENES ………………………………………….. 37
viii
PAGE
CHAPTER IV. IMMUNE RESPONSES OF SPLENIC DENDRITIC CELL SUBSETS TO
AN INTRACELLULAR BACTERIAL PATHOGEN,
LISTERIA MONOCYTOGENES …………………………………………... 60
CHAPTER V. DISCUSSION …………………………………………………………. 92
MODEL …………………………………………………………….... 94
APPENDIX ……………………………………………………………………...…… 105
REFERENCES ……………………………………………………………………….. 108
SCHOLASTIC VITAE ……………………………………………………………….. . 128
ix
LIST OF ILLUSTRATIONS
CHAPTER I
PAGE
FIGURE 1. DENDRITIC CELL SUBSETS ………………………….…………………..... 4
FIGURE 2. THE LIFE CYCLE OF LISTERIA MONOCYTOGENES …………………………... 14
FIGURE 3. CELL SURFACE, ENDOSOMAL, AND CYTOPLASM PATTERN
RECOGNITION RECEPTORS ……..……………………………………………. 19
FIGURE 4. SPLENIC ARCHITECTURE …………………………………………………. 24
CHAPTER III
FIGURE 5. GM-CSF DC AND FLT3L DC EXPRESSION OF COSTIMULATORY
MOLECULES AND CYTOKINES IN RESPONSE TO L. MONOCYTOGENES …………. 41
FIGURE 6. THE ROLE OF MYD88 AND TRIF IN SENSING LM BY FLT3L DC IN
COSTIMULATORY MOLECULE EXPRESSION AND CYTOKINE SECRETION ….…… 44
FIGURE 7. CD11B+ DC WITHIN THE FLT3L DC CULTURES ARE THE MAJOR
RESPONDERS TO LM INFECTION IN COMPARISON TO B220+ FLT3L DC …….… 49
FIGURE 8. CD11B+ DC WITHIN THE FLT3L DC CULTURES ARE MORE CAPABLE
OF PRIMING NAÏVE, ANTIGEN-SPECIFIC T CELLS THAN B220+ FLT3L DC ….… 52
FIGURE 9. CD11B+ DC WITHIN THE FLT3L DC CULTURES ARE PREFERENTIALLY
INFECTED WITH LM THAN B220+ FLT3L DC ………………………………… 55
x
PAGE
FIGURE 10. WHEN LM INFECTION IS NORMALIZED, B220+ DC ARE COMPARABLY
ABLE TO CD11B+ DC IN PRIMING NAÏVE, ANTIGEN-SPECIFIC T CELLS AND
INFECTED FLT3L DC INCREASE THEIR SURFACE COSTIMULATORY
MOLECULE EXPRESSION ……………………………………………….…….. 58
CHAPTER IV
FIGURE 11. SPLENIC DC SUBSET GATING STRATEGY ………………………….…….. 64
FIGURE 12. SPLENIC DC SUBSET DISTRIBUTION SHIFTS AFTER 48 HOURS OF
INFECTION ……………………………………………………………….…... 66
FIGURE 13. SPLENIC DC SUBSET UPREGULATION OF COSTIMULATORY MOLECULE
EXPRESSION IS DEPENDENT UPON CYTOSOLIC ENTRY BY LM AND PEAKS
AT 24 HOURS POST INFECTION ……………………………………….…..…… 69
FIGURE 14. BACTERIAL CFU IN THE LIVER …………………………………....…….. 72
FIGURE 15. SPLENIC CD8α+ AND DNDC BOTH CONTAIN LIVE LM FOLLOWING
WILD-TYPE INFECTION ……………………………………………………...... 74
FIGURE 16. SPLENIC DC DO NOT PRIME ANTIGEN SPECIFIC CD4 T CELL
RESPONSES TO LM INFECTION EX VIVO ………………………………………… 77
FIGURE 17. SPLENIC CD8α+ DC PRIME NAÏVE, ANTIGEN-SPECIFIC CD8 T CELL
PROLIFERATION AND IFN-γ PRODUCTION EX VIVO THAT IS DEPENDENT UPON
CYTOSOLIC ENTRY BY LM ……………………………………………….……. 79
xi
PAGE
FIGURE 18. LM IS ASSOCIATED WITH CD11B+ CELL CLUSTERS
IN T CELL ZONES OF THE SPLEEN DURING WILD-TYPE LM INFECTION
WHILE VACUOLE-RETAINED LM IS FOUND IN THE MARGINAL ZONES …………. 82
FIGURE 19. QUANTIFICATION OF CD11B-LM CLUSTERS …………………………….. 85
FIGURE 20. DC SUBSETS ARE FOUND IN BOTH THE MARGINAL ZONES AND
T CELL ZONES OF THE SPLEEN FOLLOWING BOTH WILD-TYPE LM AND
VACUOLAR LM INFECTION …………………………………………………… 87
FIGURE 21. CD86 EXPRESSION OF WILD TYPE LM INFECTED SPLEENS VS. VACUOLAR
LM INFECTED SPLEENS ………………………………………………………. 90
CHAPTER V
FIGURE 22. MODEL OF HOW IMMUNITY AGAINST LM IS FORMED ……………………. 94
APPENDIX
TABLE 1. TLR2, TLR4, TLR2/9, TLR7, IL-1R, CASPASE 1, AND
IFNαR ARE NOT REQUIRED IN SENSING LM BY FLT3L DC .…………………... 105
FIGURE 23. CYTOSOLIC ENTRY BY LM OCCURS IN BOTH CD11B+ AND
B220+ FLT3L DC ……………………………………………………………. 107
xii
LIST OF ABBREVIATIONS
Ab Antibody
Ag Antigen
APC Allophycocyanin
APC Antigen Presenting Cell
BrdU Bromodeoxyuridine
BSA Bovine Serum Albumin
CA Central Arteriole
CARD Caspase Recruitment Domain
cDC Conventional Dendritic Cell
CFSE Carboxyfluorescein Succinimydl Ester
CFU Colony Forming Units
CTL Cytolytic T cell
DC Dendritic Cell
DNA Deoxyribonucleic Acid
DNDC Double Negative Dendritic Cell
ELISA Enzyme Linked Immunosorbent Assay
xiii
ERAAP Endoplasmic Reticulum Aminopeptidase
Associated with Antigen Processing
FITC Fluorescein isothiocyanate
FIRE F4/80-like receptor
Flt3L Fms-like Tyrosine 3 Kinase Ligand
g Gram
GFP Green Fluorescence Protein
GM-CSF Granulocyte and Macrophage Colony Stimulating
Factor
Hly Hemolysin
hpi Hours Post Infection
IFN Interferon
IFNαR Type I Interferon Receptor
i.g. Intragastric
IL-1β Interleukin 1 Beta
IL-12p40 Interleukin 12, p40 Subunit
IL-6 Interleukin 6
InlA Internalin A
xiv
InlB Internalin B
i.p. Intraperitoneal
IRF Interferon Regulatory Factor
i.v. Intravenous
LLO Listeriolysin O
Lm Listeria monocytogenes
LPS Lipopolysaccharide
LSCM Laser Scanning Confocal Microscopy
MFI Median Fluorescence Intensity
MHC Major Histocompatibility Complex
µg Microgram
µl Microliter
µM Micromolar
mg Milligram
ml Milliliter
mRNA Messenger Ribonucleic Acid
MyD88 Myeloid Differentiation Factor 88
xv
MZ Marginal Zone
NLR NOD-like Receptor
ng Nanogram
NOD Nucleotide Oligomerization Domain
OCT Optimal Cutting Temperature
OVA Ovalbumin
PALS Periarteriolar Lymphoid Sheath
pDC Plasmacytoid Dendritic Cell
PE Phycoerythrin
PerCP Peridinin Chlorophyll Protein Complex
pg Picogram
PI-PLC Phosphatidylinositol Specific Phospholipase
PC-PLC Broad range Phospholipase
PRR Pattern Recognition Receptors
RP Red Pulp
TLR Toll-like Receptor
TRIF TIR-Domain Containing Adapter Inducing
Interferon
xvi
TNFα Tumor Necrosis Factor Alpha
WP White Pulp
xvii
ABSTRACT
Dendritic cells (DC) are vital to the development of protective immunity to many
pathogens and link the innate and adaptive immune systems. While DC as a whole have
been studied extensively and are widely known for their ability to prime naïve T cells,
there is much yet to be discovered about DC biology and function in responses to
pathogens. DC are composed of several subsets that have distinct repertoires of pathogen
sensing molecules and functions. Four subsets of DC have been identified in the spleen
and at least two DC subsets can also be generated in vitro from mouse bone marrow
using the cytokine, Fms-like tyrosine kinase 3-ligand (Flt3L). We therefore set out to
determine how distinct DC subsets respond and contribute to immune response against
the intracellular bacteria pathogen, Listeria monocytogenes (Lm).
We observed, in contrast to previous studies, that in Flt3L DC maturation
responses were independent of cytosolic entry by Lm, and responses to vacuolar Lm were
dependent on the TLR adapter protein MyD88. The CD11b+ DC population within Flt3L
DC had a higher level of maturation to Lm and uptake capacity than B220+ DC within
Flt3L DC. While CD11b+ DC were capable of priming naïve T cells, B220
+ DC were
also capable of priming T cells when the level of infection was normalized to that found
in the CD11b+ cells.
In contrast to Flt3L DC, CD8α+ DC, CD4
+ DC, and pDC subsets of the spleen
following infection with wt Lm increased costimulatory molecule expression but
responded minimally to vacuolar Lm. The DC maturation to wild type Lm peaked at 24-
48 hours post infection. We have also shown that the CD8α+ DC subset, and to a lesser
xviii
extent, CD4+ DC are capable of priming CD8 T cell responses. This was reflective of
both the maturation level and bacterial load of the CD8α+ and CD4
+ DC subsets. We also
observed that wild type Lm was found clustered with CD11b+ cells in the periarteriolar
lymphoid sheath while vacuolar Lm was found in the marginal zones of the spleen. With
this work we have developed a model of DC responses to Lm that dissects the ability of
DC to take up Lm, undergo maturation, and then prime protective T cell responses.
1
CHAPTER I
INTRODUCTION
DENDRITIC CELLS
Dendritic cells (DC) are phagocytic APC that link the innate and adaptive
immune systems. DC are particularly important for their ability to activate naïve T cells,
resulting in their clonal expansion, cytokine production, and differentiation into
functional effector and memory T cells. As a whole in mice, DC express the marker
CD11c at intermediate to high levels and are made up of several subsets. These DC
subsets also express distinguishing cell surface markers and have been attributed distinct
functions in immune responses to pathogens. DC are differentiated in the bone marrow
as well as, upon activation, in the tissue from monocytes. DC are found in the periphery
in organs such as the skin, lung, spleen, and liver, circulating in the blood, as well as in
lymphoid tissues such as the thymus and lymph nodes. Circulating “classical” DC are
found in an immature state, sampling their environment for pathogens and non-self and
are highly phagocytic. These DC also express low levels of surface costimulatory
molecules, chemokine receptors, and secrete low levels of proinflammatory cytokines. In
this state, DC are poor activators of naïve T cells. However, upon pathogenic encounter,
DC undergo a process called maturation whereby DC downregulate their phagocytic
capacity, upregulate their costimulatory molecule, cytokine, and chemokine receptor
expression, and begin to home to the nearest lymph node to interact with and activate
naïve T cells.
2
There are also resident DC found in the spleen, liver, and lymph nodes that do not
circulate in the periphery. These resident DC under non-inflammatory conditions make
up approximately 80% of the DC found in the lymph node and 100% of splenic DC (2).
In the spleen following pathogen encounter, resident DC also exhibit increased
costimulatory molecule expression, cytokine secretion, and specific chemokine receptors
to migrate from the marginal zones, which filter the blood into T and B cell zones. There
the DC will form conjugates with T and B cells for their activation.
DENDRITIC CELL SUBSETS IN THE SPLEEN
Resident DC constitute approximately 1-2% of the cells found in the resting
spleen, including T and B cells, monocytes, macrophages, and red blood cells. There
have been four different splenic subsets of CD11c+ DC described throughout the
literature (2, 54, 71, 81) (Fig. 1). These subsets have been broadly categorized into two
subtypes – conventional DC (cDC; approximately 80% of splenic DC) and plasmacytoid
DC (pDC; approximately 20% of splenic DC). CDC are made up of CD8α+CD4
-DC,
CD4+CD8α
- DC, and DC which are double negative for both CD8α and CD4 (DNDC),
while pDC are CD45R/B220+Ly6C
+. Each of these DC subsets has been attributed
different functions, which are a result of their antigen uptake, processing, and
presentation capacities. The cDC have been found in both the marginal zones as well as
the periarteriolar lymphoid sheath (PALS) of the spleen (50, 81).
The first conventional DC subtype to be discussed is the CD8α+ DC, which has
been demonstrated as important for uptake of apoptotic cells and the ability to cross-
present antigen. The factor IRF8 has been shown to be required for CD8α+ DC
3
Figure 1. Dendritic Cell Subsets. Dendritic cells (DC) are composed of several subsets
that all express CD11c. DC subsets can be isolated from the mouse spleen (among other
organs) and also generated in vitro by treating mouse bone marrow with the cytokines
GM-CSF or Flt3L. Resident splenic DC subsets include the CD8α+ DC, CD4
+ DC, DC
that are double negative for CD8α and CD4 (DNDC) and plasmacytoid DC (pDC) which
are B220+. GM-CSF DC are correlates of classical, inflammatory DC and express
CD11b. Flt3L DC are representative of two resident, splenic DC subsets (cDC and pDC)
and express CD11b+ or B220
+.
4
5
(as well as pDC) development in that IRF8-/-
mice lack CD8α+ DC (4, 96, 111). Found in
both the marginal zones and the inner white pulp of the spleen (50, 74, 89), CD8α+ DC
express DEC-205, a c-type lectin which is important for endocytosis and antigen uptake
(52, 76), CD103, and CD207 or Langerin (50, 81, 92). CD8α+ DC are enriched for the
mRNA and protein expression of MHC Class I processing components, including Tap1,
Tap2, calreticulin, calnexin, Sec61, ERp57, ERAAP, and cystatins B and C, and have
been shown to efficiently process and present antigen as well as be able to cross-present
antigens to CD8+
T cells via Class I (28, 33). These DC, more recently, have been shown
to have a higher endosomal pH than CD8α- DC, which aids in their ability to cross-
present antigens (95). This DC subtype has also been shown to produce large amounts of
IL-12p70 (59, 77), which is important for driving Th1 responses and enhancing effector
and memory CD8+ T cell responses (23, 47, 77, 78, 121) . Taken together, these studies
indicate that CD8α+ DC are particularly adept at antigen uptake, processing, and
presentation via the Class I pathway to cytotoxic T cells, and thus combating intracellular
pathogens. As such, CD8α+ DC are currently the target for several DC vaccination
studies, by targeting the antigen of choice to the marker DEC-205 or CD207 (20, 33, 50,
88).
CD8α- DC are found in both the red pulp and the marginal zones of the spleen
and are thought to traffic to the T cell zones upon antigen encounter (33, 74). These DC
make up approximately 75% of the cDC populations in the spleen and 20% of the cDC in
the LN (74). The CD8α- DC express the marker 33D1 and have been demonstrated to
have higher mRNA and protein expression levels of MHC Class II molecules than CD8α+
DC to efficiently process and antigen from extracellular pathogens to CD4+ T cells (33).
6
While several studies do not differentiate between the CD8α- DC subtypes seemingly due
to functional similarities as well as mRNA expression profiles (35, 74), the CD8α- DC
contain two DC subsets that differ in their expression of CD4 (CD4+ DC and CD4
- DC or
DNDC). While DNDC lack expression of both CD8α and CD4, they are not a precursor
DC to either CD8α+ DC or CD4
+ DC – or vice versa – and are distinct from both CD8α
+
DC and CD4+ DC in their gene expression profile (35, 54, 86). Comparison of BrdU
incorporation in CD8α+ DC, CD4
+ DC, and DNDC in vivo have demonstrated that all
three DC subtypes have a rapid turnover, half life of 1.5-3 days, and are developmentally
distinct (i.e. there is no lag in BrdU incorporation as would be expected if one population
gave rise to another) (54). Additionally, transfer experiments of CD8α- DC from Ly5.2
mice into non-irradiated Ly5.1 recipient mice, resulted in a few but an insignificant
number Ly5.2+CD8α
+ DC (86). Lastly, mice ablated of CD4
+ cells did not lack CD8α
+
or DNDC further demonstrating that CD4+ DC are not necessary for the development of
the other two cDC subtypes (54). It has also been demonstrated that Splenic CD4+
DC
and CD8α+ DC endogenously express CD4 and CD8αα, respectively, and not as a result
of pickup of the markers from lymphocytes (118).
CD8α-CD4
+ DC efficiently process and present antigen from extracellular
pathogens to CD4+ T cells. Like their DNDC counterparts, CD4
+ DC express the
myeloid lineage marker CD11b+. However unlike CD8α
+ DC, CD4
+ DC require IRF4
and not IRF8 for development (4, 111). While these DC do not cross present or directly
present antigens through the Class I presentation pathway as efficiently as CD8α+ DC,
they are capable of activating both CD4+ T cell and, to a lesser extent, CD8
+ T cell
responses (117).
7
One review has proposed that perhaps the ability of CD8α+ DC and CD8α
- DC to
present antigens to CD8 or CD4+ T cells is a factor of how the DC subtypes acquired the
antigen (117). This is built on the premise that MHC Class I and II processing is
functional in both CD8α+ DC and CD8α
- DC, and that there is likely specialized
machinery in CD8α+ DC that allow these DC to handle endocytosed antigen and have it
enter the cross-presentation pathway (98). This has been suggested as a result of a study
showing that antigen taken up via the mannose receptor enters the MHC Class I pathway,
antigens that are phagocytosed or taken up via DEC-205 enter the cross-presentation
pathway, while pinocytosed antigens enter both the MHC Class I and II pathways (16,
117).
CD8α-CD4
- DC (DNDC) also depend on both IRF4 and IRF8 for their
development (4, 111). DNDC express high levels of the myeloid lineage marker CD11b
(118), the c-type lectin Dectin-1, as well as a macrophage marker called F4/80-like
receptor (FIRE) which is expressed on some tissue macrophages and blood monocytes,
but not T or B cells (19). FIRE has been used as a marker of myeloid cells with the
potential to develop into DC and is downregulated upon DC maturation. While FIRE is
not expressed by CD8α+ DC nor CD4
+ DC, its function on DNDC has yet to be
determined. DNDC have been shown to be potent activators of CD4+ T cells much like
their CD4+ DC counterparts. It has been demonstrated in vaccination studies that when
DNDC are targeted via Dectin-1, they can generate greater Ab responses, as well as
stronger CD4+ T cell responses than CD8α
+ DC targeted via DEC-205 (20).
Due to their expression of TLR7 and TLR9, plasmacytoid DC (pDC) are most
known for their ability to produce copious amounts of type I IFN following viral
8
infection of the host and subsequent pDC activation (5, 21, 72, 75). PDC unlike other
DC and innate immune cells have endogenous IRF7 that allows these cells to quickly
respond to viral pathogens and produce type I IFN (48). As such, these DC have been
shown to be quite important in viral clearance as well as play a role in the pathology of
other diseases such as systemic lupus erythematosus and psoriasis. While pDC are
capable of priming T cells (but to a lesser extent than their CD8α+, CD4
+, or DNDC
counterparts), their main function is thought to be as a help to other DC or T cells due to
their expression of CD40L (61). These DC have been shown to have interactions with
regulatory T cells (112). A recent investigation of the differences between cDC and pDC
in MHC presentation capacities, showed that pDC continue to upregulate expression,
ubiquitinate, and turnover of MHC Class II molecules up to 48 hours after activation
(125). This is in contrast to cDC which downregulate MHC Class II molecule turnover
following activation, allowing MHC molecules loaded with peptide to remain at the
surface for longer periods of time than pDC. This continued pDC turnover of MHC
Class II molecules increased pDC ability to prime T cells with endogenous antigens but
affected the efficiency of exogenous antigen presentation.
PDC were first identified as plasmacytoid T cells then as plasmacytoid monocytes
due to their morphological similarity to plasma cells (21, 36, 38, 43). While pDC contain
mRNA for pre-T cell receptor, λ5, and SpiB as well as have D-J rearrangements of IgH,
they are not of lymphoid origin. These cells do not express CD3 or immunoglobulin but
rather high levels of the B cell marker CD45R/ B220 and the monocyte marker Ly6C (36,
72). It has been also shown that the development of pDC is independent of Notch-1 and
T cell factor -1 which are both required for T cell development (38). IRF8 along with the
9
cytokine fms-like tyrosine kinase 3-ligand (Flt3L) has been demonstrated to be critical
for pDC (among other DC subtype) development (80, 111, 116). PDC in mice express
low to intermediate levels of CD11c as well as MHC Class II and thus are classified as
dendritic cells. It has also been shown that upon viral stimulus and as a result of their
high interferon production, pDC can increase expression of surface CD8α, leading some
studies to conclude that pDC are immature precursors to conventional CD8α+ DC (22).
While there are other DC found in other lymphoid and non-lymphoid tissues, such
as the thymus, lymph nodes, liver, skin, and other epithelial surfaces, the focus of our
studies will be on DC found in the spleen following i.v. infection or their in vitro
equivalents generated from mouse bone marrow.
IN VITRO-DERIVED BONE MARROW DENDRITIC CELLS
GM-CSF DC can be generated in vitro. DC can be generated from mouse bone
marrow using in vitro culture methods and the cytokines GM-CSF or Flt3L (11, 13, 14,
51). Generated over a period of 6 to 7 days, DC can be generated in high numbers from
GM-CSF treatment of bone marrow (~90% are CD11c+) and in an immature state (low
costimulatory molecule expression) making them a tractable model for studying DC
biology. GM-CSF derived DC express the pan DC marker CD11c and are positive for
the myeloid marker CD11b (51). These DC are representative of the classical,
inflammatory DC found in the periphery that migrates to the nearest lymph node
following pathogen encounter (102). DC generated from GM-CSF require IRF4 for
development as evidenced by the lack of DC produced when treating IRF4-/-
bone
marrow with GM-CSF (111).
10
Flt3L DC can be generated in vitro. Flt3L generated DC can also be derived
from mouse bone marrow in large numbers and have been correlated with resident DC of
the spleen. These DC are more slowly generated than GM-CSF DC, over a period of 9 to
10 days. These DC also express CD11c, but contain at least 2 DC subsets (cDC and
pDC) that exclusively express either CD11b or B220. The CD11b+ population of the
Flt3L DC make up the majority of the cells cultured (approximately 60%). While in vitro
derived DC do not express surface CD8α, it has been shown that the cDC of Flt3L DC
express IRF8 and have mRNA for CD8α that is translated into protein under in vivo
conditions. The pDC within Flt3L DC, much like their in vivo counterparts, have been
shown to produce large amounts of type I IFN in response to viral pathogens such as
Vesicular Stomatitis Virus and Herpes Simplex Virus. DC generated from Flt3L require
IRF8 (much like their splenic DC counterparts) for development (111). It has also been
shown that GM-CSF treatment in vitro prevents the development of pDC generated by
Flt3L treatment (41).
Flt3L treatment first began as a therapeutic method in humans to increase immune
cell populations. Flt3L has also been used experimentally in mice where it increases
immune cell populations with numbers of DC increasing the most (27). Flt3L-/-
mice
have significantly decreased hematopoiesis, which affects the development of
hematopoietic stem cells, dendritic cells, and NK cells (80).
11
LISTERIA MONOCYTOGENES
As an intracellular, Gram-positive pathogen, Listeria monocytogenes (Lm) is a
particular concern in the food industry. Able to grow at 4° Celsius and survive
refrigeration, Lm can contaminate uncooked foods, soft cheeses, and luncheon meats.
Immunocompromised individuals, the elderly, newborns, and pregnant women, are most
susceptible to illness after eating food contaminated with Lm. Listeriosis manifests in the
form of gastroenteritis and meningitis; and because it can cross the placenta of pregnant
women, may lead to miscarriage or stillbirth, premature delivery, or infection of the
newborn. According to the Centers for Disease Control and Prevention, there are
approximately 2500 serious clinical cases of Listeriosis a year in the United States,
resulting in 500 deaths (29).
The life cycle of Lm (Fig. 2) first involves uptake by a cell into a primary vacuole,
where the bacteria utilize the cholesterol and pH dependent, pore-forming toxin,
Listeriolysin O (LLO) (40, 42) as well as a phosphotidylinositol specific phospholipase
(PI-PLC) to form pores in the vacuolar membrane and escape to the cytosol. Upon
entering the cytosol, Lm then begins to replicate and activate transcription of the actA
gene (58, 113). The product of the actA gene allows Lm to polymerize host cell actin into
comet-like tails and move around the cell. This movement also facilitates infection of
neighboring cells, where Lm escapes the secondary vacuole again using LLO and PI-PLC
as well as a second broad range phospholipase (PC-PLC) to begin the cycle again (18,
104). In this manner, Lm is spread while largely avoiding the extracellular environment,
including complement deposition and antibody responses. In humans, Lm facilitates its
own uptake via internalins expressed by the bacteria, with Internalin A (InlA) binding to
12
E-cadherin expressed by epithelial cells (39, 83) and Internalin B (InlB) binding to Met
or gC1qR on hepatocytes (63, 64) . However in mice, uptake of Lm via InlA is not
efficient due to a point mutation in murine E-cadherin where the amino acid at position
16 is a glutamic acid instead of a proline (69, 70). Thus in mice, Lm more readily infects
cells such as hepatocytes or phagocytic cells such as macrophages, and dendritic cells
that can engulf the bacteria.
Murine models of Lm infection include intravenous (i.v.), intraperitoneal (i.p.),
and oral/ intragastric (i.g.) models. Infection with Lm i.v. goes systemic, with
recoverable bacteria from the spleen (which filters the blood), lymph nodes, and liver.
I.p. infection with Lm also results in bacterial spread systemically with bacteria again
found in the peritoneal cavity, spleen, lymph nodes, and liver. A more recently described
model of infection, i.g. infection, is performed by oral administration of a bolus of
bicarbonate prior to infection with a high dose (greater than 109 cfu) of bacteria (110).
This increases the efficiency of Lm infection by neutralizing the gastrointestinal pH of the
mice. There is also a humanized mouse model of Lm infection by orally infecting mice
that express the human form of E-cadherin (hEcad) in small intestinal enterocytes that
thereby facilitates Lm uptake via InlA (70). These mice are able to be infected in an
internalin-dependent manner with higher doses of Lm. One caveat of this model is that
uptake occurs solely in enterocytes that express the hEcad. To this end, a more recent
animal model for Listeriosis is the knockin E16P mouse model (30). In this mouse
model, the glutamic acid at position 16 in the mEcad, is replaced with a proline.
Therefore, all of the cells in the “humanized” E16P knockin mouse have functional
receptors for InlA of Lm and infection through the gastrointestinal route is possible.
13
Figure 2. The Life Cycle of Listeria monocytogenes. The intracellular life cycle of
Listeria monocytogenes involves uptake of the bacterium into a cell into the endocytic
pathway. Primary vacuole escape is facilitated by the pH and cholesterol dependent
Listeriolysin O (LLO) and a phosphotidylinositol specific phosholipase (PI-PLC). Upon
cytosolic entry, bacterial replication occurs and actA gene products allow the bacteria to
polymerize host cell actin, move about the cell, and push itself into neighboring cells,
largely escaping the extracellular matrix. Once in a neighboring cell, Lm is able to
escape the secondary vacuole with LLO and a broad range phospholipases (PC-PLC) to
repeat the process again.
14
15
Listeriolysin O and cytoplasmic entry by Lm are important for protective
immunity. Central to the survival, replication, and dissemination of Lm, is the
bacterium’s ability to escape the endosomal pathway and enter the cell cytoplasm. One
of the virulence factors that Lm uses to perforate holes in the vacuole membrane is the pH
and cholesterol dependent cytolysin, LLO. Studies have shown that Lm strains that lack
the hly gene that encodes for LLO are unable to activate the endothelium like wild type
Lm can, failing to induce the same levels of TNFα and other proinflammatory cytokines
(31). It has been demonstrated previously in monocyte-derived GM-CSF DC that Δhly
Lm does not induce an increase in DC costimulatory molecule expression in comparison
to that induced by wild type Lm (15). Further, DC infected with Δhly Lm fail to prime
functional naïve CD8+ T cells and mice infected with Δhly Lm even at high doses (5x10
8
cfu) fail to provide protection against wild type Lm challenge (9, 15, 37). While this lack
of protection by Δhly Lm has simply been attributed to the restriction of Lm to the
vacuole, a more in depth investigation of the specific effects of listerial cytoplasmic entry
on DC maturation and the initiation of protective immune responses is warranted. DC
maturation with regards to costimulatory molecule induction, cytokine secretion and
chemokine receptor expression, along with migration into the T cell zones where T cell
priming occurs is likely affected by Lm gaining access to or being restricted from the
cytosol. Thus knowing how DC and, in particular, the subpopulations of DC, mature
following infection with Lm that can access the cytoplasm or is retained in the vacuole is
vital to understanding how immunity against Lm develops.
16
THE ROLE OF T CELLS DURING LM INFECTION
As a pathogen that can access the cytoplasm, Lm is processed into antigen and
presented predominantly in the context of MHC Class I to cytolytic CD8+ T cells (signal
1) which peak in their response to primary Lm infection around 7 days post infection
(17). There are also two other signals that are important for naïve T cell activation –
costimulatory molecule expression and cytokine environment. Antigen given in the
context of MHC molecules provides specificity for the particular pathogen, while
costimulation augments effector T cell responses. Cytokines provide the proper
environment for clearing the specific pathogen by driving Th1 or Th17 immune
responses important for clearing intracellular pathogens. IL-12 and type I IFN have both
been demonstrated to be quite important for CD8+ T cell activation in the context of Lm
infection and can both function as signal 3 to CD8+ T cells (23, 47, 94, 121). Type I IFN
is also important for development of CD8+ T cell memory (24). Upon activation, CD8
+ T
cells undergo activation and polyclonal expansion and traffic back to the site of infection
where they lyse and kill Lm infected cells via perforin and granzyme B production. Due
to the intracellular location of Lm, there have been several studies examining the role of
CD8+ T cells in the clearance of Lm during primary and secondary immune responses (6,
10, 17, 65, 84). T cells are required for the clearance of Lm in that SCID mice develop
chronic, lethal listeriosis (10). Using perforin and granzyme B knockout mice, it has also
been demonstrated that these molecules, while not required for protective immunity
against Lm, enhanced the CD8+ T cell response against Lm (6). Additionally, vaccination
with live Lm (as opposed to heat killed Lm vaccination) is important for priming memory
CD8+ T cell responses (65). In addition to CD8
+ T cells, there is also a robust CD4
+ T
17
cell response during Lm infection (84). While presentation to CD4+ T cells is not
required for the clearance of Lm by CD8+ T cells during primary infection, it has been
shown that CD4+ T cell help is important for effective recall response by CD8 memory T
cells (101, 106). Because DC have the unique ability to activate naïve CD8 and CD4+ T
cells, which are both important for primary and secondary immune responses, studying
how DC subsets are able to prime T cells is pivotal for understanding how adaptive
immunity against Lm is initiated.
THE ROLE OF PATTERN RECOGNITION RECEPTORS IN SENSING LM
Previous work from our lab has indicated that cytoplasmic entry of Lm is
important for induction of costimulatory molecules on GM-CSF derived Dc, and leads to
functional CD8+ T cell responses in vitro (15). Because it is an intracellular pathogen,
Lm has the capacity to interact with multiple pattern recognition receptors (PRRs) at the
cell surface, in the endosome, as well as in the cytoplasm. Sensing and subsequent
signaling through these PRRs in the context of Lm infection result in the upregulation of
costimulatory molecules, such as CD86, CD40, and CD80; and cytokine production, such
as IL-12p40, TNFα, IL-1β, and IFNα/β. At the surface of the cell (Fig. 3), Lm can
stimulate surface Toll-like receptors (TLR) 2, 4, and 5 through its peptidoglycan,
lipopolysaccharide, and flagellin, respectively (45, 49, 109). Upon entering the vacuole,
Lm can stimulate the endosomal TLRs 7 and 9, which sense nucleic acid (32). TLR
pathways signal through the adapter molecules myeloid differentiation factor 88
(MyD88) and/or TIR-domain-containing adapter inducing interferon-β (TRIF) (82, 122,
123). While most TLR signaling is exclusively MyD88 or TRIF-dependent,
18
Figure 3. Cell Surface, Endosomal, and Cytoplasmic Pattern Recognition
Receptors. As an intracellular pathogen, Lm can interact with PRRs at the cell surface,
within the endosome, and in the cytoplasm. A few of the pathways with which Lm can
interact are shown. Surface TLR (TLR2, 4, 5, 6), endosomal TLR (TLR 3, 7, 9), and
cytoplasmic receptors (NOD1, NOD2, Ipaf, among others) trigger signaling pathways
that result in the induction of maturation genes. The TLR pathways signal through the
adapter proteins MyD88 or TRIF, while NLR pathways trigger the inflammasome to
produce proinflammatory cytokines.
19
20
TLR4 signaling can occur through both adapter molecules. Downstream of these
pathways, subsequent activation and DNA binding of transcription factors, NF-κB and
interferon regulatory factors (IRFs) 3, 5, and/or 7, result in the upregulation of
costimulatory molecule and cytokine genes.
MyD88-/-
mice are quite susceptible to infection with Lm, succumbing by day 3-4
from the uncontrolled bacterial load (99). These mice have decreased levels of IL-12 and
IFNγ production, and as a consequence succumb early on to infection. IL-12/ IL-18
double knockout mice are also highly susceptible to Lm infection but not as much as
IFNγ-/-
mice but more than single knockout IL-12 and IL-18 mice (100). The IL-1/IL-18
Receptor pathway also signals through MyD88 upon ligation by either IL-1 or IL-18. As
such, Caspase 1 (which converts pro forms of IL-1 into active IL-1) production also
results in downstream MyD88 signaling through the IL-1/IL-18R. Due to the significant
production of IL-1 following Lm infection, these pathways have also been investigated as
possible pathways to sense Lm. Some studies have shown that Caspase 1-/-
mice, IL-1/Il-
18R-/-
mice, and IL-18-/-
are all susceptible to Lm early following infection (99, 115),
while for IL-18R there is also literature showing that these mice are more resistant to Lm
infection than WT mice (73). Because MyD88-/-
mice are highly susceptible to Lm,
several studies have also addressed the role of MyD88-dependent TLR in sensing Lm.
Unfortunately, no one TLR pathway has yet been identified as required to sense
Lm. TLR4 is not required for immune responses against Lm (99). More controversy
however surrounds the role of TLR2 with studies showing that it is (114) and is not
21
required for immune responses against Lm (34). There has also been one study showing
that macrophages lacking signaling through TLR3, 7, and 9 via the UNC93b1 mutation
3d had effects on their ability to produce IL-12p40 mRNA in response to wild type Lm in
comparison to macrophages infected with Δhly Lm (107). Surprisingly, Δhly Lm
infection of mutated macrophages resulted in the production of more IL-12p40 mRNA
that was not observed in wt macrophages. However, one caveat is that this study failed to
investigate the effect of this mutation on Lm virulence in an infectious mouse model.
Upon entering the cytoplasm, which is critical for the development of protective
immunity, Lm can also trigger cytoplasmic receptors, including Nucleotide
Oligomerization Domain (NOD) - like receptors, or NLRs (68). NLRs are receptors that
contain a C-terminal leucine- rich repeat sensory domain, a central nucleotide binding
domain, and a Caspase recruitment domain (CARD) at the N-terminus. CARD motifs
are found in a vast number of proteins and result in the recruitment and complex
formation of other CARD domain- containing proteins. In the case of some NLRs, these
complexes result in the formation of the inflammasome, leading to the activation of
Caspase 1 and the production of IL-18 and IL-1β. Another NLR, NOD2, senses
intracellular muramyl dipeptide found in the Gram-positive bacterial cell wall, resulting
in the activation of MAP kinases and NF-κB via the recruitment of RIP2, a serine-
threonine kinase (57). It has been demonstrated that Lm can be sensed via the NLRs,
NOD2 and IPAF (senses flagellin), resulting in the production of IL-1 and other
proinflammatory cytokines (119). While the Lm motif that is sensed by NLRs is
currently unknown, several studies hypothesize that it is possible that endosomal
membrane disruption by LLO or LLO itself in wild type Lm may cause activation of the
22
inflammasome. Unfortunately, while Lm can and has been been demonstrated to
stimulate some or all of these sensory pathways (TLR and NLR) in macrophage or
transfected cell lines, there is currently no one pathway found to be necessary and
sufficient to sense Lm. Thus, which pathways are involved in sensing Lm remains a
pertinent question to investigate.
THE SPLEEN AND DC IMMUNITY TO LM
The spleen is an important site for anti-listerial immunity. The spleen filters
the blood, removing old erythrocytes and recycling their components (such as iron and
hemoglobin), and is also a lymphoid organ that contains large numbers of lymphocytes.
Cells, particles, or pathogens that enter the spleen via the bloodstream and splenic
sinusoids first enter the red pulp (RP) (Fig. 4). The RP is composed of lymphatic tissue
that is less compact than that found in the white pulp (WP). Within the RP are CD11b+
macrophages. The RP is traversed by venous channels or sinusoids and is filled with
erythrocytes that give this zone its reddish color in the fixed or unfixed state. At the
interface of the red and white pulp is the marginal zone (MZ). This zone also contains
marginal zone macrophages that are F4/80+, MOMA-1
+, and SIGNR1
+ as well as
marginal zone B cells. Adjacent to the RP is the WP which contains compact lymphatic
tissue surrounding a central arteriole (CA). The ovoid sheath surrounding the CA is
called the periarteriolar lymphiod sheath, or PALS. The cells within the white pulp are
predominantly lymphocytes with T cell zones flanked by B cell germinal centers,
however there are also DC, plasma cells, and some macrophages found distributed
throughout (25).
23
Figure 4. Splenic Architecture. The mouse spleen filters the blood and is a lymphoid
organ where adaptive immune responses can be initiated. Areas within the spleen include
the red pulp (RP), where blood is filtered and its components (such as iron and heme)
recycled, and the white pulp (WP), where lymphocytes are located. Adjacent to the RP
are the marginal zones (MZ). The MZ contains macrophages, dendritic cells, B cells and
some circulating monocytes, whereas the WP is composed of the T cell and B cells
zones. The WP is composed majorily of lymphocytes with a few DC are scattered
throughout. Central arteries (CA) connect the MZ to the T cell zones and cells (and
potentially pathogens) migrate into the WP through these sheaths.
24
25
The importance of DC in immunity against Lm. Following i.v. infection, wild
type Lm has been previously shown to be primarily associated with CD11b+ cells as
measured by enumeration of live cfu following cell sorting and bacteria is localized to the
T cell zones (microscopy). In contrast, studies have shown that heat killed Listeria
(HKLM) and Δhly Lm, which are both retained in vacuoles, are found primarily in the
marginal zones of the spleen (85). Though DC are composed of several subsets, little has
been done to demonstrate the individual contribution of each splenic DC subset to the
immune response or their location in respect to Lm following wild type Lm or vacuolar
Lm infection. There have, however, been a few studies indicating that CD8α+ DC within
the MZ have a large role in trapping antigen and the resulting translocation of Lm to the T
cell zones in the spleen (3), as well as the presentation and cross-presentation of Lm
antigen to naïve CD8+ T cells (8, 62). There are also a few studies showing the bacterial
load of CD11c+ DC as a whole or showing bacterial load in cDC with CD8α
+ DC having
higher Lm numbers than CD8α- DC (85, 89), but have not further delineated the bacterial
load of other DC subsets within the spleen.
DC as sentinels and activators of the adaptive arm of the immune response have
been demonstrated as important in the immune response against Lm for both primary and
secondary responses (53, 126). A few studies have depleted CD11c+ cells (including the
majority of DC) using a mouse that has the diphtheria toxin receptor (DTR) gene under
the control of the CD11c promoter (DTR-tg mice) (53, 85, 126). Mice do not naturally
expression the DTR gene; therefore upon treatment of the DTR-tg mice with diphtheria
toxin, the CD11c+ cells were deleted for a span of 2-3 days. These mice upon infection
with wild type live Lm or heat killed did not induce the proliferation of adoptively
26
transferred transgenic, antigen specific CD8+ T cells, demonstrating that DC are quite
important for primary immune responses to Lm (53, 85). DTR-tg mice depleted of
CD11c+ DC have been also shown to have no viable Lm recoverable from the spleen 3
days post infection, despite having bacteria in the liver (89). In fact, DT-treated DTR-tg
mice infected with Lm showed no significant differences in bacterial liver titers in
comparison with non-DT treated wt mice at 3 days post infection. From this study, we
can conclude that the ablation of CD11c+ DC altered the dynamics in the spleen
following Lm infection in a way that was not conducive to the priming of Lm-specific T
cell responses. Another study has shown that depletion of DC also affects recall
responses of CD8+ T cells to Lm challenge in lymphoid and non-lymphoid (126). Thus
DC are key for both primary and secondary immune responses to Lm and are necessary
for listerial entry into the spleen.
One recent study which selectively inhibited Rac1 in DC populations of
transgenic mice showed that this molecule was important for CD8α+ DC uptake of
apoptotic cells. Rac1 is a GTPase that is involved in cellular endocytosis. The inhibition
of Rac1 in these mice decreased the frequency of CD8α+ DC and their ability to take up
apoptotic cells, but did not affect the frequency or apoptotic uptake ability of CD8α- DC.
This study did lend evidence of Rac1’s role in the capacity of CD8α+ DC to activate T
cells via cross-presentation following endocytosis of apoptotic cells (55). Another study
using these same Rac1 transgenic mice suggested that CD8α+ DC were subsequently
important for the presence of viable Listeria monocytogenes (Lm) in the spleen 3 days
following infection (89). While this study too strongly concluded that CD8α+ DC alone
were “required for efficient entry” of Lm into the spleen and was “crucial for [Lm]
27
spreading and proliferation” by ignoring other potential effects on other DC
subpopulations, it did demonstrate rather nicely that CD11c+ DC are important for Lm’s
presence in the spleen.
SUMMARY TO INTRODUCTION
DC are key to bridging the innate and adaptive immune systems and are
uniquely capable of providing naïve T cells with the proper signals for activation.
DC are composed of several subtypes that have been demonstrated to widely vary in
their function and TLR and surface marker expression. While dendritic cells have
been demonstrated to be important for immunity to Lm very few studies have
investigated how individual DC subsets contribute to primary immune responses
against the pathogen. To this end, we set out to investigate how individual DC
subsets, both in vitro- generated as well as those found in the spleen, respond to Lm
infection in the following ways: (1) What is the level of maturation on individual DC
subsets following Lm infection and how does listerial cytoplasmic entry affect
maturation?, (2) What pathways are involved in sensing Lm and what impact do
those pathways have on DC maturation following Lm infection?, and (3) what
capacity do these distinct DC subsets have to prime naïve antigen specific T cells?
By answering these questions of individual DC subsets, we will have further insight
into DC biology and function and demonstrate the continued potential in targeting
specific DC subsets for making better vaccines against Lm and/ or other intracellular
pathogens.
28
CHAPTER II
MATERIALS AND METHODS
Bacteria. Lm strains 10403s (wild type), DP-L2319 (vacuole-retained), DP-
L4056 (wt LmOVA), and DP-L4027 (Δhly LmOVA) were obtained from Dr. Daniel
Portnoy (University of California, Berkeley, CA). DP-L2319, which harbors deletions in
the genes encoding Listeriolysin O (LLO) and two phospholipases (Δhly ΔplcA ΔplcB),
herein referred to as “vacuolar,” cannot enter the cytosol or replicate. DP-L4027 has a
single deletion of the hly gene that encodes for LLO. Both DP-L4056 and DP-L4027
constitutively secrete the ovalbumin protein. The actA::gfp Lm strain, DH-L1245, as
previously described, has a stably integrated expression cassette containing the gene
encoding gfp from Aequorea victoria, transcribed from the actA promoter, and was
obtained from Dr. Darren Higgins (Harvard University School of Medicine). Bacteria
grown to stationary phase at 30°C in brain–heart infusion broth (BHI) were washed twice
in sterile PBS (HyClone) and resuspended in RPMI 1540 medium, supplemented with
10% fetal calf serum (HyClone), 2mM GlutaMAX (Invitrogen), and 5x10-5
M β-
mercaptoethanol (Sigma-Aldrich), at a concentration to achieve the indicated MOI or cfu.
The MOI/cfu was confirmed by plating dilutions of the inoculum on BHI agar and
enumerating cfu after 48 h at 37°C.
Mice. C57BL/6 mice were purchased from Taconic Farms (Germantown, NY) or
Charles River Laboratories (Wilmington, MA). TRIF-/-
mice and OT-I/Rag1-/-
TCR
transgenic mice specific for OVA257-264 presented by Kb were purchased from The
29
Jackson Laboratory (Bar Harbor, ME). OT-II TCR transgenic mice specific for OVA323-
339 presented by I-Ab and CD11c-DTR-GFP mice were obtained from Dr. Steven B.
Mizel (Wake Forest University School of Medicine). MyD88-/-
, TLR2-/-
, and TLR4-/-
mice were obtained from Dr. Doug Golenbock (Univ. of Massachusetts Medical School)
with the permission of Dr. Shizuo Akira (Osaka Univ., Japan). TLR2/9-/-
mice and IL-
1R-/-
mouse bone marrow were obtained from Dr. Tod Merkel (FDA/ CBER) with the
permission of Dr. Shizuo Akira. Caspase 1-/-
mice were obtained from Dr. David Chaplin
(University of Alabama-Birmingham). All mice were bred and maintained in the animal
facility at Wake Forest University School of Medicine, Winston- Salem, NC.
Generation of BMDC. Bone marrow dendritic cells were generated as
previously described with some modifications(14, 51) as follows: bone marrow was
flushed from the tibias and femurs of C57BL/6 or knockout mice. Following red blood
cell lysis, cells were washed and filtered to remove debris, then resuspended in RPMI
1640 (HyClone). For GM-CSF DC, cells were seeded in 24-well plates (Corning) with
10 ng/ml recombinant murine GM-CSF. GM-CSF DC were cultured for 6 days at 37ºC
in 5% CO2, and fresh medium with cytokine was added on days 2 and 4. Flt3L DC were
seeded in 6-well plates (Corning) with 100 ng/ml recombinant human Flt3L (Invitrogen).
Flt3L DC were cultured for 9 days at 37ºC in 5% CO2, and fresh medium with cytokine
was added on day 4. All DC were routinely >90% CD11c+
and displayed low levels of
CD40, CD80, and CD86, characteristic of immature DC.
Subset enrichment of Flt3L BMDC. Flt3L DC were grown as described above.
On day 9, Flt3L DC were gently harvested, counted, and separated into B220-positive
and B220-negative fractions using CD45R/B220 microbeads and magnetic columns
30
(Miltenyi Biotec). The resulting B220-positive (denoted as B220+ DC) and B220-
negative (denoted as CD11b+
DC) fractions were each >96% pure and both populations
remained immature as measured by CD86, CD80, and CD40 expression.
Infection of BMDC. Bone marrow dendritic cells (3x105
cells/ well) were
infected with Lm strains at the indicated MOI, were treated with 25 µg/ml Poly(I:C)
(Invivogen) or 200 µM Loxoribine (Invivogen), or left untreated. Infection was
enhanced by a brief 4 min spin of the bacteria onto the DC at 1000 rpm. At 4 h post
infection, 10 µg/ml chloramphenicol and 10 µg/ml gentamicin was added, and the cells
were cultured for an additional 20 h to allow for cytokine production and expression of
costimulatory molecules. Culture supernatants were then removed, and the cytokines IL-
6 and IL-12p40 were measured using ELISA OptiEIA kits from BD Pharmingen. IFN-β
was measured using an IFN-β ELISA kit from Invitrogen. The cells were stained with
the following fluorescently labeled antibodies in various combinations: anti-CD40 PE
(3/23), anti-CD80 PE (16-10A1), anti-CD86 PE (GL1), anti-CD11c PE Cy7 (HL3), anti-
CD45R/B220 FITC (RA3-6B2), anti-CD11b APC (M1/70) all from BD Pharmingen.
Nonspecific background staining was assessed using isotype-matched control Abs (BD
Pharmingen). Following staining, cells were fixed with 2% paraformaldehyde and stored
at 4ºC until acquisition and analysis. Surface marker and costimulatory molecule
expression were determined using a BD FACSCalibur and CellQuest Pro software for
acquisition (BD Biosciences). FlowJo software (TreeStar) was used for analysis of flow
cytometric data. GraphPad Prism software was used to generate graphs and determine
statistical significance.
31
CFSE-labeling of Lm. Wild type and vacuolar Lm strains were washed and
incubated with 0.2 nM Carboxyfluorescein succinimidyl ester (CFSE) from Invitrogen
for 30 min as described previously. Bacteria were then washed three times and
resuspended in sterile PBS. Infection and staining were carried out as described above
and cells were fixed in 4% paraformaldehyde. As a control, CFSE-labeling of Lm did not
increase the autofluorescence of the cells or cause an increase in costimulatory molecule
expression in comparison with unlabeled-Lm infections.
In vitro T cell priming assays with BMDC. Bone marrow- dendritic cells
(2x104 cells/ well) were left untreated or infected with an MOI of 1 with wild type Lm,
vacuolar Lm, or the indicated MOI of Lm-OVA in 96-well round bottom plates (Corning)
for 4 h, as described above. At 4 h post infection, chloramphenicol and gentamicin were
added, and DC were loaded with 0.1 ng/ml of preprocessed
OVA257-264 peptide
(SIINFEKL; from Wake Forest University School of Medicine Peptide Synthesis
Facility) for an additional 20 h. Peptide was not added to DC infected with Lm-OVA.
OVA-specific CD8+ T cells were isolated from the spleens
of OT-I TCR transgenic mice.
At 24 h post infection, T cells were labeled with 5 µM CFSE (BD Pharmingen),
according to the manufacturer’s instructions, and added to the
DC at a ratio of 10:1 (T
cell: DC). T cells were harvested 72 h later and restimulated by incubation with OVA257-
264 peptide (2 µg/ml) in the presence of Golgi Plug (BD Pharmingen) for 5 h. The cells
were surface stained using anti-CD8α Ab (53-6.7; BD Pharmingen) then fixed and
permeabilized for intracellular cytokine staining using the Cytofix/cytoperm kit from BD
Pharmingen,
according to the manufacturer’s instructions. Cells were stained
intracellularly using anti-IFN-γ Ab (BD Pharmingen). Following staining, cells were
32
resuspended in 2% paraformaldehyde and stored at 4ºC until acquisition and analysis.
CFSE proliferation data were analyzed, as previously described, using FlowJo software
(TreeStar). The division index represents the mean number of divisions that a cell present
in the starting population undergoes.
Sorting of Lm-infected DC for in vitro T cell priming. Flt3L DC were
separated into B220-positive and negative fractions as described above. Cell populations
were then infected with CFSE-labeled Lm-OVA for 24 hours (CD11b+ DC with an MOI
of 2; B220+ DC with an MOI of 10) and then sorted, using a BD FACS Aria, into
infected (CFSE-Lm+) and non-infected (CFSE-Lm
-) cells. Sorting had little to no effect
on maturation level. The level of infection measured by mean fluorescence intensity of
CFSE was similar for both CD11b+ and B220
+ DC subsets. As a control, CFSE-labeling
of Lm did not increase the autofluorescence of the cells in comparison with unlabeled-Lm
infections. CFSE-Lm+ and CFSE-Lm
- cells, as well as non-treated cells, for each DC
subset were then incubated with OT-I T cells for 72 hours. T cells were then restimulated
with OVA peptide in the presence of Golgi Plug for 5 hours, followed by an intracellular
cytokine stain for IFN-γ and surface stain for CD8α.
Immunofluorescent Microscopy of BMDC. Bone marrow-dendritic cells
(2x105 cells/ well) were infected in 96-well plates (Corning) with a MOI of 3 using
actA::gfp Lm. At 3 h post infection, cells were gently washed, harvested, and allowed to
adhere to sterile Poly (L)-Lysine (Sigma-Aldrich) coated glass coverslips. Cells were
then fixed overnight with 4% paraformaldehyde at 4ºC, treated with PBS containing 3%
normal goat serum (PBS-NGS), followed by permeabilization for 10 min with PBS-NGS
containing 1% NP-40 and 0.01% saponin. Polyclonal anti-Listeria Ab (BD Diagnostic)
33
was added for 30 min, followed by three washes and incubation for 30 min with Alexa
Fluor 568-conjugated goat-anti-rabbit IgG Ab (Invitrogen). Coverslips were washed and
mounted on slides with ProLong Gold Antifade containing DAPI (Invitrogen). Results
were analyzed by fluorescence microscopy using a Nikon Eclipse TE300 inverted
microscope equipped with a digital camera and Adobe Photoshop software (Adobe
Systems).
Infection and staining of splenic DC. Mice were infected with 5x104 or 5x10
5
cfu wild type Lm (10403s or DP-L4056), 5x108 cfu vacuolar Lm (DP-L2319 or DP-
L4027), or mock treated (sterile 1X PBS). At the time indicated post treatment, spleens
were harvested and digested in Collagenase IV for 30 min and then red blood cell lysis
performed to obtain a single cell suspension. Cells were stained with various
combinations of anti- CD3 (145-2C11) and anti- CD19 (1D3; both in PerCP Cy5.5 to
exclude contaminating T and B cells), anti- CD11c APC (HL3), anti- CD8α Pacific Blue
(53-6.7), anti-CD4 FITC (3/23), anti- CD45R/B220 Pacific Blue (RA3-6B2) , anti-Ly6C
FITC (), anti- CD86 PE (GL1), anti-CD80 PE (16-10A1), anti-CD40 PE (3/23), or anti -
I-Ab PE(AF6-120.1), all from BD Pharmingen. CD8α+ DC were CD3/CD19
-
CD11c+CD8α
+CD4
-; CD4
+ DC were DC were CD3/CD19
-CD11c
+CD8α
-CD4
+; DNDC
were CD3/CD19-CD11c
+CD8α
-CD4
-B220
-; and pDC were CD3/CD19
-CD11c
+ B220
+
Ly6C+. Cells were then fixed in 2% PFA and acquired using a BD FACSCanto II and
Diva software for acquisition and analysis of costimulatory molecule expression (BD
Biosciences). For bacterial enumeration in splenic DC subsets and ex vivo T cell priming
experiments, collegenase-digested splenocytes were first enriched for CD11c positive
cells using columns and CD11c microbeads from Miltenyi-Biotec. Resulting CD11c -
34
enriched cells were sorted using a BD FACS Aria using the following staining protocol,
without fixation: anti- CD3 (145-2C11) and anti- CD19 (1D3; both in PerCP Cy5.5 to
exclude contaminating T and B cells), anti- CD11c APC (HL3), anti- CD8α Pacific Blue
(53-6.7), anti- CD4 FITC (3/23), and anti- CD45R/B220 PE (RA3-6B2), all from BD
Pharmingen. For bacterial enumeration, sorted splenic DC subsets were lysed in water
and dilutions plated for bacterial counts. Cells were normalized per 103 DC or per 10
6
total splenocytes. GraphPad Prism software was used to generate graphs and determine
statistical significance.
Ex vivo T cell priming assays with splenic DC. At 24hpi, sorted splenic DC
from PBS treated, wt LmOVA infected or Δhly LmOVA infected mice were plated at
2.5x104 cells/ in 96-well V-bottom plates (Corning), as described above.
Chloramphenicol and gentamicin were added to each well, and mock treated splenic DC
of each subset were loaded with 0.1 ng/ml of preprocessed
OVA257-264 peptide
(SIINFEKL; from Wake Forest University School of Medicine Peptide Synthesis
Facility) or 10 µg/mL of preprocessed OVA323-333 peptide (courtesy of Dr. Steven B.
Mizel) for 30 min to 1h prior to the addition of T cells. Peptide was not added to DC
infected with LmOVA or Δhly LmOVA. OVA-specific CD8+ T cells were isolated from
the spleens of OT-I/Rag1
-/- TCR transgenic mice or splenocytes were isolated from the
spleens of OT-II TCR transgenic mice. OT-II splenocytes were further selected for CD4+
T cells using the T cell isolation kit for untouched CD4+ T cells (Miltenyi-Biotec) and LS
columns. T cells were labeled with 5 µM CFSE (BD Pharmingen), according to the
manufacturer’s instructions, and added to the DC at a ratio of 10:1, 5:1, or 2:1 (T cell:
DC). T cells were harvested 72 h later and restimulated by incubation with OVA257-264
35
peptide (2 µg/ml) or OVA323-333 peptide (30 µg/ml) in the presence of Golgi Plug (BD
Pharmingen) for 5 h. The T cells were then stained using anti-CD8α PerCP (53-6.7) or
anti-CD4 PerCP (RM4-5; both BD Pharmingen) then fixed in 2% PFA and permeabilized
for intracellular cytokine staining using the Cytofix/cytoperm kit from BD Pharmingen,
according to the manufacturer’s instructions. Cells were stained intracellularly using
anti-IFN-γ Ab (BD Pharmingen). Following staining, cells were resuspended in 2%
paraformaldehyde (PFA) and stored at 4ºC until acquisition and analysis. CFSE
proliferation data were analyzed, as previously
described, using FlowJo software
(TreeStar).
Staining and Laser Scanning Confocal Microscopy of Frozen Spleen
Sections. Spleens from C57Bl/6 or CD11c-DTR-GFP mice that were infected with
5x104 cfu wild type Lm (10403s), 5x10
8 cfu vacuolar Lm (DP-L2319), or mock treated
(sterile PBS) for 24h were harvested and snap-frozen in OCT media, then stored at -80ºC.
Frozen spleens were then cut into serial 8 µm sections and dried in 100% acetone for 10
min. Sections were then placed in 1X PBS until staining. Sections were blocked for 10
min in 1% BSA (Sigma), then stained for 30 min with the following primary antibodies
in various combinations: purified CD11c (HL3), purified CD3 (145-2C11), purified
CD11b (M1/70), purified CD45R/B220 (RA3-6B2) , purified CD8 (53-6.7), purified
CD4 (RM4-5), 33D1 PE (33D1; all from BD Pharmingen), DEC-205 PE (NLDC-145;
Miltenyi-Biotec), purified F4/80 and F4/80 PE (BM8; both from eBioscience), or
polyclonal rabbit anti-Listeria (BD Diagnostic), followed by the appropriate secondary
antibody: goat anti-rat Alexa Fluor 594, goat anti- hamster Alexa Fluor 594, goat anti-
hamster Alexa Fluor 488, goat anti-rabbit Alexa Fluor 405 (all from Molecular Probes) or
36
rat anti-Armenian and anti-Syrian Hamster FITC (BD Pharmingen). Spleen sections
were then mounted under coverslips with Prolong Antifade Gold Reagent without Dapi
(Invitrogen) and viewed using a Nikon Eclipse C1si Laser Scanning Confocal
Microscope using the 20x water and 60x water lenses.
37
CHAPTER III
IMMUNE RESPONSES OF IN VITRO- DERIVED DENDRITIC CELL SUBSETS TO AN
INTRACELLULAR BACTERIAL PATHOGEN, LISTERIA MONOCYTOGENES
L.M. Mitchell, C.J. Henry, J.M. Grayson, and E.M. Hiltbold
Portions of the following chapter were submitted to the journal Infection and
Immunity and are primarily found within the Results section of that manuscript.
L.M. Mitchell performed all of the experiments. Dr. C.J. Henry assisted with the in
vitro T cell priming assays. All mice were infected by Dr. J.M. Grayson. L.M.
Mitchell and Dr. E.M. Hiltbold prepared the manuscript.
38
INTRODUCTION TO CHAPTER III
Dendritic cells play a major role in the activation of adaptive immunity by
proficiently capturing, processing, and presenting antigen to naïve T cells in lymphoid
organs (2). DC are also important for protective immunity to Lm (53, 126) and are
induced to undergo maturation to prime protective T cell responses. A range of dendritic
cell subsets of distinct lineages have been identified in lymphoid organs, including the
spleen (2, 46, 54). These cells are distinguished based on their surface marker phenotype,
repertoire of pathogen sensing molecules, and specific functions. DC can be generated in
large numbers in vitro by culturing mouse bone marrow with the cytokines GM-CSF or
Flt3L (13, 14, 51). BMDC generated with GM-CSF are CD11c+CD11b
+ B220
- and are
thought to represent monocyte-derived or inflammatory DC (51, 102). We have
previously reported that these DC increase their expression of costimulatory molecules
and produce pro-inflammatory cytokines upon stimulation with Toll-like receptor (TLR)
agonists, as well as listerial infection, but only if the bacteria are able to access the
cytoplasm (15).
The in vitro generation of DC using Flt3L gives rise to two DC populations
representative of immature, steady-state DC in the spleen (87) that are
CD11c+CD11b
+B220
- (conventional) and CD11c
+ CD11b
- B220
+ (plasmacytoid) (13, 14,
51, 87). Flt3L DC are also morphologically distinct from GM-CSF DC and exhibit
differences in their development as well as TLR expression (11, 14, 41). To this end, we
sought to determine how these distinct DC subsets in vitro sense and respond to wild type
Lm and vacuolar Lm.
39
Flt3L DC respond to L. monocytogenes independent of cytoplasmic entry.
Flt3L DC contain cell populations that are CD11c+B220
+ and CD11c
+CD11b
+. Fig. 5A
and B depict the expression of these markers on Flt3L DC, as well as, a histogram (Fig.
5A) showing the response of the whole population to wild type Lm at an MOI of 1.
While the background of the starting population is slightly mature, upon further stimulus
with either TLR agonist or Lm, the whole population exhibits a marked shift in CD86
expression. Fig. 5C and D depict the pattern of costimulatory molecule expression in
Flt3L DC following infection with wild type Lm or vacuole-retained Lm compared to
GM-CSF DC as a reference. Interestingly, Flt3L DC robustly displayed increased
costimulatory molecule expression in response to both wild type and vacuolar Lm in a
dose-dependent manner. While the increase in CD86 expression induced by vacuolar Lm
at an MOI of 1 was approximately 60% of the level of induction seen with wild type Lm
at the same MOI, it was a significant increase over non-treated DC and over the levels
seen in GM-CSF DC infected with the vacuolar Lm mutant. Interestingly, the level of
costimulatory molecule expression of the GM-CSF DC was significantly lower than that
observed on the Flt3L DC in response to all stimuli tested. The pattern of CD40 in Flt3L
DC was similar to CD86 and CD80.
Our lab has recently shown that DC-produced IL-12 but not IL-23 enhances CD8+
T cell responses to Lm in vitro (47). More recently, IL-12 has been demonstrated to be
important for the formation of CD8+ T cell memory. There is also a robust type I IFN
response during Lm infection in vivo and type I IFN has also been shown to be important
not only as a signal 3 for CD8+ T cells but also for memory CD8
+ T cell development
(24, 121) . In light of these significances, we measured via ELISA the production of
40
Figure 5. GM-CSF DC and Flt3L DC expression of costimulatory molecules and
cytokines in response to L. monocytogenes is independent of cytoplasmic entry. GM-
CSF DC and Flt3L DC were each treated with wild type or vacuolar Lm at the indicated
MOI, with indicated TLR agonists, or left untreated for 24 h. (A) CD11c expression on
Flt3L DC. Gate indicates which cells were considered CD11c+. (B) CD86 expression on
Flt3L DC following infection with Lm or no treatment (nt). Number is frequency.
Surface CD86 and (D) CD80 expression on GM-CSF DC vs. Flt3L DC, assessed by flow
cytometry. (E) IL-12p40 and (F) IFN-β production, measured by ELISA. (C-D) Data
shown are compiled from three individual experiments. Error bars represent standard
deviation (SD). (E-F) Data are representative of at least three experiments. Error bars
depict SEM.
41
42
IL-12p40 and IFN-β by the Flt3L DC (Fig. 5E and F). Similar to the pattern of
costimulatory molecule induction, we observed that Flt3L DC produced equivalent levels
of IL-12p40 (Fig. 5E) in response to both wild type and vacuolar Lm. We also observed
that in Flt3L DC, IFN-β production was dependent upon cytosolic entry of Lm (Fig. 5F),
a phenomenon which has been previously demonstrated in murine bone marrow
macrophages (79, 90, 105). The GM-CSF DC, in agreement with previously published
reports, had a similar pattern of IFN-β but produced a small amount of IFN-β in response
to the vacuolar Lm at the highest MOI. Thus, we have concluded that Flt3L DC respond
robustly to both wild type and vacuolar strains of Lm with an upregulation in
costimulatory molecule expression, and thus possess a mechanism for recognition of Lm
within endocytic compartments not found in DC generated by GM-CSF. These data have
led us to postulate that Flt3L-generated DC sense and respond to vacuolar Lm via TLR
expressed within endocytic compartments such as TLR3, 7, or 9.
There is a role for MyD88, but not TRIF, in Lm-induced costimulatory
molecule expression and cytokine secretion by Flt3L DC. Upon recognition of
PAMPs, endosomal TLR3, 7, and 9 signal through the adapter molecules TRIF (TLR3)
or MyD88 (TLR7 and TLR9) (108). Thus, we wanted to determine if these molecules
were involved in DC recognition of Lm. To determine which pathways had a role in the
expression of costimulatory molecules and cytokine secretion in response to Lm, we
infected Flt3L DC derived from MyD88-/-
and TRIF-/-
mice with Lm. TLR agonists that
signal through MyD88-dependent (Loxoribine) or MyD88-independent (Poly I:C)
43
Figure 6. MyD88, but not TRIF, has a role in the response of Flt3L DC to L.
monocytogenes. Flt3L DC were derived in vitro from WT, MyD88-/-
, or TRIF-/-
mice.
DC were treated with Lm strains at an MOI of 1, with indicated TLR agonists, or left
untreated for 24 h. (A) Surface CD86 and (B) CD80 expression, assessed by flow
cytometry. (C) IL-12p40 and (D) IL-6 production, determined by ELISA. WT DC are
depicted as black bars, MyD88-/-
DC depicted as gray bars, and TRIF-/-
DC depicted as
white bars. Costimulatory molecule data shown are compiled from three individual
experiments. Error bars represent SD. ELISA data is representative of at least three
individual experiments with error bars representing SEM. Statistical analysis comparing
to WT values was performed using a Two Way ANOVA with a Bonferroni post test. * P
<0.05, ** P <0.01, *** P <0.001
44
45
mechanisms were included as controls. Fig. 6A and B show that in Flt3L DC, the
expression of CD86 and CD80 in response to both wild type and vacuolar Lm was
significantly reduced in MyD88-deficient cells. This effect was most dramatic in the
cells infected with vacuolar Lm. In contrast, TRIF-/-
DC exhibited comparable expression
of CD86 and CD80 in response to both strains of bacteria similar to that of WT DC.
Likewise, the induction of CD40 by both bacterial strains exhibited a similar dependence
on MyD88 but not TRIF. The secretion of IL-12p40 (Fig. 6C) in response to wild type
Lm was also diminished in MyD88-/-
DC, while IL-12p40 production in response to
vacuolar Lm was almost completely abrogated. IL-6 secretion in MyD88-/-
DC was
significantly reduced following Lm infection with either strain (Fig. 6D). In contrast,
cytokine secretion was not diminished in TRIF-/-
DC compared with WT DC (Fig. 6C and
D). Given that we observed a strong loss of costimulatory molecule expression in
response to vacuolar Lm in the absence of MyD88 and a decrease in cytokine secretion
induced by both strains, we have concluded that MyD88-dependent signaling is important
for the maturation response of Flt3L DC to Lm.
The CD11b+ population within Flt3L DC demonstrates the highest level of
costimulatory molecule expression in response to L. monocytogenes infection. Flt3L-
generated DC, like splenic resident DC, have previously been shown to contain at least
two DC populations (14, 87) . In our Flt3L DC cultures, we have identified two such
populations of CD11c+ DC. DC that express CD11b (cDC) make up about 60% of the
cells, and the other 40% are B220+
(pDC) (Fig. 7A and (13, 14)). We thus sought to
determine which populations within the whole Flt3L DC cultures were capable of
responding to Lm infection. To address this, we co-stained the cells for maturation
46
markers along with the subset markers CD11b or B220, gating on the population of
interest for analysis. First, we observed that there was a marked increase in CD80
expression in response to both Lm strains in the CD11b+ DC that was not seen in the
B220+ DC (Fig. 7A and B). In contrast to CD80, we observed upregulation of CD86 on
both subsets, but the level of CD86 expression on the CD11b+ cells was much higher than
on the B220+ DC (Fig. 7C and D). From these data, we have concluded that the overall
magnitude of the costimulatory molecule expression demonstrated by the mixed
population upon listerial infection can be primarily accounted for by increases on the
CD11b+ DC subset.
Several studies have demonstrated that pDC, particularly in viral infections,
provide a strong type I interferon response, which can have a positive effect on the
responses of other DC (66, 124). To determine if the B220+ DC in our cultures had any
impact on the response of the CD11b+ DC to Lm, we depleted the B220
+ cells using B220
positive selection and magnetic column purification (Miltenyi Biotec). The resulting
untouched and immature CD11b+ DC fraction was >96% pure, as were the positively
selected B220+ DC. The separated DC subset populations or whole Flt3L DC cultures
were then infected with the indicated Lm strains or treated with purified TLR agonists as
controls and allowed 24 h for maturation. Purified CD11b+ DC displayed increased
expression of CD86 when infected with either wild type or vacuolar strains of Lm at an
MOI of 1 (Fig. 7C) which was only slightly lower than that displayed by the same cells in
the mixed culture. Purified B220+ DC (Fig. 7D) also increased surface CD86 expression
following infection with either wild type or vacuolar strains of Lm, however the
magnitude of these responses were also less in comparison to cells in mixed culture.
47
We then measured the cytokine response of the two subsets that had been
separated prior to Lm infection. IL-12p40 and IL-6 were produced by CD11b+ DC in
response to both wild type and vacuolar Lm (Fig. 7E and F). IFN-β secretion by CD11b+
DC was also observed in response to wild type Lm but not to vacuolar Lm (Fig. 7G).
Although the IL-12p40 response of B220+ DC was markedly less than that observed with
CD11b+ DC, these cells did produce significant amounts of this cytokine in response to
both wild type and vacuolar Lm (Fig. 7E). Likewise, the IL-6 response was also
significantly less in the B220+ DC than the CD11b
+ DC (Fig. 7F). Surprisingly, IFN-β
was only minimally produced by B220+ DC (Fig. 7G) in response to Lm, but was
produced in response to the viral RNA mimetic, Poly I:C. Thus, we have determined that
CD11b+ DC secrete higher levels of cytokines in response to both wild type and vacuolar
Lm infection than B220+ DC. Taken together, our data suggest that CD11b
+ DC
primarily account for the magnitude of the costimulatory molecule and cytokine
responses to Lm observed in whole Flt3L DC.
CD11b+ and B220
+ Flt3L DC differ in their capacity to prime naïve CD8
+ T
cells. The ultimate measure of DC maturation is the capacity of these cells to prime and
activate naïve T cells. To determine the relative capacity of Flt3L DC to activate naïve T
cells, in vitro T cell priming assays were performed as previously described (15). First,
to evaluate primarily the costimulatory function of the DC subsets while holding antigen
concentration constant, we used wild type Lm or vacuolar Lm in combination with pre-
processed OVA peptide. DC were infected with either wild type or vacuolar Lm and
pulsed with the indicated concentrations of OVA257-264 peptide overnight. Naïve OT-I T
cells were stained with CFSE and added to the DC the next day. After 3 days of co-
48
Figure 7. CD11b+ DC within the Flt3L DC cultures are the major responders to Lm
infection in comparison to B220+ Flt3L DC. The response of CD11b
+ and B220
+
populations within Flt3L DC to L. monocytogenes. Day 9 Flt3L DC were harvested and
positively selected for B220 expression using microbeads and magnetic columns
(Miltenyi Biotec). Whole Flt3L DC, B220+, or CD11b
+ fractions were treated with Lm
strains at the indicated MOI, with indicated TLR agonists, or left untreated for 24 h. (A)
B220 vs. CD11b expression on Flt3L DC. Gates illustrate criteria for selection as B220+
and CD11b+. CD80 expression on (B) B220
+ DC and (C) CD11b
+ DC following Lm
treatment. Comparison of gated and separated (D) B220+
DC and (E) CD11b+
DC
populations for CD86 expression following Lm infection. Gated populations are depicted
as black bars. Separated populations are depicted as white bars. Data shown are
compiled from three individual experiments. Error bars represent SD. Statistical analysis
comparing gated to separated was performed using a Two Way ANOVA with Bonferroni
post test. * P < 0.05, ** P < 0.01, *** P <0.001. (F) IL-12p40, (G) IL-6, and (H) IFN-β
cytokine production of separated Flt3L DC subsets, assessed by ELISA. Data is
representative of at least three individual experiments. Error bars represent SEM.
Statistical analysis comparing B220+ to CD11b
+ was performed using a Two Way
ANOVA with a Bonferroni post test. * P <0.05, ** P <0.01, *** P <0.001
49
50
culture, the T cells were harvested, re-stimulated with peptide, and stained for IFN-γ
production by intracellular cytokine stain. Proliferation and cytokine production were
then quantitated by flow cytometry. The data in Fig. 8A-C demonstrate that whole Flt3L
DC were comparably able to induce proliferation and IFN-γ production of naïve CD8+ T
cells when infected with either wild type or vacuolar Lm. In fact, the proliferation
induced by DC infected with vacuolar Lm was slightly stronger than that induced by DC
infected with wild type Lm. This demonstrates that the costimulatory upregulation that
we observed in response to both wild type Lm and vacuolar Lm (Fig. 5) conferred
comparable activation of naive T cells.
We then wanted to determine the costimulatory capacity of each of the two DC
subsets within this population. To address this question, we separated the populations
based on B220 expression and performed similar T cell priming assays (Fig. 8D-F). In
this case, we observed that CD11b+ DC were better able to induce T cell to proliferation
(Fig. 8D and E). The major difference was that the B220+ DC were much less capable of
inducing T cells to enter division than the CD11b+ population. This may be due to the
higher level of costimulatory molecules expressed by these CD11b+ cells. Likewise, the
CD11b+ DC induced a higher percentage of the proliferated T cells to produce IFN-γ
(Fig. 8D and F) than B220+
DC when presenting pre-processed antigen.
To assess the ability of each subset to process and present bacterially encoded
antigens and activate T cells, we infected the separated DC subsets with Lm expressing
the OVA protein (Lm-OVA). When Flt3L DC were required to process and present Lm-
produced antigen (Fig. 8G-I), B220+
DC were much less stimulatory when compared to
51
Figure 8. T cell priming assays with whole and subset- separated Flt3L DC. (A-C)
T cell priming assay using whole Flt3L DC infected with wild type Lm or vacuolar Lm
plus OVA peptide. (A) Dot plot of T cell expression of CFSE and IFN-γ at OVA peptide
dose of 0.1 ng/ml. (B) Division Index and (C) Percent T cells that are IFN-γ positive
when primed with infected Flt3L DC plus peptide. (D-F) T cell priming assay using
CD11b+
DC or B220+ DC infected with wild type Lm plus OVA peptide. (D) Dot plot of
T cell expression of CFSE and IFN-γ at OVA peptide dose of 0.1 ng/ml. (E) Division
Index and (F) Percent T cells that are IFN-γ positive when primed with subset- separated
Flt3L DC plus peptide. (G-I) T cell priming assay using CD11b+
and B220+ Flt3L DC
infected with Lm-OVA (no peptide was added here). (G) Dot plot of T cell expression of
CFSE and IFN-γ. (H) Division Index and (I) Percent T cells that are IFN-γ positive when
primed with subset- separated Flt3L DC infected Lm-OVA. Data shown are compiled
from two individual experiments. Statistical analysis comparing B220+ DC to CD11b
+
DC was performed using a Two Way ANOVA with a Bonferroni post test. * P <0.05, **
P <0.01, *** P <0.001
52
53
CD11b+ DC in both driving T cells into proliferation (Fig. 8H) or the production of IFN-γ
(Fig. 8I). One potential explanation for the diminished presentation of bacterial antigens
by the B220+ DC could be that their level of direct infection was significantly lower than
the CD11b+ DC, resulting in little to no detectable antigen presentation.
The CD11b+ population within Flt3L DC is more frequently infected with L.
monocytogenes. One factor that may affect the degree of maturation of specific DC
populations is whether they become directly infected (i.e. take up bacteria). In some
cases however, it is not necessary for a cell to be directly infected by a pathogen for it to
upregulate its costimulatory molecule expression, secrete cytokines, or present its
antigens (12, 56, 60, 103). To determine the extent of infection of the DC population(s)
with Lm, we infected whole Flt3L DC with fluorescently-labeled Lm and quantitated the
amount of bacterial fluorescence associated with either the B220+ or CD11b
+ DC. Fig.
9A depicts whole Flt3L DC infected with increasing MOI of CFSE-labeled wild type Lm.
We observed that the majority of the infected DC were CD11b+. Although the B220
+ DC
population contained CFSE-labeled bacteria, it was to a lesser extent than CD11b+ DC.
This trend was quantitated in Fig. 9B as indicated by the percent of CFSE+
cells
increasing in both populations as the MOI increased. Here, we observed that a higher
percentage of the CD11b+ DC were CFSE
+ than the B220
+ DC. The same trends were
observed for CFSE-labeled vacuolar Lm. Thus we have concluded that while both
subsets are capable of being infected, CD11b+
DC are infected at a higher frequency than
B220+ DC.
Directly infected B220+
DC can prime naïve T cell responses. To address the
possibility that the diminished ability of B220+ DC to process and present listerial
54
Figure 9. CD11b+ DC within the FLt3L DC cultures are preferentially infected with
Lm than B220+ Flt3L DC. Whole Flt3L DC were infected with CFSE-labeled Lm at the
MOI indicated for 24 h. Cells were then analyzed by flow cytometry for level of
infection (CFSE expression). (A) Representative plot of CFSE versus B220 expression
(top panel) or CD11b expression (bottom panel). (B) Percent CFSE+ cells in CD11b
+
versus B220+ DC. B220
+ DC are depicted as black bars; CD11b
+ DC are depicted as
white bars. Data are compiled from two individual experiments. Statistical analysis
comparing B220+ DC to CD11b
+ DC was performed using a Two Way ANOVA with a
Bonferroni post test. * P <0.05, ** P <0.01, *** P <0.001
55
56
antigens was due to their low level of direct infection, we normalized for infection by
selecting only DC associated with CFSE-stained Lm-OVA DC from both subsets by cell
sorting. To achieve similar levels of infection, separated CD11b+ DC were infected with
at an MOI of 2 and separated B220+ DC were infected at an MOI of 10 for 24h. We then
sorted for infected cells (CFSE-Lm+ cells) and incubated both CFSE-Lm
+ and CFSE-Lm
-
DC subsets with naïve OT-I T cells for 72 h in a T cell priming assay. Mean
fluorescence intensity of CFSE-Lm+ cells for each subset was similar, indicative of
similar levels of infection on a per cell basis. T cells were then restimulated with OVA
peptide in the presence of Golgi Plug for 5 hours, followed by an intracellular cytokine
stain for IFN-γ and surface stain for CD8α. Fig. 10 depicts the T cell response to CD11b+
DC and B220+ DC normalized for Lm infection. There were no significant differences in
proliferation (Fig. 10A), IFN-γ production (Fig. 10B), or the division index (Fig. 10C) of
the naïve CD8+ T cells stimulated by either CD11b
+ or B220
+ DC when both infected
populations were compared. This indicates that Flt3L B220+ DC are fully capable of
presenting bacterially-encoded antigens and priming naïve CD8+ T cells comparably to
CD11b+ DC if directly infected. As a control, when uninfected (CFSE Lm
-) DC were
used as APC, neither the CD11b+ nor B220
+ cells were able to prime the CD8
+ T cells.
In conclusion, due to their low uptake capacity and lower frequency of direct infection,
B220+ DC are not as potent at presenting bacterial antigens and priming T cells as the
more phagocytic CD11b+ DC. However, when infected to equivalent levels, B220
+ DC
are quite capable of processing and presenting bacterial antigens and priming T cells.
57
Figure 10. T cell priming assays with CD11b+ DC and B220
+ DC infected with
CFSE-labeled Lm-OVA for 24 h and sorted for infected (CFSE-Lm+) versus
uninfected (CFSE-Lm-). (A) Dot plots of T cells primed by CD11b
+ DC and B220
+ DC
infected with CFSE-labeled Lm-OVA or non-treated cells. (B) Percent T cells that are
IFN-γ positive when primed with subset- separated Flt3L DC infected Lm-OVA labeled
with CFSE. (C) Division Index of T cells primed with subset- separated Flt3L DC
infected with Lm-OVA labeled with CFSE. (D) CD86 expression on B220+ DC that are
infected (CFSE-Lm+) at an MOI of 3 or uninfected (CFSE-Lm
-). Grey tinted line depicts
cells that were NT. Black line depicts CFSE-Lm+ cells. Dashed Dark Grey line depicts
CFSE-Lm- cells. (E) CD86 expression on CD11b
+ vs. B220
+ DC based on Lm infection
at an MOI of 3. All data are compiled from three independent experiments.
58
S
59
uSummary Statement. In conclusion, we have demonstrated that in vitro
derived Flt3L DC subsets respond to Lm quite differently in their immune response.
While Flt3L DC as a whole mature in response to Lm independent of cytoplasmic entry,
they sense vacuolar Lm in a MyD88-dependent manner. CD11b+
DC within the Flt3L
DC cultures are the major responders to Lm infection over B220+ DC. Although
cytoplasmic Lm can be found in CD11b+ DC and B220
+ DC, CD11b
+ DC were more
readily infected than B220+ DC. In T cell priming, the CD11b
+ DC were subsequently
better than B220+ DC at driving naïve OVA-specific CD8
+ T cells into proliferation and
producing IFNγ. However, when infection level normalized, the B220+ DC were
comparable to CD11b+ DC at priming CD8
+ T cells.
60
CHAPTER IV
IMMUNE RESPONSES OF SPLENIC DENDRITIC CELL SUBSETS TO AN INTRACELLULAR
BACTERIAL PATHOGEN, LISTERIA MONOCYTOGENES
L.M. Mitchell, J.G. Grayson, C.J. Henry, M.M. Westcott, J. Bates, and E.M.
Hiltbold
Portions of the following chapter will be submitted as the Results section of a
manuscript to the Journal of Immunology. L.M. Mitchell performed all of the
experiments. All mice were infected by Drs. J.G. Grayson and C.J. Henry. CD11c-
DTR-GFP transgenic mice were bred and supplied by Dr. J. Bates. M.M. Westcott
contributed to quantitation of splenic cell clustering. L.M. Mitchell and Dr. E.M.
Hiltbold prepared the manuscript.
61
INTRODUCTION TO CHAPTER IV
Infection with sub-lethal doses of wild type Lm induces the maturation of
dendritic cells in vivo (37, 85). In contrast, infection with even high doses of an avirulent
mutant Lm that cannot enter the host cell cytoplasm is not as effective at inducing DC
maturation in vivo (37, 85), and is equally ineffective in its ability to confer protection (7,
9, 44, 91). A range of dendritic cell subsets have been identified in lymphoid organs,
including the spleen (2, 46, 54). These resident, splenic DC include CD8α+, CD4
+, and
CD8α-CD4
- DC as well as the interferon-producing, B220
+ plasmacytoid DC (pDC) (2,
4), among others. While CD8α+ DC have been predominantly implicated in priming the
CD8+ T cell responses critical to protective immunity to Lm, the role of other DC subsets
in the immune response to Lm has not been defined. In particular, little has been done to
examine the maturation of individual DC subsets in the first 72 hours after infection.
The response to Lm has been well characterized in macrophages, and more recent
in vivo data support the role of DC in establishing Lm infection (89), yet it is not fully
understood how specific DC subsets respond to Lm infection. To this end, we sought to
determine how distinct DC subsets responded to both wild type and vacuolar Lm in vitro
as well as in vivo.
We therefore examined the maturation response of splenic DC subsets and their
capacity to activate naïve T cells following Lm infection. These findings offer novel
insights as to how individual DC subsets contribute to the response to an intracellular
pathogen, such as Lm and offer a potential explanation for why the vacuolar bacteria are
less potent at inducing protective immunity.
62
The number of splenic DC subsets following infection with wild type L.
monocytogenes decreases at 48 hpi. There are at least four previously described DC
subsets found in the spleen. In Fig. 11, we have shown the marker expression of total
splenocytes harvested from mock infected animals. The majority of cells in the spleen as
demonstrated by CD3 and CD19 expression are T and B cells. By excluding these
lymphocytes, we are able to observe the proportion of cells that are CD11c positive.
Further gating delineates populations of DC that are CD8α+, CD4
+, CD8α
-CD4
- (DNDC)
and B220+Ly6C
+ (pDC). From the percent of DC subsets we are thus able to estimate the
number of DC in the spleen in mock and Lm- infected animals. Our data demonstrates
that while the number of DC subtypes shifts (decreases) after 48 hours of infection with
wild type Lm (Fig. 12), the number of CD8α+ and CD4
+ DC in vacuolar Lm infected mice
remains steady. Surprisingly in vacuolar Lm- infected mice, the numbers of pDC and
DNDC at 48hpi is significantly increased. At 48 hpi, the overall number of all DC
subtypes begins to decrease as does the number of splenocytes in the wild type Lm-
infected mice and by 72 hpi, the number of splenocytes is significantly lower than mock
treated animals. In contrast, the numbers of splenocytes are significantly increased in
vacuolar mice in comparison to mock treated animals at 48 hpi and are remain high in
comparison to wild type Lm infected mice.
Splenic DC subsets mature following listerial infection. The extent of DC
maturation in specific splenic DC subsets has not been well established following i.v.
infection. We therefore determined how splenic DC subets responded to Lm infection
and how that response correlated with the development of protective immunity in vivo by
63
Figure 11. Splenic DC subset gating strategy. Whole splenocytes were stained for
CD3 and CD19 to exclude T and B cells, CD11c to delineate dendritic cells, then CD8α
and CD4, or B220 and Ly6C to delineate CD8α+ DC, CD4
+ DC, DNDC and pDC
(B220+Ly6C
+). Cells were excluded for dead, autofluorescent cells and gated on (A and
C) CD3- and CD19
- cells. (B) Cells then gated on CD8α
+ (bottom right gate), CD4
+ (top
left gate), or CD8α-CD4
- (bottom left gated) or in a separate gating strategy (D)
B220+Ly6C
+.
64
65
Figure 12. Splenic DC subset numbers decrease after 48 hours of infection. The
frequency of gated splenic DC subtypes was determined using FACSDiva analysis
software. The total number of DC subsets in the spleen for each timepoint was then back
calculated from this frequency using counts of total splenocytes harvested following
collegenase digestion using the following equation:
Number of DC subset = {% DC subset x [% CD11c+ x (% live x total # splenocytes)]}
Statistical analysis was performed by One Way ANOVA followed by a Tukey’s post test.
66
67
assessing the maturation of DC following infection with either bacteria that efficiently
induce protection (wild type Lm) versus a strain that requires a much higher dose to
induce protection (vacuolar Lm). We infected C57BL/6 mice with either 5x104 wild type
Lm or 5x108 vacuolar Lm and harvested total splenocytes at 12, 24, 48, or 72 hpi. We
examined the maturation state of the DC subsets at this time using the multistep gating
strategy described in the methods. The expression of CD86, CD80, and CD40 were
quantitated based on median fluorescent intensity compared to the level expressed by DC
from mock infected mice.
Among the cDC, we first examined the CD8α+ DC as these have been reported to
be critical for protective immunity to Lm and priming CD8+ T cell responses (8, 126).
We found that these cells strongly up-regulated the expression of CD86, CD80, and
CD40 upon infection with wild type Lm (Fig. 13) by 24 hpi. In contrast, the expression
of costimulatory molecules by CD8α+ DC in response to vacuolar Lm was barely above
the mock background at all times tested. The maturation response in the CD4+
DC
elicited by wild type Lm was significantly lower in magnitude as well as fold induction in
comparison to the CD8α+ DC (Fig. 13). However for wild-type Lm infected CD4
+ DC,
there was significant induction over the mock treated animals, and the peak of maturation
occurred at 48 hpi in contrast to the other DC subsets. Again, there was no significant
increase in costimulatory molecules on CD4+ DC in response to vacuolar Lm.
Interestingly, DNDC also did not appear to greatly upregulate CD86, CD80, or CD40 in
response to either wild type Lm or vacuolar Lm over that observed from mock infected
animals. Finally, we observed a strong maturation response in the pDC population
68
Figure 13. Splenic DC Subset Upregulation of Costimulatory Molecule Expression
is Dependent upon Cytosolic Entry by Lm and Peaks at 24 Hours Post Infection.
Splenocytes were harvested from mice infected with 5x104 wild type Lm, 5x10
8 cfu
vacuolar Lm, or mock treated with sterile PBS for 12-72h. Cells then stained for DC
subsets and surface costimulatory molecule expression. Costimulatory molecule
expression of (A) CD8α+ DC, (B) CD4
+ DC, (C) DNDC, and (D) pDC at 12-72 hours
post infection with 5x108 vacuolar Lm (white bars) or 5x10
4 wild type Lm (black bars).
Statistical analysis performed comparing to mock treated animals using a Two Way
ANOVA with a Bonferroni post test. * p<0.05, ** p<0.01, *** p<0.001
69
70
following exposure to wild type Lm (Fig. 13) that peaked also at 24 hpi. Like the B220+
DC in the Flt3L DC cultures, the absolute level of costimulatory molecule expression on
the splenic pDC was substantially lower than the cDC, but fold induction over
background was comparable. In contrast to what we observed in vitro in response to
vacuolar Lm, we did not see upregulation of costimulatory molecules
CD8α+ DC and DNDC have the highest bacterial load at 24 hours post
infection. Our in vitro data indicated that the pDC were not able to present bacterial
antigens effectively unless directly infected. Thus, we wanted to assess the level of direct
infection of the pDC and other DC subsets in vivo to address the possibility that these
cells are unable to prime protective responses to vacuolar Lm in vivo due to their level of
infection. For these experiments we infected groups of C57BL/6 mice i.v. with 5x105
wild type Lm. The higher dose was used to ensure that we could obtain detectable
numbers of bacteria in the spleen 24h post infection (when the peak of CD8α+ DC and
pDC maturation occurs). Following infection, spleens were harvested and cDC and pDC
selected by cell sorting. The sorted cells were then lysed and plated on BHI agar to
measure the number of viable bacteria associated with each subset. CFU in the liver were
also enumerated to ensure that mice had similar levels of systemic infection at 24 hpi
(Fig. 14). In Fig. 15, the data indicate that the CD8α+ DC, DNDC, and CD4
+ DC were
infected with low, but detectable numbers of bacteria, but even at the higher dose of
5x105 cfu were we not able to detect live bacteria associated with the pDC. To address
which subsets were infected with the vacuolar Lm, mice were infected with 5x108 cfu
vacuolar bacteria and the level of infection determined in splenic cDC and pDC (Fig.
71
Figure 14. Bacterial cfu in Liver. Livers were harvested from mice infected with
5x104 wild type Lm, 5x10
8 cfu vacuolar Lm, or mock treated with sterile PBS for 12-72h
and weighed. Livers then homogenized in sterile water and homogenate serially diluted
and plated on BHI for 48h at 37ºC. Colony forming units per gram of homogenate then
calculated. Vacuolar Lm depicted with blue circles. Wild type Lm depicted with black
diamonds. Students t test was used to determine significance between vacuolar Lm and
wild type cfu.
72
73
Figure 15. Splenic CD8α+ and DNDC both contain live Lm following wild-type
infection. Splenocytes were harvested from mice infected with 5x104 wild type Lm,
5x108 cfu vacuolar Lm, or mock treated with sterile PBS for 12-72h. Cells were then
positively selected for CD11c using CD11c microbeads and Miltenyi-Biotec columns.
CD11c+ cells were then sorted for DC subsets. Sorted DC subsets were then lysed in
water, serially diluted, and plated on BHI plates for 48 h at 37ºC. (A) Colony forming
units per 1000 DC subtype and (B) colony forming units of each DC per total
splenocytes from vacuolar Lm (white bars) or wild type Lm (black bars) infected mice.
Statistical analysis performed comparing wild type Lm values to vacuolar Lm infected
using a Two Way ANOVA with a Bonferroni post test. * p<0.05, ** p<0.01, ***
p<0.001
74
75
15A). In a pattern similar to the wild type Lm, we observed a low level of infection in the
cDC but no detectable vacuolar bacteria in the pDC. Thus, although we did observe
significant maturation of pDC in response to vacuolar Lm, these cells were not directly
infected. When the numbers of viable bacteria in DC from wild type Lm infected mice
were normalized to total splenocytes, we observed that there were no significant
differences between the number of viable bacteria in CD8α+ DC or DNDC per total
splenocytes (Fig. 15B). CD4+ DC have fewer bacteria than either CD8α
+ DC or DNDC
per total splenocytes; however when taking into account the relative numbers of DC in
the spleen, both CD8α+ DC are DNDC have similar numbers of bacteria.
CD8α+ DC and CD4
+ DC can prime naïve OVA specific CD8
+ T cells ex vivo.
To test the ability of sorted splenic DC subsets following infection with either wild type
Lm-OVA or vacuolar Lm-OVA to prime naïve antigen specific T cells, we performed T
cell priming assays ex vivo. While no splenic DC subset tested was able to stimulate
proliferation or IFNγ production of OVA specific CD4+ T cells (Fig. 16), we observed
that both the CD8α+
DC and, to a lesser extent, CD4+ DC were able to prime OVA
specific CD8+ T cells (Fig. 17). The CD8α
+ DC promoted T cells to enter several rounds
of proliferation as well as produce IFNγ. The CD4+ DC were also capable of inducing
proliferation but to a much lower level than that observed by the CD8α+ DC.
Interestingly, CD4+ DC did not activate the T cells to produce as much IFNγ as well as
the CD8α+ DC did. It should be noted that the T cells that did produce IFNγ in both the
CD8α+ DC and CD4
+ DC- stimulated cultures, produced the same overall level of IFNγ
as demonstrated by the similar MFI (Fig. 17A). Neither the DNDC nor the B220+ pDC
76
Figure 16. Splenic DC do not prime antigen specific CD4+ T cell responses to Lm
infection ex vivo. Splenic DC subsets were sorted from mice that were infected with
5x105 cfu wild type Lm-OVA, 5x10
8 cfu vacuolar Lm-OVA, or mock treated with PBS
for 24h, then plated in V bottom plates at a ratio of 2:1 (T cell:DC) with CFSE labeled
CD4+ OT-II T cells, specific for OVA323-339 for 72 h. An ICS was then performed for
intracellular IFN-γ production and the T cells then assessed for proliferation (dilution of
CFSE) by flow cytometry. (A) Dot plot of CD4+ T cell primed by DC subsets infected
with CFSE-labeled Lm-OVA, mock treated with PBS, or stimulated with exogenous
OVA323-339 peptide. (B) Percent T cells that are IFN-γ positive and (C) Division Index of
T cells primed by DC subsets infected with CFSE-labeled Lm-OVA, mock treated with
PBS, or stimulated with exogenous OVA323-339 peptide.
77
78
Figure 17. Splenic CD8α+ DC prime naïve, antigen-specific CD8
+ T cell
proliferation and IFN-γ production ex vivo and is dependent upon cytosolic entry by
Lm. Splenic DC subsets were sorted from mice that were infected with 5x105 cfu wild
type Lm-OVA, 5x108 cfu vacuolar Lm-OVA, or mock treated with PBS for 24h, then
plated in V bottom plates at a ratio of 2:1 (T cell: DC) with CFSE labeled CD8α+ OT-I T
cells, specific for OVA257-264 for 72 h. An ICS was then performed for intracellular IFN-γ
production and the T cells then assessed for proliferation (dilution of CFSE) by flow
cytometry. (A) Dot plot of CD8+ T cell primed by DC subsets infected with CFSE-
labeled Lm-OVA, mock treated with PBS, or stimulated with exogenous OVA257-264
peptide. (B) Division Index and (B) Proliferation Index of T cells primed by DC subsets
infected with CFSE-labeled Lm-OVA, mock treated with PBS, or stimulated with
exogenous OVA257-264 peptide.
79
80
induced the OVA specific CD8+ T cells to proliferate or produce IFNγ. This is likely to
do the level of antigen that the cells have as well as the level of costimulatory molecule
expression. While the DNDC do have measurable levels of live Lm, the level of
costimulatory molecule expression is similar to the mock background. In contrast, but
nevertheless just as detrimental, the pDC express high levels of costimulatory molecules
but are not associated with live bacteria. Thus either low costimulatory molecule
expression or too little antigen load results in lack of priming of this CD8+ T cell
response.
Following infection, wild type Lm is found clustered in the periarteriolar
lymphoid sheath while vacuolar Lm is predominantly in the marginal zones of the
spleen. Our in vitro findings with Flt3L DC showed that costimulatory molecules were
induced upon infection with both wild type Lm and vacuolar Lm. However, in vivo we
have shown that the induction of costimulatory molecules was dependent on cytoplasmic
entry of Lm. The maturation of response of DC subsets in vitro v. in vivo may be due to
the frequency of infection as well as the spatial location of wild type Lm and vacuolar Lm
to DC in the spleen. To further visualize how DC mediated immunity occurs in the
spleen, we took serial frozen spleen sections from mock treated animals, or mice infected
with 5x104 cfu wild type Lm or 5x10
8 cfu vacuolar Lm for 24h and stained to identify
zones/ cell types of the spleen (Fig. 18-20). Marginal zone (MZ) macrophages and
monocytes were distinguished by F4/80 or CD11b, T cell zones using CD3, and B cell
zones using B220 (Fig. 18). To visualize DC in the spleen we used CD11c-DTR-GFP
mice to observe where CD11c+ DC were in relation to the zones and Lm. The CD11c
+
cells in these mice constitutively express GFP.
81
Figure 18. Lm is associated with splenic DC and CD11b+ cell clusters in solely T cell
zones of the spleen during wild type Lm infection while vacuole-retained Lm is
found primarily in the marginal zones. Frozen spleen sections from (A) mock treated
C57Bl/6 or (B) CD11c-DTR-GFP (green) mice infected with 5x104 cfu wild type Lm or
5x108 cfu vacuolar Lm for 24h were stained for the indicated markers. First panel in (A)
mock is isotype control for Alexa Fluor (AF) 488, AF405, and AF594 secondary
antibodies. Arrows point to Central Arterioles. B = B cell zone; T = T cell zone; MZ =
Marginal Zone. Micrographs taken with a Nikon Eclipse C1si Laser Scanning Confocal
Microscope using the 20x water lens.
82
83
Mock treated spleens displayed bands of CD11b+ cells and F4/80
+ cells in the MZ
with few CD11b+ cells clustered within the T cell zones (Fig. 18A). Upon infection with
wild type Lm, there were increased CD11b+ cells were found in large clusters around the
PALS in addition to increased bands of CD11b+ cells in the MZ (Fig. 18B). These
CD11b+ cells within the T cell zones were associated with large numbers of Lm. There
were very few wild type Lm found in the marginal zone at this time point, with the
majority of bacteria found in the T cell zones of the spleen. In contrast to what we
observed in wild type Lm spleens, there were fewer, smaller-sized CD11b+-Lm clusters in
the T cell zones (Fig. 18B and 19). The CD11b+-Lm clusters present were again located
around the CA, but appear to contain fewer bacteria than those of the wild type Lm
infected mice. The vast majority of vacuolar Lm were found in the MZ of the spleen,
associated with CD11b+ cells there. The few vacuolar Lm that were found in the T cell
zones were either associated with the CD11b+ cell clusters or were scattered as individual
bacteria in the zone (Fig. 18B).
Following infection, wild type Lm is found associated with CD11c+ DC. DC
were also found in both the MZ and T cell zones (Fig. 18 and 20). The vast majority of
splenic DC in mock, wild type Lm infected, or vacuolar Lm infected mice did not stain
for CD8α or CD4. CD4+ DC were observed more often than CD8α
+ DC; however, CD4
+
and CD8α+ DC are both observed associated with both strains of Lm (Fig. 20B and E).
Upon wild type Lm infection, Lm was found associated with DC in the T cell zones,
whereas with vacuolar Lm infection, Lm was also found associated with DC but in the
MZ. Additionally, the vast majority of DC-Lm associations were with DC that did not
express either CD4 or, in separate sections, CD8α.
84
Figure 19. Quantification of CD11b-Lm clusters. T cell zones and the number of
CD11b-Lm clusters they contained were counted from frozen spleen sections from wild
type Lm (5x104 cfu) and vacuolar Lm (5x10
8 cfu) CD11c-DTR-GFP mice. Statistical
analysis was performed using Students t test. *p< 0.05, **p<0.01, ***p<0.001
85
86
Figure 20. Lm is associated with CD11c+ DC in both wild type Lm infected mice and
vacuolar Lm infected mice. Frozen spleen sections from CD11c-DTR-GFP (cells in
green) mice infected with 5x104 cfu wild type Lm or 5x10
8 cfu vacuolar Lm for 24h were
stained for the indicated markers. Arrows point to Central Arterioles. B = B cell zone; T
= T cell zone; MZ = Marginal Zone. Micrographs taken with a Nikon Eclipse C1si Laser
Scanning Confocal Microscope using the 20x water lens (A and D) or the 60x water lens
(B-C and E-F).
87
88
There are more CD86+ cells are found in wild type Lm-infected spleens than
in vacuolar Lm-infected spleens. We next determined if there was more mature DC
found in the spleens of wild type Lm infected mice than vacuolar Lm- infected mice. We
stained spleen sections for CD86 and observed that there are more CD86+ cells (both DC
and non DC) in the wild type Lm infected mice (Fig. 21). This is in contrast to what is
observed for vacuolar Lm infected mice where there was little CD86 staining, and while
the DC within the T cell zone in the vacuolar Lm spleens are low for CD86, these DC are
not associated with bacteria. This data suggests that consistent with our in vitro [Fig. 5
and (15)] and in vivo data (Fig. 13) that maturation of DC indeed occurs in a manner that
is dependent upon cytosolic entry.
Summary Statement. Herein we have shown that among the four splenic DC
subsets described, that the CD8α+ DC subset following infection with wild type Lm
increases expression of costimulatory molecules on its cell surface, which peaks at 24
hpi. This subset also has the highest detectable cfu of bacteria and upon incubation with
naïve OVA specific CD8+ T cell ex vivo are the most capable of priming those T cells to
proliferate and secrete IFN-γ. The CD4+ DC subset is also capable of activating naïve
CD8+ T cells to proliferate but to a lesser extent than their CD8α
+ DC counterparts.
There was also a defect in CD4+ DC to stimulate T cell IFN-γ production. CD4
+ DC had
increased costimulatory molecule expression that was dependent on cytoplasmic entry of
Lm. This expression, while increased at 24 hpi, was sustained and peaked at 48 hpi.
While DNDC and pDC did not prime CD8+ T cell proliferation or IFN-γ production, this
is likely to deficiencies in costimulatory molecule expression or antigen load,
respectively. We have lastly demonstrated that wild type Lm is in a vastly different
89
Figure 21. CD86 expression of wild type Lm infected spleens v. vacuolar Lm
infected spleens. Frozen spleen sections from CD11c-DTR-GFP (green) mice infected
with 5x104 cfu wild type Lm or 5x10
8 cfu vacuolar Lm for 24h were stained with CD86
(Alexa Fluor 594 in red) and Lm (Alexa Fluor 405 in blue). Arrows point to central
arteriole. T = T cell zone; MZ = Marginal Zone. Micrographs taken with a Nikon Eclipse
C1si Laser Scanning Confocal Microscope using the 20x water lens.
90
91
location within the spleen – in the T cell zones rather than the MZ like vacuolar Lm – and
that DC in the wild type Lm spleens have higher levels of surface CD86, consistent with
the protective properties of wild type Lm infection.
92
CHAPTER V
DISCUSSION
In summary of these findings, we have developed a model proposing how
immunity against Lm is formed and the role of individual DC subsets (Fig. 22).
Following i.v. administration of bacteria, Lm will traffic to the spleen via the blood, and
CD8α+ DC have viable Lm within 3 hours of infection with wild type Lm (89). CD8α
+
DC retain a comparable level of viable bacteria among the DC subtypes to DNDC and
CD4+ DC but not pDC at 24 hpi. The program of maturation that occurs with wild type
Lm infections is one that results in the upregulation of costimulatory molecules and
cytokine production (110) on resident DC over a period of 72 hours that peaks at 24 hpi
for both CD8α+ DC and pDC. There is also sustained costimulatory molecule expression
on CD4+ DC until 48 hpi. Substantial clustering of Lm with CD11b
+ cells observed by 24
hpi would be prime sources of antigen for CD8α+ DC and CD4
+ DC and reservoirs of Lm
replication in the spleen. With high levels of costimulatory molecules on their cell
surface and an adequate amount of antigen, both CD8α+ DC and CD4
+ DC migrate to the
T cell zone where they interact with and prime naïve CD8 (and CD4) T cells, important
for protective immune responses against Lm. In comparison, other DC populations that
have viable Lm (DNDC) and increased costimulatory molecule expression (pDC) appear
to be minor direct contributors to anti-Lm immunity.
In contrast, vacuolar Lm reaches the marginal zone and largely is walled off from
the T cell zone. Within the MZ, vacuolar Lm is scattered among the CD11b+ cells
93
Figure 22. Model of how immunity against Lm is formed. During an i.v. Lm
infection, Lm is brought to the spleen via the blood and enters the marginal zone (MZ).
In the spleen that is wild type Lm infected, CD8α+ DC contain viable bacteria by 3 hours
post infection then later in macrophages and CD8α- DC. In the hours following wild type
Lm infection, CD8α+
DC, CD4+ DC (both depicted in shades of blue), and pDC subsets
upregulate their levels of costimulatory molecule expression and cytokine secretion. DC
also begin to migrate from the MZ into the T cell zones. At 24 hpi there are large clusters
of Lm that are surrounded by CD11b+ cells, which are likely monocytic CD11b
+ DC
(from CD11c and CD11b double staining) or neutrophils. In contrast to wild type Lm
infection, infection with vacuolar Lm infection results in little to no upregulation of
costimulatory molecule expression on any DC subset over mock treated animals. At 24
hpi, there are smaller and fewer frequency of Lm clusters in the T cell zones, and the
majority of Lm is found in the MZ associated with bands of CD11b+ cells (likely
monocytes, neutrophils, or CD11b+ DC). Vacuolar Lm that is found in the T cell zones
are scattered and are also associated with CD4+ DC.
94
95
(likely macrophages, monocytes, and neutrophils), which will eventually clear Lm.
What few vacuolar Lm that make it into the T cell zones are associated with CD11b+ cells
and CD4+ DC as evidenced by microscopy and by bacterial viability assays. The DC
within vacuolar Lm– infected spleens do not upregulate costimulatory molecules or
secrete cytokines (110) to the same level as in wild type Lm infections and have
significantly lower levels of viable bacteria. The low level of DC surface costimulatory
molecules combined with smaller and fewer Lm clusters (and thus sources of antigen) in
the PALS results in inadequate T cell activate that is ultimately insufficient to prime anti-
Lm immunity. In the future as a mechanism of how movement into the T cell zone is
mediated, the chemokine receptor expression profile of CD8α+ DC and CD4
+ DC in wild
type Lm versus vacuolar Lm infections may provide vital information for how these two
DC subsets migrate into the T cell zone. It would also be interesting to visualize DC
subset clustering around antigen specific T cells that have been adoptively transferred in
as it would give us further evidence of the important role for individual subsets in
initiating protective immune responses.
Several studies, including our own have addressed the ability of Lm to stimulate
maturation of DC in vitro and in vivo as a means to understand the initiation of protective
T cell responses (15, 37, 85). With this body of work, we have extended these findings
by examining the responses of Flt3L-generated DC in combination with the responses of
splenic DC subsets to both wild type and vacuolar Lm. We have determined that Flt3L
DC sense Lm in a manner that does not depend on Lm cytosolic entry (Fig. 5) in contrast
to in vivo, where we have observed that there was significant maturation of the CD8α+
96
DC, pDC, and CD4+
DC subtypes in response to the wild type Lm infection over a period
of 72 hours but not to vacuolar Lm infection (Fig. 13).
The in vitro Flt3L DC system has allowed us the ability to study the molecular
mechanisms involved in DC sensing Lm. As an intracellular bacterium, Lm can
potentially trigger several pathogen sensing pathways in DC as it interacts with the cell
surface, is engulfed into a vacuole, and escapes into the cytosol (68). The TLR adapter
molecule MyD88, a component of most TLR pathways, has been demonstrated to be
critical in the response to Lm. Mice deficient in MyD88 are quite susceptible to Lm
infection in vivo (34, 99) and succumb earlier than mice deficient in IFN-γ or both IL-12
and IL-18 (99). Our in vitro data with Flt3L DC also support a key role for MyD88 in the
detection of Lm by DC in that both costimulatory molecules and cytokine responses were
diminished in the absence of this molecule. In fact, the response to the vacuolar Lm was
almost entirely dependent on this signaling adapter, in strong agreement with recent
findings in macrophages (67). The IL1R/IL-18R complex also signals through MyD88.
However, mice deficient in Caspase 1 (which is required for processing IL-1 and IL-18
into their active forms) are only slightly more susceptible than WT mice to Lm infection
in vivo (34, 115). We found no definitive role for Caspase 1 or for the IL-1 receptor in
the expression of costimulatory molecules or cytokine secretion following Lm infection
(using Caspase 1-/-
and IL-1R-/-
Flt3L DC), suggesting that the observed differences in the
absence of MyD88 may be attributed to TLR signaling rather than active IL-1β or IL-18
(Fig. 6). Disappointingly, after also testing TLR2, TLR7, and TLR2/9 deficient DC, we
have been unable to identify a single TLR or TLR combination that is critical for the
Flt3L DC response to Lm in vitro (Appendix Table 1). A previous study in macrophages
97
demonstrated that the responses to wild type Lm or LLO-deficient Lm was decreased
when TLR3, 7, and 9 signaling were disrupted via the Unc93b1 mutation, 3d (107).
Thus, the role(s) of these molecules in DC maturation induced by Lm may serve as
promising targets for future investigation in this arena. While we have yet to fully
understand how Lm is detected by DC, it is logical that several pathways are involved and
that the loss of any one sensor pathway might be compensated for by other pathways,
given the complexity of the bacteria.
A potential explanation for why the vacuolar bacteria may be poorer inducers of
effective T cell responses (and DC maturation) in vivo is localization of the bacteria
within the lymphoid organs. One in vivo study that attempted to address this issue used
an i.v. model of infection and focused mainly on heat-killed Lm (85). The authors
showed that following administration of killed Lm, costimulatory molecule expression
was increased on CD11c+ splenic DC, but to a lower level than when mice were infected
with wild type Lm. They further demonstrated that while wild type bacteria localized to
the T cell zones of the spleen following infection, both killed Lm and Δhly Lm were
absent from the T cell zones and localized to the red pulp and marginal zones. Thus,
although Flt3L DC in vitro may be equally responsive to wild type and vacuolar Lm, DC
may not come into contact with the vacuolar mutant as readily as wild type Lm in vivo
and may remain ignorant of the infection. This question was further addressed with our
confocal microscopy studies of spleen sections (Fig. 18-20), which demonstrated that
wild type Lm and vacuole-retained Lm were indeed found in different zones of the spleen.
Additionally, the CD11b+ cell clusters with which Lm is associated and the expression of
98
CD86 are also significantly different between wild type Lm-infected mice and vacuolar -
Lm-infected mice.
A more recent study related to the question of DC subset responses to listerial
infection used an intragastric model of infection (110). Consistent with our own data,
they demonstrated that cDC and pDC in the spleen upregulate costimulatory molecules in
response to wild type Lm. They further examined the cytokine production of these DC
subsets and reported that the IL-12, TNFα, and iNOS response was reduced in mice
infected with vacuolar bacteria. Their study, in contrast with our own studies, did not
examine the costimulatory molecule induction during the first 72 hours post infection,
rather examining the responses days 3, 5, and 7 post infections and showing that by day
5, costimulatory molecule expression is similar to those on mock infected mice. The
authors also did not examine the induction of costimulatory molecules induced by
vacuolar Lm. Thus we are the first to report the costimulatory molecule response of
splenic DC subsets following both wild type Lm and vacuolar Lm infection in the first 72
hours.
We also observed some interesting differences in the capacity of DC subsets to
take up Lm. Our results indicate that the CD11b+
DC become associated with fluorescent
bacteria much more efficiently than the B220+ pDC subset (Fig. 9). This is in agreement
with previously published reports on the relative uptake capacities of the two subsets (43,
72, 93). At least two previous studies have addressed the association of splenic DC with
live Lm in vivo. An early study used a sorting strategy to enumerate live bacteria in the
spleen following i.v. infection and found that the majority of Lm was not associated with
CD11chi
DC but primarily with CD11b+ cells (85). However, as we have previously
99
reported, CD11c+ DC are less permissive than macrophages for listerial cytoplasmic
entry, limiting bacterial growth (120), thus the first study may be an underestimate of the
total bacteria taken up by CD11c+ cells. A more recent study using an i.v. infection
model showed that CD8α+ DC are readily infected with Lm and that DC important for the
establishment of infection in the spleen. Other cell types such as CD11b+ DC and
macrophages were subsequently infected by Lm following CD8α+ DC infection (89). In
corroboration of this previously published work, we have shown that CD8α+ DC, CD4
+
DC, and DNDC, all contain viable wt Lm following infection (Fig. 15A). CD8α+ DC
trended towards higher numbers of viable bacteria per cell, however those levels were not
significantly different between CD8α+ DC and DNDC when normalized for total
splenocytes because CD8α+ DC are such a small population within the spleen (Fig. 15B).
With regards to the ability to activate T cells, the in vitro cultured Flt3L DC and
the two subsets contained within the cultures (CD11b+ DC and B220
+ DC) were able to
activate naïve, antigen specific CD8+ T cells (Fig. 8). When examined for naïve T cell
priming capacity, we found that the CD11b+ DC were superior to the B220
+ DC when
infected with Lm-OVA. One potential explanation for the diminished presentation
capacity observed in our B220+
DC was that they were infected to a much lesser extent
(Fig. 9), and as a result contained much lower levels of bacterial antigens available for
processing and presentation. Subsequently, bacteria were found in the cytoplasm of both
CD11b+ and B220
+ Flt3L DC, indicating that bacterial entry into the cytosol was not a
factor (Appendix Fig. 22). We also were able to confirm our in vitro findings by
demonstrating that cDC (CD8α+ DC, followed by the CD4
+ DC) are most capable of
priming CD8+ T cell responses to Lm-OVA but not pDC (Fig. 17).
100
The differences we observed in bacterial uptake capacity between the B220+ and
CD11b+ DC in vitro and the number of viable bacteria found in pDC in vivo are in
agreement with previous studies showing that pDC do not capture and process particulate
antigens as efficiently as myeloid DC (5, 43). Thus, the role of pDC in vivo may be to
enhance immune responses rather than direct priming of naïve T cells. We were, in fact,
able to demonstrate that B220+ DC are capable of priming naïve T cells when infected
with bacteria (Fig.10), indicating that the ability of B220+ DC to prime T cells is limited
by their uptake capacity. Other reports demonstrate that while freshly isolated pDC are
not efficient primers of CD8+ T cells, pDC activated by CD40 ligation or type I interferon
are capable of driving Th1 and Th2 differentiation of T cells (1, 8, 61, 93, 97).
Plasmacytoid DC have also been predominantly implicated in providing help and a robust
cytokine environment for other DC, promoting the expansion of T cells, and aiding in
regulatory T cell function (26, 61, 112, 124). Likewise, our studies favor a hypothesis in
which the maturation response exhibited by the CD11b+ DC within Flt3L cultures is
enhanced by the presence of the B220+ pDC. This hypothesis is supported by our data in
Fig. 7 showing enhanced costimulatory molecule expression on infected CD11b+ DC
within the whole Flt3L DC cultures and is a potential future direction of this work.
The low infection level of pDC in vitro was also observed in splenic pDC. While
these pDC did upregulate costimulatory molecule expression in response to wild type Lm,
viable bacteria were below detectable levels indicating that pDC do not have high live
bacterial titers and suggesting either (1) pDC are capable of bystander maturation without
direct infection or (2) perhaps pDC have antigen, but not large numbers of intact viable
Lm. When splenic pDC were given exogenous antigen, they were indeed able to prime
101
naïve, antigen specific CD8 and CD4+ T cells (Fig. 16 and 17). However – perhaps due
in part to their lack of viable Lm and thus a source of antigen – pDC were poor
stimulators of T cells. We have conclusively shown that Flt3L generated DC and splenic
pDC mature following infection with both wild type and vacuolar Lm (Fig. 5 and 13).
However, our results further indicate that pDC are inefficiently infected both in vitro and
in vivo and they are therefore not likely to present bacterial antigens or stimulate T cell
responses. Perhaps at higher doses of vacuolar bacteria (which have been shown to
stimulate protective immunity in some studies) these cells could become infected,
mature, and prime T cells by presenting bacterial antigens.
We are the first to report that the number of splenic DC subsets within the spleen
following infection decreases within 72 hpi when mice are infected with virulent Lm that
can access the cytoplasm (Fig. 12). This change in DC subsets is also accompanied by an
increase in costimulatory molecule expression that is dependent on Lm access to the
cytoplasm (Fig. 13), as well as, a migration of DC into the T cell zones of the spleen
(Figures 18-21). Maturation in response to wild type Lm peaks at 24 hpi for both CD8α+
DC and pDC, while increases in costimulatory molecule expression are sustained until 48
hpi for CD4+ DC. By 72 hpi, maturation (as well as number of total splenocytes and DC)
begin to decline. This decrease is not surprising, considering that the turnover of DC is
1.5-3 days and maturation in response to Lm would be a terminal event causing initially
mature cells to rapidly undergo cell death. Additionally, the signfiicant loss of
splenocytes overall by 72 hpi may be due to the efflux of T cells from the spleen to the
periphery. It is therefore likely that following 3 days and the eventual clearance of the
pathogen that resident DC numbers would return to normal levels. We have also
102
demonstrated that in vivo the maturation response to Lm was largely dependent on
bacterial entry into the cytoplasm because DC subsets from vacuolar Lm infected mice
did not increase costimulatory molecule expression. Accordingly in vacuolar Lm-
infected mice, we did not observe a decrease in number of total splenocytes or in the
number of DC subtypes (Fig. 12).
The ability to mature has implications on the ability of DC subsets to prime naïve
T cells and the initiation of protective immune responses. Similar to the maturation
response, the ability of DC subsets to prime naïve CD8+ T cells ex vivo was dependent on
cytosolic entry (Fig. 17). In addition, having great potential influence on the
development of a protective immune response are the Lm clusters within the T cell zones
in wild type Lm infections, which were not as prominent (in size or number) during
vacuolar Lm infections. It has been previously shown that responding neutrophils,
macrophages, and TNF/iNOS producing DC (TipDC) are important for controlling Lm
infection early during the immune response. Therefore, the large CD11b+ clusters, which
are likely neutrophils, macrophages, or monocytes/ dendritic cells, surrounding wild type
Lm would be significant sources of antigen to resident splenic DC.
Although we were not able to visualize associations of vacuolar Lm with CD8α+
DC, we did observe that the CD8α+ DC and CD4
+ DC were associated with both wild
type and vacuolar strains of Lm (Fig. 20). This is consistent with ability of CD4+ DC to
prime naïve CD8+ T cells ex vivo. CD11c
+ DC were also associated with wild type Lm
infections. However, the association of DC with Lm occurred in the PALS for wild type
Lm- infected mice, and the majority of DC-Lm interactions occurred in the MZ when
mice were infected with vacuolar Lm. Ultimately, the location of vacuolar Lm in the MZ
103
of the spleen would allow for the control and clearance of bacteria by neutrophils and
macrophages.
The lack large numbers of Lm found directly associated with CD8α+ DC in the
spleen via microscopy may be due in part to (1) the lower frequency of CD8α+ DC in the
spleen/ T cell zones as well as (2) the inability to distinguish CD8α expression on the
DCs by antibody in comparison to the higher level of expression on CD8+ T cells. It has
been shown that antibodies for other markers of CD8α+ DC (DEC-205 and CD207)
weakly stain sections from C57Bl/6 mice in comparison to what is observed via flow
cytometry or from sections of Balb/c and Balb/c x C57Bl/6 F1 mice (50). It is also likely
that while we are able to detect viable Lm in CD8α+ cells that this is an underestimate of
the overall level of antigen on these cells. CD8α+ DC have been demonstrated to
proficiently take up apoptotic cells as a source of antigen. Thus while we are not able to
visualize Lm near CD8α+ DC, it does not lessen the high level of costimulatory molecule
expression, bacterial load, and T cell priming capabilities of this DC subtype during wild
type Lm infections.
With the findings in this dissertation we have demonstrated that the entry of Lm
into the cytosol of CD8α+ DC and CD4
+ DC subsets results in a program of maturation
with significant implications. These two particular DC populations have been
demonstrated as important for priming CD8 and CD4+ T cells, and the activation of fully
functional CD8 and CD4+ T cell populations is important for not only primary immune
responses against Lm and its clearance, but are subsequently critical for the development
of protective immunity against Lm. These studies have highlighted that with regards to
Lm infection, both CD8α+ DC and CD4
+ DC are important responders to an intracellular
104
pathogen. This body of work also opens the door for continued investigation of the
impact of individual DC subsets on protective immune responses. Additionally, DC as a
whole are a vital component for establishing protection against pathogens such as Lm and
there remains much to be understood with regards to DC subsets, their development, and
their function.
105
APPENDIX
Table 1. TLR2, TLR2/9, TLR7, IL-1R, Caspase 1, and IFNαR are not required in
sensing Lm by Flt3L DC.
CD86 CD80 CD86 CD80
WT* Caspase 1-/-
wild type Lm +++ + wild type Lm ++++ +++
vacuolar Lm ++ + vacuolar Lm +++ ++
TLR2-/- IL-1R-/-
wild type Lm +++ + wild type Lm +++ +
vacuolar Lm ++ + vacuolar Lm ++ +
TLR2/9-/- IFNαR-/-
wild type Lm ++++ + wild type Lm ++++ ++
vacuolar Lm +++ + vacuolar Lm +++ +
TLR7-/- MyD88-/-
wild type Lm ++++ ++ wild type Lm + -
vacuolar Lm ++ + vacuolar Lm + -
*There were no significant decreases between WT and Knockout mice in any experiment other than MyD88-/- where values decreased (see Fig. 6) Values expressed in Median fluorescence Intensity
- <100
+ = 100-500
++ = 500-1000
+++ = 1000-2000
++++ > 2000
106
Figure 22. Micrographs of actA::gfp Lm- infected CD11b+ and B220
+ Flt3L DC.
Subset separated Flt3L DC were infected with actA::gfp Lm at an MOI of 3 for 3 h. DC
were then washed, harvested, and allowed to adhere to Poly (L)-Lysine coated coverslips.
DC were stained for total Lm (red channel) and DAPI (blue channel). Cytosolic bacteria
(see arrows) appear yellow (green, GFP plus red, total Lm). Non-cytosolic bacteria
appear only in the red channel. A, CD11b+
Flt3L DC infected with actA::gfp Lm. B,
B220+ Flt3L DC infected with actA::gfp Lm. Bar = 8 m
107
108
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SCHOLASTIC VITAE
Latoya M. Mitchell
Wake Forest University Graduate School of Arts and Sciences
Department of Microbiology and Immunology
5109 Gray Bldg, 1 Medical Center Blvd • Winston-Salem, NC 27157
Work 336.716.3301 • Fax 336.716.9928
EDUCATION
Wake Forest University Graduate School of Arts and Sciences, 2009
Winston-Salem, North Carolina
Ph.D. Candidate, Microbiology and Immunology
Advisor, Elizabeth Hiltbold Schwartz, Ph.D.
Furman University, 2004
Greenville, South Carolina
B.S., Biology
Advisor, Min-Ken Liao, Ph.D.
RESEARCH EXPERIENCE
Wake Forest University Graduate School of Arts and Sciences, 2004-2009
Dissertation: Dissecting the roles of distinct dendritic cell subsets in the
immune response to the intracellular bacterial pathogen, Listeria
monocytogenes
TEACHING EXPERIENCE
Wake Forest University Graduate School of Arts and Sciences, 2008
Designed and taught 3 hour lecture, titled Immunology I, and test
questions for post-baccalaureate students, introducing them to the
concepts of immunology before attending medicine school
Wake Forest University Graduate School of Arts and Sciences, 2007
Designed and taught lecture, titled Innate Immunity II, in the
Fundamentals of Immunology course for first year biomedical graduate
students
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ADVANCED COURSES
Introduction to Professional Development
Microscopy Research Techniques
Tutorial in Medical Microbiology
AFFILIATIONS
American Association of Immunologists, 2005- 2009
American Society for Microbiology, 2006-2009
Beta Beta Beta National Biological Honor Society, 2004
Charter Member of Pi Nu Chapter
PROGRAMS & PROFESSIONAL FORUMS
Graduate School Representative at the Fayetteville State University Graduate
School Fair, Nov. 2008
Graduate School Representative at the NC Alliance Meeting Graduate School
Fair, Oct. 2008
Graduate School Representative at the Ronald E. McNair Symposium Graduate
School Fair, Jan. 2008
Attended the NIH/ NIAID Bridging the Career Gap Workshop in Bethesda, MD,
2007
FUNDING
Salary, tuition, and travel supported by a Grant Supplement to NIH/ NIAID
Parent Grant 1R01 AI057770-01A1
AWARDS
Keystone Symposium on Dendritic Cells Minority Scholarship, 2009
American Association of Immunologists Trainee Abstract Award, 2008
American Association of Immunologists/ FASEB- MARC Program Travel
Award, 2006, 2007, and 2008
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CONFERENCE ABSTRACTS
Mitchell, L.M., J.M. Grayson, C.J. Henry, and E.M. Hiltbold. 2009. Differential
Responses of Dendritic Cell Subsets to Listeria monocytogenes in vitro and in
vivo. Keystone Symposium on Dendritic Cells.
Mitchell, L.M., J.M. Grayson, and E.M. Hiltbold. 2008. Dendritic Cell Subsets
Differentially Sense and Respond to Listeria monocytogenes Infection both In
Vitro and In Vivo. American Association of Immunologists Annual Meeting/
Experimental Biology.
Mitchell, L.M., J.M. Grayson, and E.M. Hiltbold. 2007. The Effects of Listeria
monocytogenes on Dendritic Cell Subsets. American Association of
Immunologists Annual Meeting.
Mitchell, L.M. and E.M. Hiltbold. 2006. Activation of Plasmacytoid Dendritic
Cells by the Intracellular Pathogen Listeria monocytogenes. American
Association of Immunologists Annual Meeting.
INVITED ORAL PRESENTATIONS
Mitchell, L.M., J.M. Grayson, and E.M. Hiltbold. 2008. Dendritic Cell Subsets
Differentially Sense and Respond to Listeria monocytogenes Infection both In
Vitro and In Vivo. American Association of Immunologists Annual Meeting/
Experimental Biology.
Mitchell, L.M. and E.M. Hiltbold. 2006. Activation of Plasmacytoid Dendritic
Cells by the Intracellular Pathogen Listeria monocytogenes. American
Association of Immunologists Annual Meeting.
PUBLICATIONS
Mitchell, L.M., C.J. Henry, J.M. Grayson, and E.M. Hiltbold. Differential
Responses of Murine Bone Marrow-Derived Dendritic Cell Subsets to an
Intracellular Bacterial Pathogen. Infection and Immunity. Manuscript under
review.
Mitchell, L.M., J.M. Grayson, C.J. Henry, M.M. Westcott, J. Bates, and E.M.
Hiltbold. The Role of Splenic Dendritic Cells in the Immune Response to Listeria
monocytogenes Infection. Manuscript in Preparation.
Ahmed M., L.M. Mitchell, S. Puckett, K.L. Brzoza-Lewis, D.S. Lyles, and E.M.
Hiltbold. 2009. M protein mutant vesicular stomatitis virus stimulates maturation
of TLR7-positive dendritic cells through TLR-dependent and -independent
mechanisms. J Virol [Epub ahead of print on Jan 14]
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Henry, C.J., D. Ornelles, L.M. Mitchell, K.L. Brzoza-Lewis, and E.M. Hiltbold.
2008. IL-12 Produced by Dendritic cells Augments CD8 T cell Activation through
the Production of the Chemokines CCL1 and CCL17. J Immunol 181 (12): 8576-
84.
Henry C.J., J.M. Grayson, K.L. Brzoza-Lewis, M.M. Westcott, L.M. Mitchell,
A.S. Cook, and E.M. Hiltbold. Dendritic Cell produced IL-12/ IL-23 Increases
the Protective Capacity of Memory CD8+ T Cells. Infection and Immunity.
Manuscript under review.