THE IMPORTANCE OF INFLAMMATORY CHEMOKINE RECEPTORS IN THE IMMUNE RESPONSE TO LEISHMANIA INFECTIONS

DISSERTATION

Presented in Partial Fulfillment of the Requirement for the Doctor of Philosophy

Degree from the Graduate School of The Ohio State University

By

Joseph James Barbi

*****

The Ohio State University 2008

Dissertation Committee: Approved by:

Dr. Abhay R. Satoskar, Advisor ______

Dr. Richard Mortensen Abhay R. Satoskar, Advisor

Dr. Virginia Sanders Graduate Program in Microbiology

ABSTRACT

Within the genus Leishmania there are several member species that are responsible for a spectrum of human diseases. The so called leishmaniases range in severity from life-threatening to self resolving, but all pose significant global health concerns. As the leishmania organisms are obligate intracellular parasites of phagocytic cells, elimination of this invader by an infected host depends largely upon the ability of the host to activate the microbiocidal defenses of parasite-harboring cells. From the study of host responses to leishmania infection, it was shown that both parasite- and host-factors contribute to leishmaniasis disease outcome. Among the factors of the host immune system employed to control parasite replication and spread are the chemokines. These small molecules interact specifically with g-protein coupled receptor molecules on target cell surfaces. As chemokine-chemokine receptor interaction can result in cellular migration and activation, it is not surprising that chemokines and their receptors play important roles in both disease and homeostatic processes.

ii The two-pronged objective of these studies was to evaluate the relevance of certain chemokine receptors to the disease outcome of

Leishmania major infection and to elucidate the mechanisms regulating the expression of a crucial chemokine receptor, CXCR3. In the following chapters we report the results of our inquiries into the importance of certain chemokine receptors to the host response during cutaneous leishmaniasis, a comparison of the roles played by the chemokine receptor CXCR3 in distinct manifestations of leishmaniasis, as well as our detailed dissection of the mechanisms regulating this particular chemokine receptor in murine T cells.

In summary, we found that functional CXCR3 signaling is required for murine resistance to Leishmania major, while that of CCR3 and CCR5 does not significantly influence the outcome of infection. Also, CXCR3 appears to mediate this resistance to L. major by recruiting IFNγ producing T cells to the site of infection. Interestingly, CXCR3-mediated T cell recruitment was found not to be important in the response to experimental Leishmania donovani infection. Studies of CXCR3 induction by T cells both in vivo and in vitro demonstrate that T cells from L. major-resistant mice strains dramatically up- regulate CXCR3 upon activation or challenge while T cells from an L. major- susceptible strain fail to do so. Further in vitro studies revealed that this differential CXCR3 expression stemmed in part from disparate IL-10 production and receptiveness. Additional studies into the requirements of T cells to induce

CXCR3 upon activation revealed that unlike CD4+ T cells, CD8+ T cells do not

iii require IFN-γ or the signaling molecule STAT1. Here we also present the results of further studies into the factors regulating CXCR3 induction by T cells.

These studies have improved our understanding of the role played by chemokine receptor-ligand pairs in anti-parasite immune responses. Also, these studies have revealed the identity of several aspects of CXCR3 regulation. These findings hold relevance, not only in the study of the anti- leishmania response, but they are also important for the development of therapies for numerous autoimmune diseases that depend upon CXCR3 activity.

iv

DEDICATION

For my Family

v

ACKNOWLEDGMENTS

Here I would express my gratitude to the many individuals who have aided me in both the completion of the aforementioned studies and my academic and scientific training to date. Particularly, the following individuals and their indicated contributions are recognized.

Firstly, I thank my mentor Dr. Abhay R. Satoskar for all the exciting opportunities and sound guidance he has provided throughout my Ph.D. studies. His high expectations and great enthusiasm for discovery are mostly to thank for the completion of these studies and my accomplishments under his lead.

Drs. Richard Mortensen, Virginia Sanders, and Paula Bryant: for the illuminating and helpful discussions of my work made at my committee meetings and candidacy exam. Also, for making the resources of their laboratories available to me, and furthermore for their thoughtful and constructive guidance.

vi Fellow Satoskar Lab members:

Dr. Lucia E. Rosas, Dr. Hisashi Hitori: for training, advice, guidance, and in the case of L.E.R., initial observation of the CXCR3-/- mouse phenotype during L. major infection.

Dr. Claudio Lezama-Davilla: for critical reviews of manuscripts and interesting discussions of results.

Tracy Keiser, Nickolas Zorko, Neeti Bhardwaj, Nicole Stauffer, Gabriel

Lockhart: for technical assistance and advice.

Steve Oghumu and Dr. Ian Fisher: for assistance in RNA extraction and molecular biology protocols.

Drs. Bao Lu, Craig Gerard, Andrew Luster, J. Farber, and Joan Durbin: for supplying various knockout mice critical for these experiments.

Dr. Luis I Terrazas: for giving me several opportunities to present and disseminate my findings.

Drs. Arlene Sharpe, Cox Terhorst, Harvey Cantor, Jon Serody, Jenny Ting,

Lishan Su, and Drew Pardoll for interesting discussions of my results.

Allison Flannigan for supplying IL-4R-/- mice.

Drs William C. Florence and Raj Nepal: for assistance in the standardization of antigen presentation assays.

Dr. Dwight D. Bowman, Dr. James B. Russell, Dr. Benjamin Lucio, and Ms.

Elizabeth Fogarty: for priceless research experience in the early stages of my growth as a scientist.

vii The Ohio State branch of Sigma Xi: for a grant-in-aid of student research.

The Herta Camerer Gross Memorial fellowship fund.

The Department of Microbiology at the Ohio State University: for financially supporting my training by way of an extended teacher‟s assistantship.

To all co-authors and contributors who have provided skills, reagents and/or training in the individual studies and articles that constitute this work.

And to Sarah Walker: for frequent advice, constant moral and technical support, and critical manuscript review.

viii

VITA

1981………………………………………...Born – Geneva, New York

1999………………………………………...Valedictorian – Lyons Central High School. Lyons, New York

1999-2003………………………………….B.S. Biology – Cornell University, Ithaca, New York

2003-present……………………………….Graduate Teaching and Research Assistant, The Ohio State University, Columbus, Ohio

PUBLICATIONS

1. Butkus MA, Starke JA, Bowman DD, Labare M, Fogarty EA, Lucio-Forster

A, Barbi J, Jenkins MB, Pavlo M. Do iodine water purification tablets

provide an effective barrier against Cryptosporidium parvum? Military

Medicine, v. 170 issue 1, 2005, p. 83-6.

2. Rosas LE, Barbi J, Lu B, Fujiwara Y, Gerard C, Sanders VM, Satoskar AR.

CXCR3-/- mice mount an efficient Th1 response but fail to control

Leishmania major infection. European Journal of Immunology. 2005

Feb;35(2):515-23.

ix 3. Rosas LE, Keiser T, Barbi J, Satoskar AA, Septer A, Kaczmarek J,

Lezama-Davila CM, Satoskar AR. Genetic background influences

immune responses and disease outcome of cutaneous L. mexicana

infection in mice. International Immunology. 2005 Oct;17(10):1347-57.

4. Rosas LE, Satoskar AA, Roth KM, Keiser TL, Barbi J, Hunter C, de

Sauvage FJ, Satoskar AR. Interleukin-27R (WSX-1/T-cell cytokine

receptor) gene-deficient mice display enhanced resistance to

leishmania donovani infection but develop severe liver

immunopathology. The American Journal of Pathology. 2006

Jan;168(1):158-69.

5. Rosas LE*, Snider H* Barbi J*, Keiser T, Satoskar AA, Papenfuss T,

Durbin J, Radzioch D, Glimcher LH, Satoskar AR. Cutting Edge:

STAT1 and T-bet play opposite roles in determining outcome of

visceral leishmaniasis caused by L. donovani. The Journal of

Immunology. 2006 Jul;177(1): 22-5. (*these authors contributed

equally to this work)

6. Barbi J, Oghumu S, Rosas LE, Carlson T, Lu B, Gerard C, Lezama-Davila

C, Satoskar AR. Lack of CXCR3 delays development of hepatic

inflammation but does not impair resistance to Leishmania donovani.

The Journal of Infectious Disease. 2006 Jun;195(11):1713-7.

x 7. Lezama-Davila CM, Isaac-Marques AP, Barbi J, Oghumu S, Satoskar AR.

17 -Estradiol increases Leishmania mexicana killing in macrophages

from DBA/2 mice by enhancing production of nitric oxide but not pro-

inflammatory cytokines. The American Journal of Tropical Medicine

and Hygiene. 2007 Jun;76(6):1125-7.

8. Barbi J, Oghumu S, Lezama-Davila C, Satoskar AR. IFN and STAT1 are

required for CXCR3 expression by CD4+ T-cells, but not CD8+ T-cells.

Blood 2007 Sep;110(6): 2215-6.

9. Barbi J, Snider HM, Satoskar AR. The role of chemokines in

leishmaniasis. Advances in Immunobiology of Parasites: Research

Signpost.

FIELDS OF STUDY

Major Field: Microbiology

Focus: Immunology

xi

TABLE OF CONTENTS

Section: Page:

Abstract ...……………………………………………..……...... ii Dedication ..………………………………………………..……… v Acknowledgments ..……………………………………………… vi Vita ..……………………………………………………………….. x List of Figures ..………………………………………………….. xiv Abbreviations ..………………………………………..………….xviii

Chapter 1. Chemokines And The Leishmaniases: An Introduction And Review Of The Current Literature …….….………………...... 1 1.1 Summary …...……………………………………….…...... 1 1.2 Background…………………………………………...... …… 2 1.3 Innate Immunity And Leishmania ...…………….…....…… 4 1.4 Chemokines In The Adaptive Response To Leishmania ……………………………………... 10 1.5 Distinctions Between The Leishmaniases……………… 19 1.6 Conclusion……………………………………….…...……. 23

Chapter 2. The Role Of Inflammatory Chemokine Receptors In The Host Response To L. major Infection. ..…………………...….... 25 2.1 The Role Of CXCR3 In Cutaneous Leishmaniasis caused By L. major………………………………………...... 25 2.2 The Role Of CD8+/CXCR3+ T Cells In The Response To L. major:…………………..……....…….. 37 2.3 Non-T Cells And CXCR3 Function During L. major Infection…………..……………………………….…... 41 2.4 CXCR3 And Antigen Presenting Cells………….………. 44 2.5 CXCR3-Ligand Mediated Killing Of Intracellular L. major By Macrophages As A Mechanism For Resistance……………………...... ….. 46 2.6 The Role Of CCR5 And CCR3 In The Host Response To L. major Infection ...…….……..……. 55 2.7 Materials And Methods ..……………………………...….. 60

xii Chapter 3. Regulation Of CXCR3 By T Cells………………………. 67 3.1 Differential Induction Of CXCR3 by T Cells Of L. major-Resistant and Susceptible Mice………………………………………….. 66 3.2 IFNγ And STAT1 Signaling In CXCR3 induction By CD4+ And CD8+ T Cells ...…………………..... 80 3.3 Materials And Methods………………………………...… 87

Chapter 4. The Role Of CXCR3 In Visceral Leishmaniasis….……. 90

Chapter 5. Future Directions And Concluding Remarks………….. 101 5.1 PI3kinase Gamma As A Regulator. Of CXCR3 Expression In T Cells…………………………….. 102 5.2 CXCR3 Induction And Other Aspects Of The TCR Signaling Cascade ...………….....…... 107 5.3 CXCR3 Expression On Memory T Cells ...………...…... 108 5.4 Materials And Methods……………………………..….… 112 5.5 Concluding Statement ...………………………….…..…. 113

List Of References ...……………………………………………..…… 114

Appendices ...……………………………………………………..…… 136

Appendix A: FIGURES 1-23 ..……………………………………. 137

Appendix B: FIGURES 24-33…………………………………….. 180

Appendix C: FIGURES 34-39…………………………………….. 200

Appendix D: FIGURES 40-43……………………………………. 212

xiii

LIST OF FIGURES

Fig. 1… Course of L. major infection in CXCR3+/+ and CXCR3–/– mice …………………………………………………………………… 138

Fig. 2… Infection of CXCR3-/- and CXCR3+/+ mice with an intermediate dose of L. major promastigotes in the ear pinna ...... 140

Fig. 3…Histopathology of L. major-induced lesions inCXCR3+/+ and CXCR–/– mice ………………………………………….…..………... 142

Fig. 4…Antibody responses in L. major-infected CXCR3–/– and CXCR3+/+ mice …………………………………………………….... 144

Fig. 5…Analysis of in vitro parasite-specific proliferation of dLN cells of CXCR3-/- and CXCR3+/+ mice …………………………………. 146

Fig. 6…Analysis of in vitro cytokine production by LmAg stimulated lymph node cells from L. major-infected CXCR3+/+ and CXCR3–/– mice……………………………………………………………………. 148

Fig. 7…Quantification of IFN-γ and IL-4 gene transcript levels in lesions of L. major-infected CXCR3+/+ and CXCR3–/– mice …… 150

Fig. 8…Analysis of CXCR3-expressing CD4+ and CD8+ T cells in lesions from L. major-infected CXCR3+/+ mice …………….…….. 152

Fig. 9…Enumeration of CD4+ and CD8+ T cells in lesions from L. major-infected CXCR3+/+ and CXCR3–/– mice ………………..... 154

Fig. 10…IFNγ production by T cells from CXCR3+/+ and CXCR3-/- mice infected with L. major …………..………………………...... 155

xiv Fig. 11…IFNγ production by T cells of the CXCR3+/+ and CXCR3-/- dLN and lesion during an in vitro APC assay ………...…………… 157

Fig. 12…CXCL10 responsiveness of CD4 and CD8 T cells from C57BL/6 mice infected with L. major .……………………………… 159

Fig. 13...Flow cytometric analysis of non-T cell populations in CXCR3+/+ and CXCR3-/- lesions following L. major infection ………..………………………………………………………………… 160

Fig. 14…Determining the role of CXCR3 in DC migration to LN tissues ……..…………………………………………………………... 162

Fig. 15…In vitro infection rate and infectivity of BMDM from wild type, CXCL9-/- and CXCL10-/- and CXCR3-/- C57BL/6 mice ...... …….. 164

Fig. 16…Indirect assessment of NO production by in vitro infected BMDM from C57BL/6 WT, CXCL9-/- and CXCL10-/- and CXCR3-/- mice………………...... 166

Fig. 17…CCR3 is expressed by the BMDM of C57BL/6 mice ………………………………………………………………………….. 167

Fig. 18…Analysis of the L. major-induced lesion growth and parasite burdens of infected C57BL/6 WT, CXCL9-/-, and CXCL10-/- mice ………………………………………………………………………….. 168

Fig. 19…Analysis of the cytokines made by the dLN cells from L. major-infected C57BL/6 WT, CXCL9-/-, and CXCL10-/- mice ex vivo upon re-stimulation by L. major antigen…………………………..... 170

Fig. 20…Course of L. major infection in CCR5+/+ and CCR5-/- mice ……………………………………. …………………………………… 172

Fig. 21…Analysis of ex vivo cytokine production by LmAg stimulated lymph node cells from L. major-infected CCR5+/+ and CCR5–/– mice …….……………...... 174

Fig. 22…Course of L. major infection in CCR3+/+ and CCR3-/- Mice on a BALB/c background ……………………………………………. 176

Fig. 23…Analysis of ex vivo cytokine production by LmAg stimulated lymph node cells from L. major-infected CCR3+/+ and CCR3–/–

xv mice…………………………………………………………………….. 178

Fig. 24…C57BL/6 T cells express higher levels of CXCR3 than those of BALB/c mice both in vivo and in vitro……..……………………... 181

Fig. 25…BALB/c and C57BL/6 derived T cells produce comparable levels of IFNγ, and despite the greater amounts of Th2 cytokine produced by BALB/c T cells, blocking IL-4 signaling did not alter CXCR3 expression by BALB/c…...... 182

Fig. 26…IL-10 suppresses CXCR3 expression by BALB/c T cells, but not those of C57BL/6…………………………..…………………….. 185

Fig. 27…BALB/c and C57BL/6 derived T cells express comparable levels of IFNγ receptor upon in vitro stimulation………………….. 187

Fig. 28…Transgenic BALB/c T cells do not up-regulate CXCR3 upon antigen-specific stimulation unless removed from stimulus and endogenous cytokines……………………….………………………. 189

Fig. 29…Flow cytometric analysis of CD49b+ (DX5+) NK cells in the lesion and LN of C57BL/6 and BALB/c mice during infection by L. major:…………………………………………………………………… 191

Fig. 30…IFN- and STAT1 are required for efficient induction of CXCR3 on CD4+ but not CD8+ T cells …………………….……….. 193

Fig. 31…TNF-alpha is not required for CXCR3 expression by CD8 or CD4 T cells …..………………………………………………………... 195

Fig. 32…STAT1 controls CXCR3 and T-bet gene transcription in CD4+ but not CD8+ T-cells ..…………………………………………. 197

Fig. 33…STAT1-independent mechanisms for CXCR3 induction by CD8+ T cells ………………………………………………………….. 199

Fig. 34…CXCR3-/- mice and wild type C57BL/6 control parasite burdens comparably and are both resistant to L. donovani infection …………………………………………………………..……………… 201

Fig. 35…CXCR3-/- mice show delayed liver granuloma formation, but eventually develop granuloma counts comparable to CXCR3+/+ mice ………………………………………………………..………………… 203

xvi

Fig. 36…Kinetics of the antibody response in L. donovani-infected CXCR3-/- and CXCR3+/+ mice at 15, 30, 45, and 60 days after infection ………………………………………………………….…… 205

Fig. 37…Kinetics of the cytokine response of LdAg-stimulated splenocytes from infected CXCR3+/+ and CXCR3-/- mice .…….. 206

Fig. 38…The liver infiltrate of CXCR3-/- mice induce cytokine mRNA as efficiently as that of CXCR3+/+ mice ……………………………. 208

Fig. 39…The CXCR3-ligands are not significantly reduced in CXCR3- /- mice infected with L. donovani …………………………………… 210

Fig. 40…Inhibiting PI3Kgamma suppresses CXCR3 induction following in vitro T cell activation …………………………………... 213

Fig. 41…CXCR3 uniquely requires PI3Kgamma for expression by T cells ……………………………………………………………………. 215

Fig. 42…PLC and CXCR3 regulation…………………………….... 217

Fig. 43…Expression of CXCR3 on memory T cells generated during L. major infection…………………………….……………………….. 219

xvii

COMMONLY USED ABBREVIATIONS

LN = lymph node dLN = draining lymph node i.p. = intraperitoneal i.d. = intradermal s.c. = subcutaneous

APC = antigen presenting cell

DC = dendritic cell

NK = natural killer cell

Treg = regulatory T cell

LFP = left foot pad

ELISA = enzyme linked immunosorbence assay

BMDM = bone marrow derived macrophages

IP10 = interferon gamma induced protein of 10 kDa (CXCL10)

MIG = CXCL9

ITAC = CXCL11

CL = cutaneous leishmaniasis

VL = visceral leishmaniasis

PMN = polymorphonuclear cell

TLR = toll-like receptor

xviii STAT1 = Signal transducer and activator of transcription 1

Tbet = Th1-specific T box transcription factor

GPCR = g-protein coupled receptor

xix

CHAPTER 1

CHEMOKINES AND THE LEISHMANIASES:

AN INTRODUCTION AND REVIEW OF THE CURRENT LITERATURE.

1.1 SUMMARY:

Studying the diseases caused by members of the genus, Leishmania have greatly expanded our understanding of several elements of the immune system. One such element is chemokine biology. The breadth of literature concerning the role of chemokines in the leishmaniases is impressive and at times convoluted (reviewed in references 1 and 2). Certain chemokines are required to resist parasitism, while others are not, or do so through unclear mechanisms. To shed light on this topic I shall summarize significant findings in the field, mostly gleaned from work in the mouse model, and human patients.

The results of these studies will be discussed along with the questions they inspire.

1

1.2 BACKGROUND:

THE CHEMOKINE SUPERFAMILY: The chemokines are structurally similar molecules ranging in size from 8-15 KDa. Structurally, they have a characteristic tetra-cysteine motif and are classified and systematically named based on the amino acid sequence at their N-termini (3). Chemokines function by binding specific G-protein coupled receptors (GPCR) and are not only involved in the mobilization and activation of immune cells, but these molecules also mediate homeostatic localization of various cells in their proper anatomic sites (4,5). Therefore, the chemokine and receptor pairs are often classified as either homeostatic or inflammatory in nature. In our studies we have explored the roles of various inflammatory chemokine receptors during leishmanial infection.

Chemokines have also been grouped functionally according to the cytokines produced by the cells they attract (6,7). Although some contend that the Th1/Th2 divisions are not absolute (8), the culminating research supports the association of chemokine production and chemokine receptor expression with one dominant arm of the adaptive immune response. The biological roles of the chemokines have been expanded beyond those of simple chemoattraction. Some “non-traditional” functions of chemokine-chemokine receptor interaction are highlighted by the study of chemokines in the leishmaniases.

2 THE LEISHMANIASES: Leishmania are obligate intracellular parasites transmitted by the bite of female sand flies of the genus Lutzomyia or

Phlebotomus. During a blood meal, motile, elongate promastigotes are deposited in the host dermal tissue. These flagellated parasites are taken up by phagocytes. Within these cells the parasites transform into the ovoid amastigote form. These protozoa then replicate protected within the acidic parasitophorous vacuole (9).

Different species of leishmania parasites cause strikingly distinct disease manifestations. Cutaneous Leishmaniasis (CL) caused by L. major or L. mexicana typically presents as a localized skin lesion ranging in form from nodular to ulcerating. However, complications of L. mexicana and L. aethiopica infection can result in diffuse cutaneous leishmaniasis (DCL) – a condition characterized by poorly contained lesions and an inefficient Th1 response that result in disseminated non-ulcerative skin lesions.

Mucocutaneous leishmaniasis, caused by L. braziliensis in the western hemisphere also results in poorly contained infection that spreads gradually usually causing extreme facial disfiguration (10). Generally, control of leishmania infection is associated with a strong Th1 response while susceptibility is by and large linked to elements of the Th2/humor response.

During the response to infection by the various leishmania species, the skin (or lesion) and skin-draining lymph nodes serve as the pathologically and

3 immunologically important tissues. However the tissue tropisms of other leishmania species differ significantly.

Visceral Leishmaniasis (VL), caused by L. chagasi and L. donovani, chiefly affects the organs of the recticuloendothelial system and in humans is fatal if not treated. This mortality associated with VL stems from secondary infection and complications from profound hepatosplenomegaly (9,11).

A trait common to all these parasites is their penchant for modulating aspects the host immune system to favor their own persistence (reviewed in references 12,13). Both extracellularly, and intracellularly, leishmania parasites exploit host defenses and among these are the chemokines.

1.3 INNATE IMMUNITY AND LEISHMANIA

The interplay between leishmania parasite and host defenses is a complex one. To best appreciate the multi-faceted interaction, it is useful to follow the invading organism from its arthropod vector through the host tissues.

When the pool-feeding sand fly introduces the promastigotes into the dermis, the parasites encounter a harsh environment in which they cannot persist. In order to survive the parasite must be taken up by phagocytic cells.

Paradoxically, while this step may be the first in the destruction of the parasite, it is also requisite for establishment of parasitism. Chemokines produced by macrophages permit phagocyte location by the promastigote and speed their eventual uptake. It has been shown that certain CC- family chemokines actually promote the uptake process (14). Interestingly, CCL3, CCL4, and

4 CCL5 were also shown to bind uncharacterized surface molecules on the parasite and elicit promastigote chemotaxis toward the chemokine source (15).

These observations may explain how promastigotes locate and enter macrophages, their desired cells of residence. Also of note, these studies suggest the existence of a G-protein coupled receptor (GPCR) molecule on the promastigote capable of interacting with host chemokines– a hitherto unknown phenomenon.

Although the leishmania parasites are adept at exploitation of host immune factors, including the chemokines, the host immune system is actually subverted before any parasite-specific factor comes into play. The sand fly saliva itself enhances parasite uptake by macrophages (16), reduces NO production (17) and alters the cellular composition of the initial infiltrate to favor parasite survival (18-20). Particularly, the saliva of the sand fly vector attracts

PMN cells and monocytes (16,21). Teixeira and colleagues have shown that salivary gland extract from Lutzomyia longipalpis induces CCL2 expression and can promote recruitment of potential host cells to the site of parasite deposition.

Interestingly, these studies showed that heightened CCL2 levels caused increased macrophage recruitment to the site of extract injection in susceptible mice, but not in resistant mice (22), perhaps indicating the existence of

CCL2/monocyte-independent parasite control mechanisms. Prior to the recruitment of these inflammatory cells, however, the key immunological events probably involve the parasite‟s interaction with cells patrolling the dermis.

5 SENTINELS OF THE SKIN: In the skin tissues, resident cells play an important role in the initiation of a chemokine cascade. These include keratinocytes, dendritic cells (DC), tissue macrophages and other cells capable of detecting pathogen products and initiating immune cell recruitment (23).

Typically, resident cells “sound the alarm” after detecting pathogen associated molecular patterns via Toll-like receptors (TLRs). Triggering TLR3 has been shown to induce the CXCR3 ligands, CXCL9 and CXCL11 – chemokines associated with a Th1 response. Also activation of TLR2 or TLR4 induces

CXCL8, CCL5, and CCL2 (24,25). These chemokines signal through receptors expressed by neutrophils and monocytes/macrophages that perpetuate the recruitment cascade at the site of infection.

It is not surprisingly, given these findings, that TLR4 is required for murine resistance to L. major (26). Interestingly though, the expression of chemokine receptors and many chemokines do not differ significantly in TLR4- deficient and wild type mice infected with L. major (27) suggesting that the main contribution of this activation pathway to the host response is not induction of protective chemokines.

Once inside macrophages, the parasite can alter the function of these immune cells in various ways. For instance, STAT1 signaling within leishmania-infected macrophages is sabotaged, as is phagocyte adherence to the extracellular matrix (28,29). Chemokine gene expression is not exempt from such immunomodulatory activity. Chemokines induced by leishmania

6 infection differ according to the species of parasite and may partially explain the differences in disease severity (as will be discussed later). But in general, infection of macrophages with L. major induces PMN-attractants including

CXCL1 (KC) and CXCL2 (MIP-2) induced in the skin following L. major infection

(30). These recruit the first infiltrating cells to the infection site. Also, the human chemokine CCL2 and its mouse homolog are induced by infection

(31,32). Interaction of this chemokine with its receptor, CCR2 results in monocyte, NK cell, DC as well as PMNs.

NEUTROPHILS AS TROJAN HORSES

As mentioned above, some of the initial chemokines made by cells residing at the site of experimental L. major infection include CXCL1 and

CXCL2 (30). Similar events transpire during in vitro infection of human and murine macrophages (31,32). Production of these chemokines results in the infiltration of PMN cells (30), which can also serve as host cells for leishmania during the early phases of infection (33).

Leishmania promastigotes are ill-equipped for long term extracellular survival in the mammalian host (34). An obstacle facing the promastigote is obtaining intracellular shelter. Compounding this, macrophages, the preferred cellular host of leishmania, do not infiltrate the skin in large numbers until 2-3 days post-infection. However, numerous PMN cells infiltrate the inflammatory site less than 24 hours post infection, and despite the cytotoxic nature of these cells, once infected, PMNs do not kill the parasites (20,35). Additionally,

7 harboring parasites has been shown to prolong the life of the ordinarily short- lived neutrophil, (36) which may increase the likelihood of parasite uptake by macrophages as they phagocytose the parasitized granulocytes. Such uptake by macrophages is immunologically “silent”, as opposed to the activating event that is typified by pathogen phagocytosis (37). PMNs are taken up by macrophages and anti-inflammatory mediators, such as TGF-β are released causing macrophage inactivity and silent uptake of parasite (33,38). Thus, through PMNs acting as “Trojan Horses”, Leishmania can gain entry into their host cells without triggering macrophage killing response. In support of this notion, prolonged influx of PMN cells at the site of infection is characteristic of a host susceptible to L. major (39).

Infected PMNs also release heightened CCL4 (MIP1-1β), a macrophage chemoattractant (33), perhaps luring in the next wave of host cells.

Promastigotes also modify host cell recruitment by releasing a chemoattractant of their own origin. van Zandbergen and colleagues found that cultured promastigotes secrete a protein of about 10-50 kDa, that in vitro promotes PMN chemoattraction. Also, promastigote contact causes increased production of

CXCL8, and decreased release of CXCL10. While the former chemokine is critical for neutrophil recruitment, the latter recruits T-cells and NK cells – sources of Th1 cytokines such as IFNγ (20). Therefore, this “leishmania chemotactic factor” can selectively enrich the cellular infiltrate at the infection

8 site with potential host cells and reduce the number of cells capable of promoting its demise.

Coupled with the PMN cell-attracting function of sand fly saliva, parasite and vector specific factors appear to cooperate to promote PMN-promastigote contact (16). These findings imply that PMN infection represent an important and likely chemokine-dependent conduit in the establishment of parasitism facilitated by parasite- and host-derived chemoattractants.

CHEMOKINES AND MACROPHAGES:

Both resident macrophages as well as monocytes recruited to the site of parasitism, participate in the immune response by producing chemokines that recruit other leukocytes to the site of infection. In addition, macrophages can directly mediate the killing of the parasites they harbor. This microbicidal action is increased by exposure to the cytokine IFNγ supplied by T-cells and NK cells

(40), but another macrophage activating signal is provided by certain chemokine-chemokine receptor interaction. Specifically, CCL2 and CCL3 (MIP-

1alpha) aid in macrophage killing of several leishmania species both in vitro and in vivo (14,41). CCL2 also synergizes with IFNγ to promote of further parasite killing (42), and lower expression of this chemokine may result in the development of DCL instead of the milder LCL following L. mexicana infection

(41). Also, CXCL10 also mediates in vitro killing of L. amazonensis (43). Prior to this work, expression of CXCR3 on inflammatory macrophages was not a

9 certainty. However, the specific role and importance of CXCL10 for in vivo parasite killing and control remain ill-defined.

In addition to resident macrophages and dendritic cells which initiate the chemotactic cascade, monocytes/macrophages recruited from the circulation are also important in leishmania. Monocytes are attracted to the sand fly bite by components of the vector‟s saliva. During later phases of the response, following the first waves of PMN cells, recruitment of monocytes as well as several other cell types is mediated by CCR2-binding chemokines.

Whether incoming macrophages are more likely to be the next wave of parasite hosts or the added killing machines needed to properly contain the invader likely depends upon the cytokine milieu at the site of infection.

Specifically, the Th1-dominated response is associated with resistance to several leishmania species (44). Cells of the adaptive immune system are needed to supply potentially macrophage activating cytokines. Chemokines are involved in both their generation and mobilization.

1.4 CHEMOKINES IN THE ADAPTIVE RESPONSE TO LEISHMANIA:

The notable contributions of chemokines are not limited to the innate immune response (1). Aside from their microbicidal activity, the phagocytic sentinels of the dermis bridge the innate and adaptive immune responses.

10 DENDRITIC CELLS: Dendritic cells in the skin (called Langerhans cells) migrate to the lymph nodes draining the cutaneous lesion during CL. The chemokines that mediate such relocation preferentially use the chemokine receptor CCR2. Guided by the ligands for CCR7 and CCR2, these cells transport parasite-derived antigens to the dLN (1). There these APCs, particularly dendritic cells (DCs), stimulate CD4 and CD8+ effector T-cells, the cells needed to successfully combat infection (45), Mice genetically deficient in this molecule mount a poor response to L. major, and display characteristics of a Th2-dominated, susceptible phenotype stemming from an inability of DCs to rendezvous with T-cells in the LN and activate them by presenting parasite- derived epitopes (39). This important chemokine-mediated juncture in the host response is also hindered by the actions of the parasite. A similar requirement for CCR7-mediated DC migration to the splenic T-cell rich zones has been demonstrated during visceral leishmaniasis caused by L. donovani (46).

Following contact with L. major, CCR2 and CCR5 are down-regulated by

DCs and their responsiveness to CCL2 and CCL3 is diminished. Interestingly,

CCR7 was up-regulated in these same cells (47). Such an altered receptor expression profile would still allow infected cells to migrate from the periphery to the dLN. This shuttling of infected DCs and phagocytes may be responsible for the spread of parasite to the secondary lymphoid organs and persistence of the parasites there (48).

11 Additionally, DCs influence the outcome of leishmaniasis through their ability to secrete CXCL10, a chemokine important for several aspects of the immune response to these parasites. CXCL10 is up-regulated in the dLN of L. major-resistant strains of mice, but not in susceptible mice. In fact, production of this chemokine by DC is seen only in resistant mice. One function of this chemokine is the retention of T-cells in the DC-T-cell cluster within the lymph node (49). Also a particular subset of DC is known to produce heightened levels of CXCL10 in the dLN (50).

NK CELLS AND THE DRAINING LN:

CXCL10 can also recruit and activate natural killer (NK) cells, potential sources of the cytokine IFNγ (51,52). These cells are known for their ability to recognize infected, stressed or otherwise altered cells through a culmination of inhibitory and stimulatory signals originating from the interplay of various cell surface molecules (53,54). Also, NK cells are capable of driving the Th1 response through interaction with DC. IFNγ production by NK cells induces IL-

12 secretion by DCs and therefore perpetuates a Th1 promoting loop (55,56).

NK cells and their Th1-priming activities have been suggested to play a role in immunity to Leishmania. They produce IFNγ and infiltrate the site of infection early during leishmaniasis (57). Martin-Fontecha and colleagues have shown that CXCL10 in the dLN can contribute to Th1 priming in these organs by recruitment of NK cells from the blood in a CXCR3-dependent manner (58).

12 Once there, they interact with DCs, push the cytokine balance towards a Th1 response, impacting the differentiating T-cells accordingly (58).

Despite these convincing reports as well as the fact that NK cells appear in infected tissue shortly after infection by several leishmanial species (1), the significance of these findings are uncertain. Mice devoid of NK cells, are more susceptible during the early phases of leishmaniasis, but are resistant in the long term (59). It remains to be seen if such temporary resistance can translate to heightened resistance later on in the human host. Also, Th1- priming of the LN cannot depend solely on CXCR3 since we have found that mice lacking CXCR3 are not deficient in dLN levels of IFNγ during CL (60 and

Chapter 2).

EFFECTOR CELL MIGRATION:

Another important aspect of the adaptive immune response to leishmania is the generation and mobilization of effector T-cells. These lymphocytes provide macrophage activating IFNγ, as well as other activating stimuli.

CXCR4 and CCR7 expression on T-cells is characteristic of a naïve phenotype, and responsiveness to the ligands specific for the latter

(CCL19/CCL21) mediates lymph node homing (61,62). When activated by

APCs, naïve T-cells give rise to effector and memory T-cells. A switch in chemokine receptor expression profile accompanies this transition. Expression

13 of lymph node homing receptors is lost in favor of those mediating cell traffic to the periphery (61,62).

The secondary lymphoid tissues are not the only ones that possess a characteristic recruiting axis. Other anatomic locations rely on their own chemokine-chemokine receptor pairs to mediate tissue-specific homing of certain cells. For instance activated CD4+ T cells are recruited to the skin in response to several inflammatory models by CXCR3 and CXCR6-CXCL16 signaling and are important for leukocyte recruitment to the inflamed liver as well.

Expression of chemokine receptors is not only an indicator of its tissue- specific destination, also the chemokine receptor expression profile is characteristic of the cytokine environment in which the cells are activated and the type of immune response the T cells themselves can promote (63-65).

Receptors linked to Th2 promoting cells include CCR3, CCR4, and

CCR8 while CCR5, CCR1, and CXCR3 are Th1-associated (6,7). These CD4+

T-cells, capable of activating macrophages with IFNγ, must reach the site of infection in order to participate in the containment and killing of the leishmania parasite (44,66). Similarly, CD8+ T-cells are required for primary and secondary resistance against CL (45,67).

Historically, the Th1 arm of the adaptive immune response has been associated with resistance to various forms of leishmania (reviewed in reference 44). Therefore, one would expect that “Th1 chemokines” would all

14 mediate recruitment of IFNγ-producing cells and thereby contribute to resistance. However, such a generalization is not accurate in light of recent discoveries. The dichotomy of Th1/Th2 immune responses and resistance/susceptibility has proven to be more complex than originally thought.

This is evident in a survey of investigations concerning the role of various chemokines during leishmaniasis.

CCR1

CCR1 is expressed on monocytes, lymphocytes, neutrophils and eosinophils, and despite being associated with the Th1 response, does not appear beneficial to the host during the early phases of infection. Mice deficient in CCR1 do not experience hindered T-cell or macrophage migration and are actually more resistance to L. major than CCR1-competent mice (68). Such findings suggest

CCR1 and its ligands (including CCL3) do not contribute to L. major resistance, possibly due the role of these molecules in neutrophil homing to the infection site.

CXCR3

Unlike CCR1, another Th1-associated chemokine receptor, CXCR3 has been shown crucial for resolution of CL. This receptor is expressed on DC subsets, neutrophils, NK cells, CD4+ Th1 cells, CD8+ T cells and B cells (69,70).

Three well studied ligands of CXCR3 include CXCL9 (MIG), CXCL10 (IP-10), and CXCL11 (I-TAC) (71,72). Induction of these chemokines are common during Th1 dominated immune responses (73). Receptor and ligands play a

15 critical role in recruitment of NK cells, neutrophils and T-cells to inflammatory sites in several disease models including allergic and autoimmune conditions

(74-76) and a variety of infectious agents (77-79).

Expression of CXCR3 in CD4+ T-cells depends upon T-cell activation and IFNγ (80) - and therefore the Th1 response. Also, expression of the ligands for this receptor depends on the signaling of this cytokine through the

STAT1 pathway used by IFNγ (73). Furthermore, expression of this chemokine receptor contributes to autoimmune diseases typified by an overactive Th1 response (81-83).

It was known that in patients suffering from CL, CXCL9 and CXCL10 are both produced in the lesion (84). Also, elevated levels of CXCL10 in the dLN during leishmaniasis is seen in strains of mice that usually heal their lesions

(47). These observations coupled with the role of these ligands in NK cell activation make CXCR3-mediated leukocyte recruitment a likely contributor to host resistance to CL. Therefore, using CXCR3 deficient mice, I and members of our group have evaluated the role of CXCR3 in CL caused by L. major and our findings are to be presented in detail (Chapter 2).

Briefly, normally resistant mice (C57BL/6) genetically deficient in CXCR3 fail to control parasite growth at the lesion site despite limiting parasite growth elsewhere, such as the dLN. This heighten susceptibility coincided with concurrent deficiencies in lesion IFNγ levels as well as CD4 and CD8 T-cell recruitment to the lesion (60). Therefore, recruitment of T-cells to the lesion

16 site, Th1 establishment there, and an efficient anti-leishmanial immune response depend upon CXCR3. Work by others have since bolstered the role of CXCR3 as a skin homing mechanism for T-cells (85) and its importance in mediating the migration of Th1 cells to the periphery (86).

CCR5

Recently, the role of CCR5 in leishmaniasis has become increasingly complicated. Recognized as a Th1-associated chemokine receptor, its up- regulation by CD4+ T-cells requires IL-12 (87). It was expected that this chemokine receptor should mediate resistance to leishmania. - especially since one of its best studied ligands, CCL5, has been shown to aid host resistance to

L. major (88). However, as will be discussed later, we and others have found

CCR5‟s role in leishmaniasis to be a convoluted one.

Despite its Th1-association (7,89) and CCR5 expression on skin-homing

T-cells (90), CCR5 deficient mice are not overly susceptible to L. major (39) or

L. donovani (91) . However, some report that CCR5-/- mice actually have lower parasite loads than wild type controls when infected with L. major (92) suggesting a role in CL susceptibility.

Naturally occurring regulatory T-cells (Tregs) are known to accumulate in the lesion tissue of L. major infected mice and the expression of CCR5 by these cells and in vitro responsiveness to CCR5-ligands have been shown. These findings suggest that CCR5-mediated Treg recruitment contributes to the suppression of Th1 immunity and the persistence of parasitism in these animals

17 (92). Supporting this notion Brazilian patients carrying a mutation that renders

CCR5 non-functional are reported to display less severe manifestations of

Leishmania braziliensis than their CCR5-competent counterparts (93).

However, the role of naturally occurring Tregs in exacerbating CL, no matter how intuitive, is not clearly defined. In fact, a recent study found that a significant amount of the immune-suppressing cytokine, IL-10 was in fact made by non-Treg cells with a Th1 phenotype. Furthermore, the suppression of the

Th2 response by Tregs actually contributed to CL resistance (94). How Tregs appear to mediate a Th2-specific suppression in leishmaniasis or any model is not known. In light of these observations, it is clear that the roles of both CCR5 and Tregs in leishmaniasis require further elucidation.

CCR3

The chemokine receptor CCR3 is expressed highly by eosinophils. The role of this receptor and its traditional ligands in the recruitment of eosinophils to the lung during allergic airway inflammation is well studied. However, CCR3 is expressed by macrophages and Th2 cells as well (202). Given this association it stands to reason that CCR3‟s contribution, if any to the disease outcome of

CL would be one leading to host susceptibility. Further suggesting that CCR3 signaling promotes a counterproductive response to Leishmania infection is the finding that the ligands of this receptor and the Th1-associated CXCR3 antagonize each other‟s normal signaling (204). Interestingly I found that deletion of CCR3 from susceptible mice did not enhance their resistance to L.

18 major infection. This suggests that either the Th2-linked receptor does not in fact promote susceptibility or that any such susceptibility promoting effects are relatively small compared to strain-dependent determinants of susceptibility.

1.5 DISTINCTIONS BETWEEN THE LEISHMANIASES:

Despite beginning their host-interactions under quite similar circumstances, the different species of leishmania cause disease manifestations that differ markedly. Also, the immune factors important for host resistance to these different parasites differ as well. For instance, IL-12-driven

CD4+ Th1-type lymphocytes play a critical role in immunity to L. donovani (95).

However, the clear Th1-Th2 pattern of disease development demonstrated for

L. major is not observed in VL caused by L. donovani in mice and humans because Th2 cytokines such as IL-4 and IL-13 do not determine susceptibility to the parasite (96). In fact, IL-4-/- and IL-4Rα-/- mice are more susceptible to L. donovani than wild type mice, and respond poorly to anti-leishmanial drug therapy indicating that IL-4 may play a disease protective role during VL

(97,98). Given the different roles played by these cytokines in VL and CL, one might expect that chemokines too might have disease-specific roles in the responses to these diseases. Indeed, recent work by our group and others in the mouse models of L. major and L. donovani confirmed such a notion.

19 Another point of distinction between the role of chemokines in cutaneous and visceral diseases is the importance of CCR7 and its ligands. CCL21 and

CCL19, are important for the migration of DC from the marginal zone of the spleen to the periarteriolar lymphoid sheath region (PALS). This anatomical region hosts the critical interaction of T-cell and APC. DCs from L. donovani chronically infected mice fail to make this migration (46). Also, mice deficient in

CCL19 and CCL21 generate less IL-12, and experience diminished hepatic T- cell recruitment and granuloma formation (109). Interestingly CCR7 expression by DCs as well as the responsiveness of these cells to CCL21 is actually enhanced by L. major infection (47), suggesting a very different role for CCR7 in responding to skin-dwelling leishmania species.

Infection by L. amazonensis, which unlike L. major presents a non- healing phenotype in many mouse strains seems to suppress certain chemokines made in response to other leishmania parasites. Compared to an

L. major infection, L. amazonensis caused lower expression of multiple chemokines including CCL5, MIP1-alpha and –beta, MIP-2. CCR5, CCR1, and

CCR2 expression were also hindered by L. amazonensis (110). It has been suggested that this modulation of chemokine/chemokine receptor expression after infection is a mechanism by which more virulent parasites may subvert the

Th1 response and promote their own persistence (1).

20 Chemokines may play different roles in leishmaniasis caused by members of the same parasite species. Two genetically distinct isolates of L. braziliensis induce unique chemokine profiles. Using an in vivo air-pouch model, it was found that within hours of infection, the more pathogenic strain induced high levels of CCL2, CCL3, CXCL1, and the chemokine receptors

CCR2, CXCR2, and CCR1. The less virulent strain induced CXCL10 only

(111). Interestingly, though, the relationship between L. major virulence and chemokine induction appears to be the opposite of that which has been reported for L. braziliensis. In vitro macrophages showed greater induction of

CCL2 and CXCL1 by infection with less virulent parasite compared to more pathogenic L. major strains (32).

Following infection by L. donovani, the cells of the murine liver produce high levels of CXCR3-ligand (99). Their interaction with CXCR3 regulates recruitment of T-cells into the liver during both infectious and non-infectious diseases (100,101) implicating this receptor-ligand axis as an important part of leukocyte trafficking to the liver. Furthermore, patients suffering from VL show elevated levels of CXCL9 and CXCL10 in their sera during active infection

(102), suggesting a crucial role for CXCR3 and its ligands in VL. Based on these observations, we investigated the role of CXCR3 in the response to L. donovani infection.

21 Despite the suggestive observations mentioned above, and the documented importance for CXCR3 in CL, murine resistance to the visceral L. donovani does not depend upon this chemokine receptor. During CL caused by

L. major, CXCR3 is needed for lesion-homing T-cells to reach the infection site and promote a Th1 response (60). However, CXCR3 is not required for leukocyte recruitment to the liver and spleen or the control of parasite growth in these organs during L. donovani infection. Also, despite being initially stymied, granuloma formation in the liver of CXCR3 deficient mice was normal sixty days post infection (103).

Such a discrepancy may be due to the different tissues involved in CL and VL. Chemokine receptors play organ-specific roles in regulating cell traffic.

Recruitment of naïve lymphocytes to the secondary lymphoid organs is mediated by the action of CCR7 and its ligands (36) whereas CCR9 mediates early T cell localization in the thymus (104). Similarly, among the so-called inflammatory chemokine receptors, CCR4 and CCR10 are involved in recruitment of certain T-cell subsets to the skin (105,106). Our findings suggest that although CXCR3 is necessary for optimal recruitment of T-cells to skin, other mechanisms efficiently recruit them to the spleen and liver tissue during

VL.

A possible liver-specific recruitment axis involves CXCL16, a ligand for

CXCR6, which is key for leukocyte recruitment to the liver in several disease models (107,108). CXCL16 mediated recruitment may be a dominant

22 chemokine for liver specific homing in VL. Studies combining neutralization of the CXCR3 ligands and CXCL16 should prove enlightening in this respect.

These findings, while demonstrating the distinct immune factors required to resist various leishmania infections, have also greatly complicated our concept of the chemokine‟s role in leishmaniasis.

1.6 CONCLUSION:

Chemokines are key in the immune response to many infectious agents and the leishmania species are no exception. These molecules not only mediate the cellular events needed to control the parasite, but they also are exploited by the parasite. From our in vivo studies of chemokine receptor knockouts it seems that of the “inflammatory chemokines” several appear particularly important for resisting leishmania while others appear dispensable for an effective response.

Still others may have complex and ill-defined roles in this model. Also, the importance of a given chemokine varies from one leishmanial species to another, as do the chemokines elicited by infection.

Lastly, much of this subject remains to be elucidated, leaving vast potential for future study and innovation. For instance, the factors regulating chemokine receptor expression are still being explored, and while we report several previously unknown intricacies of CXCR3 regulation here, still more work remains to be done. Also, the role of chemokines in the memory response to Leishmania infection has not been explored. As yet no widely useful vaccine

23 exists to prevent Leishmania infection. With the revelation that a large percentage of antigen-independent central memory T cells express CXCR3, it is likely that at least this chemokine/chemokine receptor axis will attract a great deal of research attention from those interested in developmenting future vaccine strategies. Additionally understanding chemokine receptor regulation will provide the basis for potential therapies modulating chemokine or chemokine receptor expression to the host advantage or to combat chemokine- dependent immunopathologies. Manipulation of chemokine receptor expression can be used to either enhance or inhibit cellular responses. These are certainly exciting avenues of research in the field of chemokine and parasite biology.

24

CHAPTER 2

THE ROLE OF INFLAMMATORY CHEMOKINE RECEPTORS IN

THE HOST RESPONSE TO L. MAJOR INFECTION.

2.1 The Role of CXCR3 in Cutaneous Leishmaniasis caused by L. major.

Abstract:

Chemokines play a critical role in recruitment of leukocytes to the site of infection, which is essential for host defense. We analyzed the role of CXC chemokine receptor 3 (CXCR3) in the control of cutaneous leishmaniasis using

CXCR3–/– C57BL/6 mice. We found that Leishmania major-infected CXCR3–/– mice mount an efficient Th1 response as evident by markedly increased serum levels of Th1-associated IgG2a and significant production of IFNγ and IL-12 by cells of the draining lymph node (dLN), restrict systemic spread of infection, but fail to control parasite replication at the site of infection and develop chronic non-healing lesions. Furthermore, the inability of CXCR3–/– mice to control cutaneous L. major growth was associated with lower levels of IFNγ in their

25 lesions as compared to CXCR3+/+ mice. CXCR3-/- mice also recruited fewer

CD4+ and CD8+ T cells to the lesion than CXCR3+/+ mice, and this deficiency in T cell recruitment likely accounts for this reduced cytokine production in

CXCR3-/- mice. These results demonstrate that CXCR3 plays a critical role in the host defense against cutaneous leishmaniasis caused by L. major.

Furthermore, they also suggest that the susceptibility of CXCR3–/– mice to L. major is due to impaired CD4+ and CD8+ T cell trafficking and decreased production of IFNγ at the site of infection rather than to their inability to mount a parasite-specific Th1 response.

Introduction:

Leishmania species are obligate intracellular parasites that are of extensive public health importance in the tropical and subtropical regions of the world (9).

In humans, cutaneous leishmaniasis caused by Leishmania major commonly manifests as a self-healing skin lesion followed by the development of long-term immunity. Several studies have shown that an IL-12-driven Th1 response and

IFN-γ production are critical for the control of L. major infection in genetically resistant mice, such as CBA/J, C57BL/6, and C3H (112-114, 66). On the other hand, the development of non-healing lesions in susceptible BALB/c mice is associated with the preferential induction of a Th2-like response associated with production of IL-4 and IL-10 (66, 116). Recent studies have shown that chemokines play a critical role in the development of innate as well as acquired

26 immunity against a variety of viruses, bacteria, fungi, and parasites (119, 120,

68). The protective role of chemokines has been attributed to their ability to activate leukocytes and facilitate their recruitment to the site of infection (121,

122) distinct patterns of chemokine secretion have been observed in polarized

Th1 and Th2 cells, suggesting that Th1 and Th2 subsets may differentially contribute to leukocyte migration into the site of inflammation (126). CXC chemokine receptor 3 (CXCR3) is expressed on natural killer (NK) cells, certain

T cells, B cells, and micro-vascular endothelial cells in S/G2–M phase of their life cycle (69). Three CXC chemokines, CXCL9 (Mig), CXCL10 (IFN-inducible protein 10, IP-10) and CXCL11 (IFN-inducible T cell alpha chemoattractant,

ITAC), signal via CXCR3 (72, 71), activate Ras/extracellular signal-regulated kinase (ERK), Src, and the phosphatidylinositol 3-kinase/Akt pathway, and mediate their biological functions such as cell migration and proliferation (127).

Many clinical studies have implicated a role for CXCR3 in the pathogenesis of several inflammatory diseases such as multiple sclerosis, psoriasis, and rheumatoid arthritis by facilitating the recruitment of pathogenic T cells to the site of inflammation (128-131). Moreover, experimental studies using a cardiac transplantation model show that CXCR3–/– recipients and anti-CXCR3 Ab- treated wild type mice exhibit prolonged survival of a cardiac allograft associated with a marked decrease in recruitment of CD4+ and CD8+ T cells, but not macrophages, into the transplanted organ (81). Moreover, recent studies have shown that CXCR3-signaling chemokines also regulate early

27 granuloma formation by neutrophils following Mycobacterium tuberculosis infection (77) and eosinophil recruitment in early delayed-type hypersensitivity

(132). Previous studies have shown that CD4+ Th1 cells preferentially express

CCR5 and CXCR3, which may regulate migration of these cells into the site of inflammation (130, 133, 6). Nevertheless, others have shown that CD4+ Th2 cells also express high levels of CXCR3 (134, 8). Furthermore, expression of

CXCR3 on individual peripheral CD4+ memory T cells does not correlate with their ability to produce IFN-γ (8, 80). The critical role of CXCR3 in Th1 trafficking has been suggested in a recent study which showed that blockade of

CXCR3 with anti-CXCR3 Ab diminishes the recruitment of Th1 cells into the peritoneal cavity following adjuvant-induced peritonitis (134). However, it remains to be determined whether CXCR3 regulates development of the host- protective Th1 response during infection with intracellular pathogens. The goal of this study was to explore the in vivo role of CXCR3 in regulation of host immunity against an intracellular protozoan parasite, L. major, which causes cutaneous leishmaniasis. Therefore, we analyzed the course of L. major infection and immune responses in L. major-resistant C57BL/6 mice lacking

CXCR3 gene (CXCR3–/–) and compared them with that in wild-type

(CXCR3+/+) counterparts. Our results demonstrate that CXCR3 plays a critical role in the control of cutaneous L. major infection.

28 Results and discussion:

The novel observation in this study was that CXCR3–/– C57BL/6 mice were susceptible to cutaneous L. major infection and developed chronic non-healing lesions following inoculation with 2x106 L. major stationary phase promastigotes

(SPP; Fig. 1A). Furthermore, footpads from both CXCR3+/+ and CXCR3–/– mice contained a comparable number of parasites during the early phase of infection; however, as infection progressed, footpad parasite loads increased rapidly in CXCR3–/– mice, which displayed 4–5 log more parasites in their footpads than CXCR3+/+ mice (Fig. 1B). Injection of a more physiological parasite number in the ear pinna of CXCR3+/+ and CXCR3-/- mice yielded similar results. CXCR3-deficient mice developed larger lesions containing heavy parasite burdens that did not resolve unlike CXCR3 lesions (Fig. 2A and data not shown). Also we compared CXCR3-/- and CXCR3+/+ lesions after an extended period of infection. We found that after 15 weeks of infection wild type lesions harbored few viable parasites, barely above the limit of our assay‟s sensitivity, but CXCR3-/- lesions still experienced extensive parasitism.

Histological analysis revealed that by week 8 post-infection, footpads from CXCR3–/– mice showed extensive s.c. tissue destruction that was marked by diffuse inflammatory infiltrate comprised of heavily parasitized macrophages, whereas those from CXCR3+/+ mice displayed preserved skin and an inflammatory infiltrate predominantly comprised of lymphocytes and

29 macrophages containing few or no parasites (Fig. 3). Interestingly, despite their inability to control parasite replication at the site of inoculation,

CXCR3–/– mice were able to control parasite loads in their lymph nodes and prevent the systemic spread of infection as efficiently as the CXCR3+/+ mice (Fig. 1C) .Similarly, no parasites were detectable in spleens from both groups at week 8 post-infection (data not shown).These findings demonstrate that CXCR3 plays a crucial role in the host defense against L. major by regulating the immune response required for controlling parasite growth at the site of infection.

Several studies have implicated a role for chemokines in regulation of the immune response during Leishmania infection (41, 32, 36, 31, 52, 39, 135,

84). For example, patients suffering from cutaneous leishmaniasis (CL) show a significant increase in levels of CCL2 (MCP-1) and CCL3 (MIP-1a) mRNA in their lesions (41). Similarly, infection of mouse and human macrophages with L. major promastigotes induces expression of several chemokines such as CCL2,

CXCL8 (IL-8), and MCAF (32, 48, 31). Studies using mouse L. major model have shown that the draining lymph nodes from disease resistant C3H and

C57BL/6 mice contain significantly higher amounts of CCL2, CXCL10 (IP-10), and XCL1 (lymphotactin) mRNA than those from susceptible BALB/c mice (52,

135). L. major-infected C57BL/6 mice also display increased levels of CXCL10 in their lesions (27). On the other hand, C57BL/6 mice lacking the CCL2- receptor CCR2 show decreased migration of Th1-inducing dendritic cells from

30 the site of infection to the lymph nodes, mount a Th2 response, and develop non-healing lesions (52). Conversely, CCL2–/– BABL/c mice show impairment of Th2 development and IL-4 production, and become marginally resistant to L. major (123). Finally, one study found that administration of recombinant

CXCL10 to L. major-infected BALB/c mice results in activation of NK cells, which are a major source of IFN-γ, which plays an important role in early resistance and development of subsequent Th1 response. As IFN-γ -producing

Th1-type CD4+ T cells are indispensable for the development of protective immunity against L. major (66), we determined whether enhanced susceptibility of CXCR3–/– mice to L. major is due to their inability to mount a Th1-like response by determining serum titers of Th1-associated L. major Ag (LmAg)- specific IgG2a at different time points and measuring IL-12 and IFN-γ production by LmAg-stimulated lymph node cells at weeks 3 and 8 post- infection. Throughout the course of infection, both groups displayed similar titers of Ag-specific Th1-associated IgG2a Ab (Fig. 4A, B). At weeks 3 and 8 post-infection, LmAg-stimulated lymph node cells from L. major infected

CXCR3+/+ and CXCR3–/– mice displayed comparable proliferation responses

(Fig. 5) and produced significant amounts of IL-12 and IFN-γ (Fig. 6A,B), however, levels of both these cytokines were significantly higher in lymph node cell supernatants from CXCR3–/– mice at week 8 (Fig. 6A, B).

31 At both time points, only basal levels of IL-4 were detectable in the lymph node cell culture supernatants from both groups (Fig. 6C). At week 8 post- infection, the lymph nodes from L. major-infected CXCR3–/– mice also contained significantly more CD4+ and CD8+ T cells, suggesting that these cells may be responsible for increased IFN-γ levels observed in CXCR3–/– mice (Fig. 5B, C). Nonetheless, these results demonstrate that the lack of a systemic Th1 development or enhancement of a Th2 response is not responsible for the susceptibility of CXCR3–/– mice to L. major. Furthermore, they also indicate that CXCR3 is not required for recruitment of effector T cells into draining lymph nodes during L. major infection. Because CXCR3 is believed to play a role in the recruitment of IFN-γ -producing CD4+ Th1 and

CD8+T cells into the site of inflammation (133), we quantified levels of IFNγ and

IL-4 gene transcripts in lesions by real-time RT-PCR. At weeks 3 and 8 post- infection, lesions from L. major-infected CXCR3–/– mice contained significantly less IFN-γ mRNA than those from similarly infected CXCR3+/+ mice (Fig. 7A).

There were no significant differences in levels of IL-4 mRNA between the groups (Fig. 7B). Next, we enumerated T cell populations in lesions from L. major-infected CXCR3–/– and CXCR3+/+ mice by flow cytometry. CXCR3 was expressed on significant proportions of CD4+ and CD8+ T cells recruited to the site of infection in the wild-type (CXCR3+/+) mice (Fig. 8). Similar differences were noted at week 8 post-infection (data not shown). Interestingly, the majority of CD4+ cells recruited to lesions during L. major infection did not express

32 CXCR3, suggesting that CXCR3-independent mechanisms may be involved in the trafficking of CD4+ T cells to site of infection (Fig. 8A). Alternatively, it is possible that a significant proportion of CD4+ T cells lose expression of CXCR3 after their migration into the site of infection. Such a phenomenon has been reported upon the re-challenge of CXCR3-expressing T cells in an antigen- specific model of eye inflammation (138). Ongoing studies in our laboratory are examining the possibility that similar events may occur in our model and also investigating the factors mediating down-regulation of CXCR3 expression in T cells.

At weeks 3 and 8 after L. major infection, flow cytometric analysis of lesion-derived cells showed that the lesions from CXCR3–/– mice contained fewer CD4+and CD8+ T cells than those from CXCR3+/+ mice (Fig. 9). While the proportion of CD4+ cells in lesions from L. major-infected CXCR3–/– mice was marginally lower as compared to similarly infected CXCR3+/+ mice, the difference in actual number of CD4+ cells between the groups was bigger, as lesions from L. major-infected CXCR3–/– mice contained fewer lymphocytes, probably due to impaired recruitment. Because CXCR3 regulates trafficking of

CD4 Th1 cells into the site of inflammation, it is perhaps not surprising that lesions from L. major-infected CXCR3–/– mice contained fewer CD4+T cells.

Interestingly, the lack of CXCR3 also reduced migration of CD8+ T cells into lesions of L. major infected CXCR3–/– mice (Fig. 9). While CD4+ Th1 cells are involved in the host defense against L. major (9), CD8+ T cells recruited to the

33 site of infection also play a critical role in resolution of primary L. major infection

(67, 136). Furthermore, CXCR3 has been shown to facilitate trafficking of CD8+

T cells into the site of inflammation (81, 137).

Taken together, these findings show that the lack of IFN-γ production at the site of infection, possibly due to impaired recruitment of both CD4+ and

CD8+ T cells, is responsible for the inability of L. major-infected CXCR3–/– mice to control L. major replication in skin.

Indeed, the draining lymph nodes from L. major-infected CXCR3–/– mice contained significantly more CD4+ and CD8+ T cells at week 8 post-infection

(Fig. 5B, C). This observation supports our hypothesis that CXCR3 is required for efficient migration of CD4+ and CD8+ T cells from the draining lymph nodes to infected skin during L. major infection. In fact, in ongoing studies in our laboratory we have found that the ear lesions from L. major-infected susceptible

BALB/c mice contain significantly fewer CXCR3-expressing CD4+ and CD8+ cells than to those from similarly infected resistant C57BL/6 mice (see Chapter

3).

These observations are apparently at odds with a recent report that

CXCL10 treatment of CD4+ T cells in vitro promotes IFN-γ production. In that study it was found that the chemokine induced higher IL-12R expression and that in turn resulted in greater amounts of the Th1 promoting cytokine IFNγ

(137). In the dLN environment, an abundance of CXCL9 and CXCL10 has been documented (49, 58). Therefore it stands to reason that while cells of the

34 dLN in a WT mouse would produce high amounts of IFN-γ, CXCR3-/- dLN cells should prove deficient in IFN-γ production. Since this was not seen in our in vivo experimental system, it would appear that, at least in the dLN CXCL10- driven IFNγ production is not a significant mechanism for augmenting the Th1 response. In the periphery, such might not be the case. Perhaps in the tissues, where activating signals from cellular neighbors are fewer, CXCL10-mediated increases in IL-12 responsiveness may provide more critical stimuli for IFNγ.

Indeed, our in vivo results demonstrate that differences in IFNγ exist at the site of infection in CXCR3+/+ and CXCR3-/- mice (Fig. 7). However, we did not assess IL-12 receptor expression. While the high levels of IFNγ seen in the dLN suggest that a Th1cell trafficking defect is responsible for the disparity in the wild type and knockout lesions, this notion may not be exclusive of the existence of an IL-12-mediated effect on IFNγ production in certain tissue contexts. Further experimentation will shed some light on this issue.

To determine if T cells lacking CXCR3 are inherently less able to produce IFNγ, we isolated CD4+ and CD8+ T cells from the LN and lesion of

CXCR3+/+ and CXCR3-/- infected with L. major and assessed their IFNγ- producing capacity upon polyclonal stimulation with plate-bound anti-CD3/anti-

CD28 antibody.

While we found that CD4+ T cells of both groups made comparable levels of IFNγ, CD8+ T cells from CXCR3-/- mice made significantly less of the cytokine than wild type CD8+ T cells (Fig. 10). This suggests that

35 CD8+/CXCR3+ T cells may be a high-IFNγ producing T cell subset or it may indicate that the CD8+ T cells recruited to the lesion by CXCR3-independent means may be less active.

In an effort to confirm these experiments with a more physiologically accurate approach, we used a BMDM-based in vitro T cell activation assay in which either L. major or L. major antigen extract was presented to CXCR3+/+ and CXCR3-/- derived T cells by BMDM.

When LN-isolated T cells from infected CXCR3+/+ and CXCR3-/- mice were used in these experiments, the results supported the findings from our previously described polyclonal activation results. LN cells of both groups made similar amounts of IFNγ (Fig 11A).These experiments also revealed that the T cells recruited to the CXCR3-/- lesion were less capable of parasite antigen-specific IFNγ production than those recruited to the L. major-induced lesion of wild type mice (Fig. 11B). Therefore these findings support the notion that the T cells missing from the CXCR3-/- lesion are the source of resistance- promoting IFNγ.

It should be noted that in these APC-assays, BMDM from both

CXCR3+/+ and CXCR3-/- mice functioned equally well as the APC component

(data not shown). CXCR3+/+ T cells incubated with BMDM from either group produced comparable levels of IFNγ (Fig. 11) suggesting that differences in cytokine production arose specifically from the T cells. Also, while we did not examine CXCR3-/- macrophages for altered co-stimulatory molecules (i.e.

36 CD86, CD80, etc.), these results suggest that macrophages lacking CXCR3 are equally capable APCs as such cells from their wild type counterparts.

Future studies by our group will utilize a both CXCR3-deficient animals also possessing transgenic TCRs recognizing epitopes of the model antigen ovalbumin (OVA). T-cells from these double mutants will be used to assess the antigen specific response to L. major expressing the model OVA epitopes. This will involve breeding CXCR3-/- mice with commonly used transgenic lines, OT-1 and OT-2. In vivo tracking studies using a transgenic CXCR3-GFP reporter animal (currently in development) will be used to further characterize the behavior of these CXCR3-expressing cells during parasitic infection.

2.2 The role of CD8+/CXCR3+ T cells in the response to L. major:

Also of note are our findings regarding CXCR3 responsiveness to its ligand

CXCL10 in vitro. CD4+ and CD8+ T cells were FACS purified and immunomagnetically purified from the LN of L. major infected mice and their in vitro responsiveness was assessed using a Transwell migration assay

(Corning). We found that CD8+ T cells migrated much more readily to rCXCL10 than CD4+ T cells. This shows that CD8+ T cells were much more responsive to CXCL10 than CD4+ T cells (Fig. 12)

Given theses results and that of the mitogenic in vitro stimulation of

CD4+ and CD8+ T cells, it may be tempting to conclude that CD8+/CXCR3+ T cells are responsible for supplying the IFNγ lacking in CXCR3-/- mice infected

37 with L. major. However, the results of these experiments should be viewed with caution given the strength of this polyclonal stimulus. This experimental stimulus is typically much more intense that physiologically accurate stimulation traditionally supplied by antigen presenting cells. It is possible that CD4+ T cells from CXCR3-/- mice express artificially high levels of IFNγ in response to anti-CD3 and anti-CD28, yet would be less capable under more physiological conditions. While the differential production of IFNγ by mixed T cells in response to BMDM-presented antigen suggests that under such conditions,

CXCR3-/CD4+ T cells produce less IFNγ, it does not prove this conclusively.

Furthermore, it might be inferred that since CXCR3-/CD4+ T cells made equal amounts of IFNγ compared to WT CD4+ T cells, these cells are less important for resistance to L. major than CD8+ T cells. While we have not addressed this question directly, adoptive transfer experiments performed by our group suggest the answer. To evaluate the importance of the STAT1 signaling pathway in the response to L. major, we adoptively transferred

STAT1+/+ or STAT1-/- splenocytes to Rag2-/- recipient mice before a high dose

L. major challenge (2x106 promastigotes). Not surprisingly, the STAT1-/- reconstituted animals proved highly susceptible to the parasite. Furthermore, to identify the T cell subset responsible for mediating this STAT1-dependent resistance, we seeded Rag2-/- mice with either STAT1+/+ CD4 cells and CD8 cells; STAT1-/- CD4 and CD8 cells; STAT1-/- CD4 and STAT1+/+ CD8 cells; or

STAT1+/+ CD4 and STAT1-/- CD8 cells. As expected, the all wild type cell

38 seeded animals resisted L. major infection while the all knockout group proved highly susceptible. Interestingly, mice that received STAT1-/- CD4 cells were nearly as susceptible to L. major as the all STAT1-/- group while the STAT1+/+

CD4/STAT-/-CD8 cell recipients controlled infection as well as the all wild type group (ARS and JB unpublished data). These findings suggest that in C57BL/6 mice, CD4+ T cells are primarily responsible for mediating immunity to L. major in a STAT1-dependent manner.

In other studies, we discovered that CXCR3 expression by CD4+ T cells is wholly dependent upon STAT1, while CD8+ T cells express CXCR3 both with and without STAT1 (see Chapter 3). Since unlike STAT1-competent CD4+ T cells, CD8+ T cells did not mediate resistance to L. major in our adoptive transfer studies, it seems that the CXCR3+/CD8+ population or any CD8+ T cell population for that matter is not sufficient to effectively mediate resistance to L. major in vivo.

In contrast to the results of the mitogenic stimulation experiments, these findings suggest that CD8+ T cells have limited importance for experimental CL.

Yet, they also appear to contradict a study that found CD8+ T cells to be crucial for resistance to a low dose of L. major (67). It is possible that the difference in parasite dose that exists between these models could give rise to a disparate requirement for CD8+ T cells. Indeed challenging T cell-reconstituted Rag2-/- mice with low doses of L. major parasite would address this issue. In all, these

39 results serve to demonstrate the importance of proper T cell mobilization for optimal resistance to L. major.

The fact that T cells recovered from the lesions of L. major-infected

CXCR3-/- mice produced less IFNγ than those from wild type mice further implicates these cells as the source of the cytokine. Also, since CXCR3-/- derived LN T cells make either slightly less or comparable amounts of IFNγ as wild type LN cells emphasizes the degree to which IFNγ-producing T cells must be accumulating in these tissues in order to produce the elevated IFNγ levels seen in L. major –infected dLN populations. This suggests that many cells must be accumulating in the LN of CXCR3 mice to the apparent expense of T cell recruitment to the lesion.

If CXCR3-deficient T cells do not efficiently home to the lesions tissue, they may not effectively exit the LN or they may re-circulate, fail to enter or remain in the lesion, and accumulate in the dLN. Such an activated T cell

„bottle-neck‟ would explain both the increased levels of IFNγ and the greater cellularity of CXCR3-/- LNs draining the L. major-induced lesion. However, recently a hitherto unimagined role was uncovered for CXCR3 in the dampening the adaptive immune response at the LN level. These authors found that CXCR3-mediated recruitment of cytotoxic CD8+ T cells to the inflamed LN resulted in the killing of antigen carrying APCs and consequently, a suppressed cellular activation with the LN (140). The absence of such killing

40 might be responsible for more cells residing in the CXCR3-/- LN during CL. To address this one would assess dLN DCs for evidence of cell death.

It is indeed noteworthy that despite theoretically lacking this anti- inflammatory safeguard, CXCR3-/- mice still fail to efficiently marshal an effective local immune response able to control parasite growth during CL. This only serves to highlight the importance of CXCR3-mediated T cell recruitment to the lesion during CL as a necessary element of the immune response.

In conclusion, L. major-infected CXCR3–/– C57BL/6 mice mount an efficient Th1 response and limit systemic spread of infection, but are unable to control L. major growth at the cutaneous site of infection. Moreover, increased susceptibility of CXCR3–/– mice to L. major is associated with markedly impaired trafficking of CD4+ as well as CD8+ T cells to the site of infection and reduced levels of IFN-γ in their lesion.

41

2.3 Non-T cells and CXCR3 function during L. major infection:

In the previous section we presented the results of studying the importance of

CXCR3-mediated T cell signaling during L. major infection. Here we investigate the role, if any, CXCR3 has on cell types other than T cells. Quite interestingly, we found that non-T cell recruitment was not lacking in CXCR3-/- mice. Also we report that while the killing of internalized parasite by macrophages depends upon non-redundant CXCL9 and CXCL10 activity, such was not the case during in the in vivo infection model. The results presented here serve to bolster the notion that CXCR3 mediates its anti-leishmanial activity through recruitment of

IFNγ-producing cells.

CXCR3 and NK cells:

NK cells express CXCR3 and the CXCR3-binding chemokines CXCL9,

CXCL10, and CXCL11 (in humans), which are known to enhance NK cell migration and activity (51-56). Previous studies have shown that NK cells produce IFN-γ and play a role in host immunity against L. major during early phase of infection (57). Also, NK cells express CXCR3 and readily respond to

CXCL10. Hence, it is possible that impairment of NK trafficking to the site of infection may be responsible for enhanced susceptibility of CXCR3–/– mice to

L. major. In the experiments described in Chapter 2, we found that at week 3 post-infection, although lesions from CXCR3–/– mice contained lower amounts of IFN-γ gene transcripts as compared to CXCR3+/+ mice, both groups displayed comparable parasite loads in their footpads. These findings suggest that the recruitment of NK cells to the site of infection may be impaired in the absence of CXCR3, but this deficiency does not enhance their susceptibility to

L. major during early-phase infection.

Interestingly, LN cells from L. major-infected CXCR3-/- mice actually produced more IFNγ when reactivated ex vivo compared their wild type counterparts (Fig. 6). This finding seems contrary to a report that NK cells recruited by CXCR3-dependent mechanisms to the LN prime the Th1 response there (58). Indeed we have shown that by week 3 post infection, IFNγ production is not lacking in the absence of CXCR3. However, it is known that during the latter stages of leishmaniasis, NK cells, having promoted resistance initially, become dispensable for disease control in mice (59). It would be worthwhile, nonetheless, to examine the dLN very soon after infection to determine if these organs contain fewer NK cells. It is possible that some

CXCR3-independent NK recruitment mechanism is responsible for the fact that the negative consequences of CXCR3-deficiency do not become evident until the later stages of L. major infection when NK cells become unimportant for mediating resistance (approximately eight weeks post infection).

Certainly though, the observation that IFNγ is not lacking in the CXCR3- deficient dLN indicates that, Th1 cytokine producing cells arise by, or are primed by NK-independent mechanisms (possibly under the influence of APCs in the dLN). Their abundance in the dLN and coincident scarcity in the lesion suggest that these Th1 cells exist, but do not traffic properly in the absence of

43 CXCR3. Instead of migrating to the tissues, these evidently Th1-primed cells may become trapped in the LN. The question of how NK cell migration is affected by CXCR3-deficiency has yet to be properly studied.

In Part 1 of this chapter, we demonstrated that CXCR3 deficiency during

L. major-induced CL resulted in low numbers of CD4+ and CD8+ T cells in the lesion. These results suggest that fewer T cells are recruited to the lesion in the absence of CXCR3 signaling. This deficiency in lesion homing was apparently unique to T cells. Neutrophils (GR1+), NK cells (NK1.1+), and macrophages

(CD11b+) were actually detected at higher frequencies in the lesions of

CXCR3-/- mice compared to those of wild type mice (Fig. 13A-C).

The finding that recruitment of myeloid phagocytes (GR1+ and CD11b+ cells) is not affected by CXCR3-deficiency was corroborated by additional studies using the thioglycollate-induced peritonitis model. In these experiments, we found that both CXCR3+/+ and CXCR3-/- mice recruit similar numbers of macrophages and granulocytes to the peritoneal cavity following injection of thioglycollate (Fig. 13D).

2.4 CXCR3 and Antigen Presenting Cells:

CXCR3 is expressed by numerous cell types, among them a subset of DC.

These so called plasmacytoid DC (pDC) are recruited to the lymph node from the circulation during inflammation (163). The inability of DCs to effectively traffic to the dLN during infection would theoretically result in a dampened

44 adaptive immune response that would certainly favor parasite persistence in the host.

Such a concept was demonstrated by Sato and colleagues. They found that mice lacking CCR2 had fewer skin dwelling DC (Langerhans cells) migrating from the lesion site to the dLN. These mice generated a sub-optimal

T cell response to L. major infection that was also skewed in favor of the Th2 response permitting L. major persistence (39).

The fact that certain DCs migrate in response to CXCR3 ligands and that deficiencies in mature DC migration to the dLN causes a poor response to L. major lead us to explore the ability of CXCR3-/- DC to deliver antigen to the dLN. Particularly, we investigated whether the sentinel DCs residing in the dermis of CXCR3-/- and wild type mice could migrate to the LNs draining inflamed skin tissue with similar efficiency.

To this end we painted a mixture of unconjugated FITC with a chemical irritant (5% FITC in 1:1 acetone/dibutylphthalate), on the ears and shaved rumps of WT and CXCR3-/- mice as described previously (39). After twenty four hours, we excised the LNs draining the painted skin and examined them for the presence of FITC+ cells. Viewing histologically prepared dLN sections by fluorescence microscopy we found that both wild type and CXCR3-/- mice harbored comparable numbers of FITC+ cells within them (Fig. 14A) indicating that the loss of the chemokine receptor did not impede the trafficking of skin- deposited FITC dye to the dLN by DC. Therefore, CXCR3 is not requisite for

45 the migration of DCs from the skin to the dLN where they stimulate T cell activation by presenting antigen to naïve T cells. Also, flow cytometric analysis of the L. major-lesion draining LN revealed that CXCR3-/- mice recruited dendritic cells to that site as efficiently as wild type mice (Fig. 14B). Given these results, and since CXCR3-/- mice do in fact mount a robust immune response to L. major, we believe that CXCR3 is not involved in the delivery of antigen to the LN during CL.

To further investigate the possibility of CXCR3 signaling contributing to the initiation of an adaptive immune response, we employed an in vitro antigen presentation assay. As previously described in Chapter 2, we found no difference in antigen presentation or T cell activation ability in the wild type and

CXCR3-/- macrophages. These results suggest that CXCR3-/- macrophages are capable APCs, and that deficiencies in T cell activation are not responsible for the high and persistent parasite burdens of the CXCR3-/- mouse during CL.

This finding was expected since we found high amounts of IFNγ and numerous cells in the dLN of CXCR3-/- mice infected with L. major indicating no deficiency in T cell activation by APC there. However, macrophages can promote the anti- leishmania response not just through their T cell activating functions, but also directly through the killing of internalized parasite. In the following section, we investigate the role of CXCR3 and its ligands in the leishmanicidal functions of macrophages.

46 2.5 CXCR3-ligand mediated killing of intracellular L. major by macrophages as a mechanism for resistance:

Based on our findings described in the preceding section, we propose that the primary cause of the susceptibility of CXCR3-/- mice to L. major lies in their reduced T cell trafficking to and Th1-promotion within the lesion. Supporting this, are the results presented in this section. As mentioned previously, non-T cells were not deficient in their trafficking to the L. major-induced lesion (Fig.

13). Among these were CD11b+ cells that likely consist of mostly macrophages.

With report that macrophages also express CXCR3 and that its ligands can promote killing of internalized parasite, we set out to investigate whether these observations could be seen in the L. major model.

Introduction:

The CXC chemokine receptor 3 (CXCR3) is expressed on various cells including activated T cells (72). Signaling through CXCR3 by its specific ligands CXCL9 and CXCL10 mediate recruitment of such cells during cell mediated immune response (73). Previously, we showed that CXCR3 is important for mediating T cell recruitment and local Th1 initiation in the

Leishmania major lesion (60). CXCR3 signaling has been shown to have functions other than chemotaxis. For instance, CXCR3 signaling on CD8+ T cells enhances the activity of these cells in a model of viral response (149).

Also, CXCL10 has been shown to mediate intracellular killing of leishmania

47 parasites by macrophages (43). However, despite the demonstration of

CXCR3 expression by macrophages, the mechanism by which CXCL10 activates these cells for parasite killing remains unknown. Furthermore, it is not known if other cutaneous species are susceptible to such chemokine mediated killing mechanisms.

In vitro killing assays revealed that BMDM from CXCL10-/- and CXCL9-/- mice were similarly less equipped to eradicate intracellular parasites than wild type cells. Interestingly, CXCR3-/- and wild type macrophages eliminated intracellular parasites comparably, suggesting that this CXCR3-ligand killing effect was not mediated through their canonical receptor CXCR3.

However, while our in vitro killing assays suggested that lack of either

CXCL9 or CXCL10 would result in susceptibility of the host to L. major, CXCL9-

/- and CXCL10-/- effectively controlled parasite group, induced a Th1 response, and recruited leukocytes to the lesion as well as wild type controls. These results indicate that despite non-redundant roles in in vitro parasite killing, individual CXCR3 ligands may play redundant roles in T cell recruitment – which appears to be the determining factor for CXCR3-mediated murine resistance to L. major.

Results and Discussion:

48 In vitro killing of L. mexicana and L. major by bone marrow derived macrophages is reduced in the absence of CXCL9 or CXCL10.

CXCL10 appears to mediate in vitro killing of L. amazonensis by murine macrophages (43), and the chemokine has been shown to be important for optimal CD8 activation (149). Because of these observations, as well as the importance of CXCR3 in host resistance to L. major (60), we investigated its importance in the elimination of intracellular Leishmania promastigotes by macrophages. To this end we grew BMDM from wild type C57BL/6 mice and

CXCL10 knockout (CXCL10-/-) mice on circular cover-slips within the wells of a

24-well tissue culture plate prior to infection by L. major promastigotes.

Following infection, these macrophages were stimulated with either rIFNγ and

E. coli LPS, rIFNγ alone, or nothing. At specified time points, culture media was sampled and the cover-slips were stained and mounted on glass slides. Upon microscopic examination, the percentage of macrophages harboring intact parasite was determined for each sample.

As expected, a higher percentage of the activated BMDM from CXCL10-

/- mice were infected than similarly treated cells of wild type mice (Fig. 15). To determine if CXCL10 was unique in its ability to promote parasite killing, cells lacking the CXCR3 ligand CXCL9 were similarly infected and stimulated as above. These CXCL9-/- derived cells were also infected more readily than wild type cells and were therefore deficient in their ability to destroy intracellular parasites (Fig. 15).

49 The ability of CXCL10 to promote leishmania killing by macrophages has been previously documented (43). However, whether this affect is mediated by its interaction with the chemokine receptor CXCR3 remains to be determined.

Interestingly, BMDM from CXCR3 deficient mice (CXCR3-/-) were as efficient as wild type derived BMDM in their ability to kill intracellular L. major (Fig. 15).

Similar results were obtained for cells infected in vitro with another agent of CL,

L. mexicana (data not shown). These results suggest that the individual

CXCR3 ligands play an important role in mediating the elimination of intracellular parasites since deficiency in just one results in markedly diminished parasite killing.

CXCL9 and CXCL10 mediate NO-production by L. mexicana and L. major infected BMDM in vitro. One mechanism by which macrophages eliminate intracellular parasites including L. major is nitric oxide (NO) production by the inducible nitric oxide synthase (iNOS) (150). Also, in vitro CXCL10-mediated parasite killing was shown to involve NO production by macrophages (43). We hypothesized that CXCL9 and CXCL10 mediate macrophage killing of L. major though an NO-dependent mechanism and elimination of these chemokines would result in reduced NO production in addition to the heightened parasitism observed above.

To explore this we subjected culture supernatants from our in vitro killing assays to a Griess reaction assay. CXCL10 and CXCL9 deficient cells both

50 produced significantly lower levels of NO when infected with L. major or L. mexicana and stimulated for 24 hours or longer. (Fig. 16 and data not shown).

In these experiments CXCR3-/- derived cells induced levels of NO similar to wild type controls.

Furthermore, analysis of the cytokines made by these activated macrophages during stimulation was carried out by using a sandwich ELISA.

We found that CXCL9 and CXCL10 deficient macrophages made levels of pro- inflammatory cytokines similar to those released by wild type cells. This was evident in the similar concentrations of TNF-alpha and IL-12 seen in the culture supernatant after infection and stimulation. Interestingly, the CXCR3-ligand knockout derived macrophages made more immune suppressive IL-10 than the efficient killing cells of the wild type and CXCR3-/- derived macrophages (data not shown). The significance of this observation remains to be tested.

In all, these findings support the notion that parasite killing by macrophages may be an important mechanism by which CXCR3-signaling contributes to L. major resistance in addition to recruitment of IFNγ-producing cells to the site of infection.

Since the CXCR3 ligands do not appear to mediate killing effects through

CXCR3, we then investigated how the ligands mediate such killing. Since the ligands of CXCR3 are known to interact with another chemokine receptor,

CCR3, we investigated the role of CXCR3 in these chemokine-mediated affects.

51

CCR3 is expressed on activated C57BL/6 BMDM:

Others have reported that macrophages can express the chemokine receptor

CCR3 (202). Also, it is known that the CXCR3-ligands interact with this receptor and inhibit the signaling of its traditional ligands, including the eotaxins

(203, 204). Therefore, since in our in vitro model, CXCL9 and CXCL10 appeared to exert an effect on macrophages independent of CXCR3, we hypothesized that they do so through the CCR3 receptor – either directly by supplying a hitherto unrecognized stimulatory signal, or by blocking the signaling of CCR3-ligands. To investigate this possibility, we first assessed

CCR3 surface expression by in vitro derived mouse macrophages. Flow cytometric staining of C57BL/6 BMDMs with FITC-labeled monoclonal anti-

CCR3 antibody revealed significant expression of CCR3 by these cells (Fig.

17). Therefore it is possible that the CXCR3-ligands are acting through CCR3 to mediate their pro-leishmaniacidal effects.

Supporting this notion is our finding that, while not altering the disease outcome of experimental CL (i.e. lesion growth, severity of necrosis or cytokine polarization), CCR3-deficiency did result in slightly reduced parasite burdens in susceptible BALB/c mice (described in detail in this chapter). While this result could have arisen from the disruption of Th2-cell trafficking to the lesion site, it could also have arose from enhanced parasite killing by macrophages lacking

CCR3. Since to our knowledge it has not been demonstrated that CCR3

52 signaling inhibits the killing capacity of macrophages, experiments are underway to investigate this possible function of CCR3.

Importance of CXCL10 and CXCL9 for resistance to L. major in vivo.

Based on these in vitro findings, we expected that CXCL10 and CXCL9 would contribute to L. major resistance and play non-redundant roles in controlling the infection. Indeed in some systems, the CXCR3 ligands have been shown to elicit unique or differential responses from CXCR3-expressing cells (154, 206).

To investigate whether the same principal of non-redundancy holds true in the

L. major model, we infected CXCL9-/- and CXCL10-/- mice with L. major and monitored lesion development.

Unexpectedly, neither CXCL9-/- nor CXCL10-/- mice were remarkably susceptible to L. major infection. These knockouts did not develop significantly larger lesions than wild type mice, while, as previously reported, the lesions of

CXCR3-/- mice were significantly larger than wild type lesions (Fig. 18A).

Furthermore, at six and nine weeks post-infection, the lesions of CXCL9-/-,

CXCL10-/- and wild type mice harbored comparable parasite loads (Fig. 18B) suggesting that these mice were similarly resistant to L. major infection.

Supporting these findings, we found that mice lacking CXCL9 or CXCL10 were capable of mounting an anti-L. major Th1 response. dLN cells from infected wild type as well as CXCL9 and CXCL10-deficient mice, when re- stimulated ex vivo with L. major antigen produced comparably low levels of IL-4

53 (data not shown). Also levels of the Th1-promoting cytokine IL-12 were comparable across the groups at the time points tested. Interestingly, by nine weeks post infection, cells of the CXCL9-/- dLN produced significantly less IFNγ than wild type mice. Even though this might be taken as an indication of a defective Th1 response, no impediment to parasite growth was observed in this group. It maybe that since the same cells produced less IL-10, the resultant phenotype of these mice is neither more nor less susceptible to L. major (Fig.

19). As before CXCR3-/- dLN cells produced significantly higher levels of IFNγ than wild type mice despite the heightened susceptibility of these mice.

Also, we examined the cellular composition of the ear lesion and dLN of

CXCL9-/-, CXCL10-/-, and wild type mice during L. major infection. We found that the ligand knockout mice harbored similar proportions of CD4+ and CD8+ T cells in both sites and that these cells displayed comparable levels of the activation marker CD69 (data not shown). Interestingly CXCR3+ T cells were found in each. This suggests that in contrast to the CXCR3-ligands‟ contributions to in vitro parasite killing, the CXCR3 ligands may act redundantly to mediate T cell recruitment to the site of infection during CL.

Furthermore these results demonstrate that despite individually enhancing parasite killing by BMDM, each CXCR3 ligand is not required for proper lesion homing of leukocytes, Th1 priming, and resultant resistance to L. major.

54 It is interesting that even though CXCL9 and CXCL10 proved to be individually important for in vitro control of L. major infection by macrophages, each ligand proved nonessential for infection control in vivo. Most likely the

CXCR3 ligands compensate for the absence of one since CXCL9-/- and

CXCL10-/- mice are resistant to L. major infection. This is significant since it distinguishes the role of CXCR3 and its ligands in the leishmania model from other experimental systems in which deletion or blockade of CXCL9 and

CXCL10 resulted in the discovery of unique affects or importance for each (154,

206). Additionally, these results further the notion that T cell recruitment demonstrated previously (60) is the primary mechanism by which CXCR3 signaling promotes L. major resistance in mice.

The observation that CXCR3 ligands are found in larger amounts in receptor deficient mice than their wild type counterparts (156) makes even clearer the importance of receptor and ligands for the immune response. If indeed our CXCR3-deficient mice also produced elevated CXCL9 and CXCL10 during experimental CL, then most likely the CXCR3-independent effects of these chemokines would become more important. Yet without functional

CXCR3-signaling, the deficient local Th1 response is not offset by any infection controlling mechanism mediated through the CXCR3 ligands.

2.6 The Role of CCR5 and CCR3 in the Host Response to L. major infection.

55 In this section we present findings from our studies of CCR5- and CCR3- deficient mice infected with L. major. We hypothesized that the Th1-associated

CCR5, like the similarly reputed CXCR3, would prove important for mediating the anti-L. major response. We also predicted that CCR3-deficiency in normally susceptible mice will result in enhanced resistance since this chemokine receptor has been linked to the Th2 response. To our surprise, resistant mice lacking CCR5 were not more susceptible to L. major than their wild type counterparts. Also, CCR3-deficient animals did not experience an overly different disease outcome than wild type controls. These findings suggest that

CXCR3-mediated signaling is unique in its significant role in mediating control of L. major.

CCR5 and L. major

CCR5 is a C-C chemokine receptor that binds CCL3 (MIP-1alpha), CCL4

(MIP-1beta), and CCL5 (RANTES). It is expressed on dendritic cells, macrophages, and polarized CD4+ Th1 cells. Although CCR5 has been shown to regulate leukocyte trafficking, it is also believed to play a role in the induction of IL-12 production from dendritic cells required for Th1 development (142).

Nevertheless, our observations in the present study that CXCR3–/– mice are susceptible to L. major suggest that CCR5-mediated mechanisms are unable to compensate for the lack of CXCR3 and mediate immunity against these parasites.

56 In fact, we found that CCR5-/- mice controlled L. major infection as efficiently as wild type mice. Footpad lesions of both CCR5-/- and CCR5+/+ mice developed to similar thickness and were controlled in both groups (Fig.

20A). Also, these lesions proved to contain comparable parasites burden (Fig.

20B). These findings were in stark contrast to those obtained from our comparison of CXCR3-/- and wild type mice during L. major induced CL.

Cytokines produced by cells of the dLN were measured by re-stimulating

LN cells ex vivo with L. major antigen extract and subjecting culture supernatant to a sandwich ELISA. At week 5 post infection we found that dLN cells of both

CCR5-/- and CCR5+/+ mice produced comparable levels of IL-12, IFNγ, IL10

(Fig. 21) as well as barely detectable amounts of IL-4 (not shown). Similar trends were also observed at week 9 post infection (data not shown).

Similarly, a previous study showed that even though CCR5–/– C57BL/6 mice showed decreased migration of Langerhans cells to their draining lymph nodes during L. major infection, they mounted a Th1 response and controlled the infection (39).

These results suggest that either 1) both CCR5 and CXCR3-mediated T cell homing cooperate in Th1 cell recruitment to the lesion during L. major- induced CL or 2) CXCR3-mediated T cell recruitment is the dominant recruitment axis directing Th1 cells to the skin. Recent findings support the latter. It was reported that CCR5 plays a role in promoting susceptibility to L. major. Particularly the chemokine receptor recruited regulatory T cells (Tregs)

57 and the action of these cells suppressed the anti-leishmanial response (92). It is possible that the lack of phenotypic difference we observe with these mice is the combined result of both reduced pro-inflammatory Th1 cells and coincident diminished immunosuppressive Treg cell migration – both subpopulations have been shown to rely on CCR5 signaling for tissue localization. It should be noted however, that CXCR3 has also been implicated as a mediator of both Th1 and

Treg cell trafficking, yet in the L. major model the most important population for determining disease outcome are the former CXCR3-expressing cell type.

CCR3 and L. major:

In addition to these two Th1-linked chemokine receptors, we also assessed the importance of CCR3 in murine resistance to L. major. CCR3 is expressed on eosinphils and other granulocytes, as well as on Th2 polarized cells and therefore it has a reputation as a Th2 indicator. The ligands for this chemokine receptor include CCL5 (RANTES), CCL7 (MCP-3) and CCL11 (eotaxin) (207).

CCR3 has an interesting relationship with CXCR3. Particularly, the ligands for CCR3 can be blocked from signaling through their receptor by the ligands for CXCR3. Also since the Th2-response is linked to parasite persistence and exacerbated disease in the L. major model. To investigate if

CCR3 contributes to L. major susceptibility were utilized mice of a super- susceptible genetic background (BALB/c) that lacked CCR3.

58 L. major-susceptible BALB/c mice deficient in CCR3 (CCR3-/-) were infected with a high dose of L. major in the LFP. We hypothesized that the elimination of this Th2 chemokine receptor would impart these mice with heightened resistance to L. major-either through reduced Th2 cell homing to the lesion or loss of interference of CXCR3-ligand interaction. However, lesion development in L. major-infected CCR3-/- mice was comparable to that of wild type mice. Both groups developed large and eventually ulcerative foot pad lesions by week 5 or 6 post-infection (Fig. 22A). Interestingly, CCR3-/- mice did have reduced parasite burdens relative to wild type BALB/c mice infected with

L. major, but both groups were still very high and significantly larger than that seen in resistant strains (Fig. 22B). Also suggesting that elimination of CCR3 did not alter L. major susceptibility was the finding that the cytokines generated in response to L. major infection and ex vivo re-stimulation were not markedly different from those seen in wild type mice. Specifically, levels of IL-12, IFNγ,

IL-10 and IL-4 were similar in the culture supernatants of CCR3+/+ and CCR3-/- dLN cells (Fig. 23).

Given these findings, it is tempting to exclude CCR3 as a potential susceptibility promoting factor during L. major infection. It is possible, however, that negating CCR3 signaling yields an effect masked by the super-susceptible nature of the BALB/c genetic background which harbors multiple factors responsible for their increased susceptibility to CL compared to more resistant strains (44). Perhaps experiments using CCR3-deficient C57BL/6 mice might

59 allow for such an intermediate amelioration of L. major infection. If eliminating the CCR3 side of the chemokine-interplay with CXCR3 and its ligands results in more efficient Th1 lesion homing (and perhaps more importantly, a decreased

Th2 presence there), we might expect smaller lesions and reduced parasite loads in the CCR3 deficient C57BL/6 animals. It too may be that the phenotype gained by knocking out CCR3 in these mice would be insufficient to affect the response to L. major. In which case it may be appropriate to conclude that the antagonism between CXCR3 and CCR3 does not account for the differences in

L. major-resistance seen between CXCR3-/- and CXCR3+/+ mice.

Lessons gleaned from these experiments with chemokine receptor knockout mice have improved our understanding of which recruitment mechanisms are important in the anti-L. major response. These results should be useful in developing treatments, vaccines or adjuvants aimed at treating CL in humans.

2.7 Materials and methods:

Animals: CXCR3 gene-deficient C57BL/6 mice were generated as described previously (81) and gifted by Drs. Bao Lu and Craig Gerard. CCR5 knockout mice were purchased from the Jackson Labs. Also, CCR3 deficient BALB/c mice and CXCL9 and CXCL10 deficient mice on a C57BL/6 background were gifts of Dr. Andrew Luster Wild-type BALB/c and C57BL/6 mice were purchased from Jackson Labs. All experiments were performed using 8–10-

60 week-old, sex-matched mice maintained in a facility at The Ohio State

University according to the guidelines for animal research.

Parasites and infection protocols: L. major (LV39) was maintained by serial passage of amastigotes inoculated s.c. into shaven rumps of BALB/c mice.

Amastigotes isolated from the lesions were grown to SPP as described previously (68). For left footpad infections, mice were infected by inoculating

2x106 L. major SPP into the hind footpad. Intradermal infections were carried out by injecting 1x104 promastigotes in the ear pinna of tribromoethanol- drugged mice. In either model, lesion growth was analyzed by measuring the increase in footpad or ear thickness with a dial-gauge micrometer at weekly intervals up to 8 weeks after infection.

Quantitation of parasite loads: Mice were sacrificed at weeks 2 and 8 after infection and parasite burdens in the infected footpad or ear were determined by limiting-dilution analysis as described before (68). The results were expressed as mean parasite titer per footpad.

Leishmania-specific ELISA: Peripheral blood was collected from L. major- infected CXCR3+/+ and CXCR3–/– mice at 2-week intervals. Serum samples were analyzed for the levels of Leishmania-specific Th1-associated IgG2a and

61 Th2-associated IgG1 Ab by ELISA as described previously (68). LmAg used for

ELISA was prepared by repeated freeze-thawing of L. major SPP.

T cell proliferation assay and cytokine analysis:

The lesion-draining lymph nodes were removed from L. major-infected mice at indicated time points after infection. T cell proliferation assays were performed as previously described (68). Single-cell suspensions were prepared by gentle teasing of the draining lymph nodes and live cells were enumerated by Trypan blue exclusion. To the wells of a 96-well flat-bottom tissue culture plate (Costar,

Cambridge, MA), 3x105 lymph node cells were added. Cells were stimulated with 20 µg/ml LmAg (prepared by repeated freezing and thawing of L. major promastigotes) or supplemented medium as a negative control. The proliferation response was measured by Alamar Blue Assay (Biosource

International, Camarillo, CA) as described previously (145). Following

o incubation at 37 C for 72 h in 5% CO2, supernatants were collected and frozen at –20oC for subsequent analysis. IFN-γ, IL-12p70, IL-4, and IL-10 were evaluated from culture supernatants by ELISA as described previously (68).

Reagents used for cytokine ELISA were all purchased from BD PharMingen

(San Diego, CA).

In vitro polyclonal stimulation of T cells: CD4+ or CD8+ T cells were either

FACS sorted or isolated by immunomagnetic beads from the dLN and lesion of

62 L. major-infected mice. Between 1x105 and 5x105 of these cells were incubated with plate-bound anti-CD3e and anti-CD28 antibody (Biolegend). Culture supernatants were sampled and analyzed by sandwich ELISA.

In vitro Transwell Migration Assay:

CD4+ or CD8+ T cells were FACS sorted from dLN cell suspensions from L. major-infected mice. 1x105 T cells were seeded in the top chamber of a

Transwell plate (Corning). The bottom chamber was filled with serum-free

o RPMI with 200nM rCXCL10. After 2 hours of incubation at 37 C, 5% CO2, cells having migrated to the bottom chamber were enumerated microscopically.

Wells of media alone (no chemokine) was used as a control.

Quantitative real-time PCR:

Quantitative real-time PCR was performed as described previously (146).

Briefly, a common master mix [LightCycler-FastStartDNA SYBR Green I

(Roche, Mannheim, Germany), 2 mM MgCl2, 0.5 µM gene-specific primer], and

1.5 µl of cDNA for a final reaction volume of 15 µl was used. Each transcript was quantified using the following cycling protocol: 95 oC for 10 min, followed by

35 cycles of 95oC denaturing for 15 s, gene-specific annealing temperature for 2 s, and 72oC extension for 20 s. The concentration of gene-specific cDNA was quantified by comparison to a standard curve of the genes pecific PCR product diluted 1:10 for concentrations ranging from 1 ng/ml to 1 fg/ml. After each real-

63 time reaction, a melting curve was generated and samples were run on a 1.2% agarose gel to ensure that only one gene-specific PCR product was generated.

Real-time PCR was preformed using the Rotogene 2000 Real-time Cycler

(Phenix Research Products, Hayward, CA). The following primers were used: b- actin, 5‟-TACAGCTTCACCACCACAGC-3‟ and 5‟-

AAGGAAGGCTGGAAAAGAGC-3‟ (annealing temperature 60oC, 206-bp product); IL-4, 5‟-TCAACCCCCAGCTAGTTGTC-3‟ and 5‟-

CGAGCTCACTCTCTGTGGTG-3‟ (annealing temperature 60oC, 128-bp product); IFNγ, 5‟-CACGGCACAGTCATTGAAAG-3‟ and 5‟-

GCTGATGGCCTGATTGTCTT-3‟ (annealing temperature 60oC, 198-bp product),

Histopathology:

Footpads from L. major-infected CXCR3–/– and CXCR3+/+mice were excised at week 8 after infection, and fixed in decalcifying solution F (Stephens Lab,

Riverdale, NJ) for7 days. The tissues were processed and embedded in paraffin, and 4- to 8-µm sections were cut. The sections were hydrated and stained by routine hematoxylin/eosin staining for histopathological analysis.

Isolation of cells from the ear lesions and phenotypic analysis of cell populations by flow cytometry. Cells from the ear lesions of L. major-infected

CXCR3–/– and CXCR3+/+ mice were isolated as described previously (67).

64 Briefly, two sheets of ear were separated and incubated with dermal side down on complete RPMI 1640 at 37oC for 6 h. The cells spontaneously migrating out of the dermis were collected and filtered through 70-µm pore size cell strainer for flow cytometric analysis. Flow cytometric analysis was carried out on cells pooled from the lesions of four to five mice to enumerate different T cell populations. Cells (1x105–2x105) were labeled using FITC- or PE-conjugated antibodies (PharMingen, R&D systems, Biolegend).Flow cytometry analysis was performed using a FACSCalibur flow cytometer and CellQuestPro software

(Becton Dickinson, Mountain View, CA). Lymphocytes were gated according to forward and side scatter and at least 10,000 events were acquired and analyzed. The numbers of CD4+ and CD8+ cells per ear lesion were calculated from total numbers of cells using appropriate percentages from the flow cytometry.

Derivation of Bone Marrow Macrophages: Bone marrow progenitor cells were flushed from the femurs and tibias of adult, sex and age matched mice and washed in completed RPMI plus 10%fetal bovine serum, 0.1% pent-strept. antibiotic and 0.001% betamercaptoethanol. After hemolysis, progenitor cells were washed and cultured for 7 days in the presence of 10% L929 cell-free supernatants (containing CFS-1). Non-adherent cells were discarded and the

BMDMs were scraped and plated for in vitro assays.

65 In vitro killing assay: The ability of BMDM to kill internalized parasites was determined by an in vitro killing assay described previously (150). Briefly,

BMDM from various mouse strains were adhered (in triplicate) to circular glass cover slips in 24-well plates prior to co-incubation with L. major promastigotes

(1:10 cell to parasite ratio). After washing away non-phagocytosed parasite with warmed PBS, we stimulated the macrophages with either recombinant

IFNγ or IFNγ and E. coli LPS (Roche). At various time points post-stimulation, supernatants were harvested and the adherence BMDMs were washed, cold- methanol fixed and Geimsa stained according to the manufacturer‟s protocol.

Mounted cover-slips were then examined by a blinded observer under 1000X bright-field microscopy. Between 300-400 macrophages were counted per replicate and the percentage of macrophages that contained intact parasite, as well as the average burden of infected cells were determined.

NO assay and Cytokine ELISA: Supernatants collected from in vitro assays were examined for NO content indirectly by Griess reaction and cytokine levels by sandwich ELISA as described previously (68).

Statistical significance: Statistical comparisons between lesion sizes, cell numbers, and cytokine concentrations were made using Student's unpaired t- test. Differences in titers were determined using the Mann-Whitney U-prime test. Differences yielding P values<0.05 were considered significant (*).

66

67

CHAPTER 3

REGULATION OF CXCR3 BY T CELLS

3.1 Differential Induction of CXCR3 by T cells of L. major-resistant and susceptible mice.

Abstract:

CXCR3-mediated trafficking of effector T cells can result in pathology or effective cell-mediated immunity against numerous pathogens including

Leishmania major, a cause of cutaneous leishmaniasis. Several well- characterized mice strains respond differently to this disease. Th1-biased,

C57BL/6 mice control infection while BALB/c mice mount an infection exacerbating the Th2-dominated response. We hypothesized that resistant mice strongly express CXCR3 on T cells, while susceptible animals do not.

Using flow cytometry and real-time PCR we assessed CXCR3 expression both in vivo and in vitro. Here we report that C57BL/6 T cells, but not those of

BALB/c, mice dramatically up-regulate CXCR3 at the protein and transcript

68 level during both L. major infection and following mitogenic stimulation. This strain-dependent CXCR3 expression disparity resulted from differential production of IL-10 and expression of the IL-10 receptor, but not IL-4, IFNγ or

Tbet levels. These results further define the mechanisms regulating CXCR3 expression by T cells.

Introduction:

Leishmania major is one of several species of intracellular parasite responsible for the disease cutaneous leishmaniasis (CL). In humans, CL symptoms include localized, self-resolving lesions at the infection site. However, healing and long-term immunity to re-infection are often achieved by immune- competent hosts (11,66).

Experimental murine L. major infection has demonstrated that mice strains which mount a predominately Th1-biased response (i.e. C57BL/6,

CBA/J) efficiently resolve infection while the decidedly Th2 skewed BALB/c strain fails to control the parasite and displays severe morbidity (44). Numerous differences in the immune responses mounted against L. major by these mice are known (44, 208). Among them is the differential production of the chemokines CXCL9 and CXCL10 by DC of C57BL/6 and BALB/c mice (49).

69 The receptor for these ligands, CXCR3 (71, 72) is expressed on several cell types (86) and is highly up-regulated by activated T cells with a Th1 bias

(71). CXCR3 expression on CD4+ T cells requires the Th1 transcription factor

Tbet and the cytokine IFNγ (86, 80). While both CXCR3 and its ligands are associated with elements of the Th1-immune response (73), it is not known if the receptor is differentially expressed by T cells of C57BL/6 and BALB/c mice.

Previously, we demonstrated an important role for CXCR3-mediated T cell recruitment in the immune response to L. major (12). L. major-resistant

C57BL/6 mice did not control infection without intact CXCR3. These CXCR3-/- mice had diminished T cell presence and IFNγ production in their lesions (13).

Therefore we hypothesize that the degree of inherent L. major-resistance for a mouse strain correlates with its capacity to express CXCR3 on T cells.

To test this, we infected C57BL/6 and BALB/c mice with L. major and sampled lesion and draining LN (dLN) cell populations to assess CXCR3 expression by flow cytometry. Also, we stimulated C57BL/6 and BALB/c derived T cells in vitro to further dissect chemokine receptor expression. We found that the L. major-induced lesion and dLN of BALB/c mice contain lower proportions of CXCR3+ T cells than their C57BL/6 counterparts. Also, we show that C57BL/6 derived T cells induce CXCR3 more frequently and more intensely than those of BALB/c mice upon polyclonal stimulation in vitro.

Unexpectedly, while T cell-derived cytokines affected receptor induction,

IFNγ levels did not determine the extent of CXCR3 expression. Instead,

70 differential induction was found to be due in part to the production of and receptiveness to the cytokine IL-10. These results illustrate a previously unrecognized aspect of CXCR3 regulation on T cells; namely that in addition to the requisite IFNγ/Tbet signal, CXCR3 expression is influenced by non-Th1 factors such as IL-10.

Results and Discussion:

C57BL/6 mice express higher levels of CXCR3 than BALB/c mice during L. major infection.

Previous studies revealed that L. major-susceptible and resistant mice differentially express the CXCR3 ligands (49), however differences in receptor expression by the T cells of these mice has until nown escaped investigation.

To investigate CXCR3 expression, mice of both backgrounds were infected intradermally with L. major promastigotes. At weekly intervals, leukocytes of the lesion and the LN draining it were analyzed for surface CXCR3 expression.

Following staining for CXCR3 and CD4 or CD8, we found by flow cytometry that three weeks post-infection, the dLNs of C57BL/6 mice contained higher percentages of CXCR3-positive CD4+ and CD8+ lymphocytes than those of

BALB/c mice (Fig. 24A). Comparing cells explanted from L. major-induced lesions yielded similar results (Fig. 24B). This trend persisted for the duration of the seven week infection course with C57BL/6 mice consistently having a larger

71 percentage of their CD4+ and CD8+ compartments also expressing CXCR3 in the dLN and lesion (Fig. 24C and data not shown, respectively). After seven weeks, C57BL/6 lesions began healing while BALB/c lesions began to ulcerate, necrose and develop secondary infections preventing further monitoring.

Differential presence of CXCR3+ T cells in the lesion and dLN of these mice could be attributed to differences in CXCL9 and CXCL10 and resultant disparate cell recruitment to the tissues in question. However, we found that

CXCR3+ cells were not sequestered in the spleen or peripheral blood of

BALB/c mice (data not shown). Based on these results, we hypothesized that the C57BL/6 T cells up-regulate CXCR3 more efficiently than those of BALB/c mice. To test this notion, we activated T cells of both strains in vitro to determine if they indeed differentially regulate CXCR3.

T cells of L. major-resistant mice induce higher levels of CXCR3 than susceptible mice upon mitogenic stimulation.

Resting T cells from C57BL/6 and BALB/c mice were incubated with plate bound stimulating antibodies for 48 hours. Then cells and conditioned media were removed from the stimulus and the activated T cells were rested prior to

CXCR3 surface expression analysis. Flow cytometry revealed that prior to stimulation, neither group expressed considerable surface CXCR3, as reported previously (data not shown). However, following in vitro stimulation, as in the inflamed LN, C57BL/6 T cells efficiently up-regulated CXCR3 while those of

72 BALB/c mice did not (Fig. 24D). This trend in CXCR3 expression was further observed at the mRNA level by real time PCR analysis suggesting differential regulation of the chemokine receptor beyond the level of surface protein (Fig.

24E). Similar results were obtained when CD4+ and CD8+ cells were separately purified and stimulated (data not shown).

Yoon et al have described differences in CXCR3 transcript in the stressed ocular tissues of C57BL/6 and BALB/c mice. However, it was unclear if this observation resulted from differential recruitment of CXCR3+ cells or from differences in CXCR3 regulation (158). Given our findings, differential CXCR3 expression may partially explain this phenomenon.

A released Th2 associated factor regulates CXCR3 expression by T cells.

Since the cytokine IFNγ plays an important role in CXCR3 induction by CD4+ T cells, (80), we sampled the culture supernatants of C57BL/6 and BALB/c derived T cells and measured the concentrations of Th1 and Th2 cytokines by

ELISA. As expected, the Th2-prone BALB/c T cells produced significantly greater amounts of IL-4 than cells from C57BL/6 mice (Fig. 25A left panel).

Also, BALB/c T cells produced higher levels of IL-10 than C57BL/6 T cells (Fig.

25A middle panel).

Interestingly, both groups produced high amounts of IFNγ during activation (Fig. 25A right panel). Furthermore, comparable induction of Tbet mRNA was observed in activated BALB/c and C57BL/6 T cells (Fig. 25B). This

73 observation mirrors that seen in the dLN since this site in both resistant and susceptible mice harbors similar percentages of IFNγ-producing T cells during the early stages of L. major infection (159). These results suggest that although

Th1-associated factors such as IFNγ and Tbet may be required for CXCR3 induction by T cells (86), they are not the sole determinants of CXCR3 expression. Indeed, a superseding inhibitory factor appears to suppress

CXCR3 expression in BALB/c derived T cells.

Suspecting that a Th2 cytokine might suppress CXCR3 in BALB/c derived T cells, we washed activated T cells of the cytokines released during in vitro activation (i.e. the conditioned media) prior to resting. These washed T cells of BALB/c mice induced CXCR3 modestly, though less than C57BL/6 isolated T cells which were not affected by washing (data not shown). This finding confirmed that a soluble factor made during T cell activation is partially responsible for low BALB/c CXCR3 levels.

IL-10, but not IL-4 or IL-13 regulates CXCR3 in BALB/c derived T cells

Since BALB/c derived T cells secreted high levels of IL-4 (Fig. 25E), and since this cytokine antagonizes Th1 immunity, we hypothesized that this noted Th2 cytokine was suppressing CXCR3 in BALB/c T cells. To test this we stimulated

BALB/c and C57BL/6 T cells in the presence of 10μg/ml IL-4 neutralizing antibodies. Compared to isotype control (rat IgG) treated BALB/c derived T cells, IL-4 neutralized cultures expressed comparably low levels of surface

74 CXCR3 as detected by flow cytometry (Fig. 25C). As expected, C57BL/6 T cells were unaffected by IL-4 neutralization (Fig. 25C). Furthermore, T cells from BALB/c genetically lacking the IL-4 receptor alpha chain (IL-4Rα-/-) induced similarly low levels of CXCR3 compared to wild-type controls (Fig.

25D). Therefore, we conclude that IL-4 does not suppress CXCR3 on BALB/c

T cells. Furthermore, since IL-13; another Th2-associated cytokine, shares the

IL-4Rα chain (209), this cytokine can also be eliminated as a candidate suppressive factor.

Since BALB/c cells also released markedly higher levels of IL-10 during stimulation, we investigated whether the cytokine regulates CXCR3. To this end we blocked IL-10 signaling by including anti-IL-10 receptor antibodies in the culture media of BALB/c- and C57BL/6-derived T cells. Flow cytometric analysis showed that CXCR3 expression by BALB/c T cells was greater than that of isotype control treated cells (Fig. 26A). Not surprisingly, C57BL/6 derived T cells were unaffected by IL-10R blockade (data not shown).

Furthermore, BALB/c mice deficient in IL-10 (IL-10-/-) expressed CXCR3 more readily than WT controls (Fig. 26B). Corroborating these observations, intracellular flow cytometric staining of in vitro stimulated T cells revealed that while IL-4+ T cells of both strains were found to co-express CXCR3, IL-10+ T cells did not significantly co-express CXCR3 (data not shown).

75 Further demonstrating the role of IL-10 in CXCR3 suppression, IL-10-/- mice contained greater percentages of CXCR3+ T cells in the dLN three weeks after L. major infection (data not shown).

We expected that stimulating C57BL/6 T cells in the presence of recombinant IL-10 (rIL-10) would suppress the robust levels of CXCR3 expressed by the strain. Interestingly, administration of rIL-10 to C57BL/6 had no effect on CXCR3 surface expression (Fig. 26C). This led us to investigate whether in vitro stimulated T cells from C57BL/6 and BALB/c mice differentially responded to IL-10.

It has been reported that the Th1 responses of BALB/c mice are suppressed more dramatically by IL-10 and the activity of regulatory T cells than C57BL/6 mice (210). Therefore it was not surprising that at both the mRNA and protein level, BALB/c T cells expressed more IL-10R than their C57BL/6 counterparts (Fig. 26D, E). Any differences in IL-10 responsiveness downstream of the IL-10R (i.e. STAT3-mediated signaling events) in C57BL/6 and BALB/c derived T cells are unknown. These results explain why exogenous IL-10 does not suppress CXCR3 expression by the T cells of

C57BL/6 mice.

Since the receptiveness of BALB/c and C57BL/6 derived T cells to the

CXCR3 expression promoting cytokine IFNγ might also be different, as was the case for IL-10, we analyzed expression of the IFNγ receptor on activated T cells from these mice. Interestingly, RT-PCR analysis found no significant deficiency

76 was found in the fold induction of this receptor by in vitro activated BALB/c derived T cells compared to those of C57BL/6 mice (Fig. 27).

Since the aforementioned experiments were carried out using a mitogenic stimulus (plate bound CD3/CD28 cross linking antibodies), we attempted to validate our findings in more physiologically relevant scenarios.

First, we repeated our cross-linking antibody experiments with reduced concentrations of stimulating antibody. This lower intensity stimulus has in the past been used to mimic natural T cell activating signals, but in a polyclonal manner (80). Our results in this model mirrored those seen in the high intensity stimulation experiments. This was true for both the relative expression levels of

CXCR3 by T cells of the two strains and the production of CXCR3 expression suppressing factors in the supernatant of BALB/c derive T cells (data not shown). Furthermore, when transgenic T cells of BALB/c genetic background, reactive to OVA peptide (DO11) were activated in vitro by OVA-pulsed BMDM, we also found that the T cells needed to be washed before significant CXCR3 up-regulation could occur (Fig. 28). These findings lend some validation to our in vitro-intensive studies of CXCR3 regulation. Also the corroborative findings obtained from in vivo infection models also suggest that our observations reflect physiologically relevant relationships rather than artifacts of an artificial model.

In conclusion, CXCR3 expression in mice correlates with the ability to mount an effective immune response to L. major. Also, we found that CXCR3 induction in T cells is suppressed in L. major-susceptible strains by T cell

77 derived IL-10. To our knowledge, this is the first report of differential CXCR3 regulation by the T cells of these commonly used lab mouse strains. Also, in IL-

10 production and receptiveness we have identified a mechanism that in part accounts for this strain-dependent discrepancy. While this cytokine has been observed to inhibit CXCR3 expression by human eosinophils (211), no such role is known for T cells. Interestingly, in murine colitis forced expression of IL-

10 results in amelioration of this Th1- (and CXCL10-) mediated disease (160).

These observations are particularly interesting in light of recent studies of the

IL-10 producing capacity of Th1 cells as a means of limiting their own inflammatory potential (94). One such consequence of T cell-derived IL-10 may be to limit CXCR3-mediated cell homing – a recruitment axis critical for Th1 trafficking in numerous disease models.

Even though we have shown an important role for IL-10 in controlling

CXCR3 expression between the mouse strains C57BL/6 and BALB/c, simply blocking the cytokine‟s signaling did not equalize the CXCR3 expression of

C57BL/6 and BALB/c T cells. Therefore other factors are likely to influence

CXCR3 expression by these cells. Exploring particularly the relative level and activity of transcription factors may prove interesting.

It has been shown that the master Th1 transcription factor Tbet is crucial for CXCR3 expression by T cells (86, 176). Also our data suggest that the amount of Tbet expressed is not predictive of actual CXCR3 expression level in

T cells. We found that GATA-3 levels were more predictive of CXCR3 surface

78 expression (data not shown). Since these factors antagonize each other at different levels of interaction, this could indicate that while Tbet expression is needed for CXCR3 as a prerequisite, the interaction of transcription factors with their accessory molecules could determine CXCR3 expression. Also the ability of T-bet and GATA-3 to bind to the CXCR3 promoter may differ between these mouse strains. In which case, the relative levels of the factors or their mRNA transcripts may not prove critical or predictive for CXCR3 expression. Further study of transcription factor activity will prove enlightening in this regard. Such studies, likely to involve promoter binding as well as reporter assays are to be undertaken in our lab.

In addition to comparing CXCR3-expressing T cells in these two mouse strains, we also examined CXCR3 expression by NK cells (CD49b+) in vivo.

This cell type has also been shown to be an important source of IFNγ and promoter of the Th1 response (52, 58) even though their importance for long term resistance is questionable (59). Flow cytometric analysis of lesion and LN cell populations revealed that C57BL/6 mice have more CXCR3-expressing NK cells in these tissues compared to BALB/c mice infected with L. major (Fig. 29).

These results suggest that NK cells of L. major-infected C57BL/6 induce

CXCR3 more readily than those of BALB/c mice. However, one must approach such results cautiously. It may be that NK cells are simply attracted to the LN and lesion sites more efficiently in C57BL/6 mice. Investigating CXCR3-

79 induction by NK cells in vitro (as we have done with T cells) will resolve this matter.

Also, BALB/c mice have been reported to lack the NK1.1+ subpopulation of NK cell present in C57BL/6 mice. Indeed using flow cytometry we have not consistently found significant numbers of NK1.1+ cells in the dLN or lesion of

BALB/c mice, yet C57BL/6 mice do possess these cells which can express significant amounts of CXCR3. These NK1.1+/CXCR3+ cells comprise a significant percentage of leukocytes in the C57BL/6 lesion throughout the L. major infection course (Fig. 30). The functional consequences of CXCR3 expression by NK cells as well as the differences between NK1.1+ and CD49b+

NK cells have yet to be determined. Both seem to express CXCR3, but the importance of this observation has yet to be investigated. Certainly in light of our observation that NK cells are not deficient in the lesions of susceptible

CXCR3-/- mice during infection suggests that CXCR3 expression by NK cells of

C57BL/6 mice is not as critical for mediating resistance as T cell expression of

CXCR3. The same might not be the case for the BALB/c model, however such speculation does little but beg further investigation.

In conclusion, a disparity exists between T cells and probably NK cells of the C57BL/6 and BALB/c genetic backgrounds. The more L. major-resistant strain expresses CXCR3 to a greater extent than its susceptible counterpart.

For T cells, this was observed consistently both in vitro and in vivo.

Furthermore, through these studies we have uncovered that CXCR3 induction

80 by native T cells is suppressed by IL-10 and most likely other factors. To find what other factors are responsible for strain-dependent differences in CXCR3 expression further inquiry is needed to expand upon our findings.

3.2 IFNγ and STAT1 signaling in CXCR3 induction by CD4+ and CD8+ T cells.

Abstract:

CXCR3 regulates migration and function of T cells during inflammation.

Interferon-γ has been shown to control expression of CXCR3 on activated T cells, but its role in regulating CXCR3 on CD4+ versus CD8+ T cells is not clear.

In the preceding section, we described the differential expression of CXCR3 by

T cells of different mouse strains. We also identified a factor suppressing

CXCR3 expression as the cytokine IL-10. In this section, we report our findings concerning the roles of IFN-γ and STAT1 in the regulation of CXCR3 on CD4+ and CD8+ T cells of C57BL/6 mice. It was observed that blockade of IFN-γ as well as STAT1 deficiency prevented an increase in CXCR3 expression on CD4+ but not CD8+ T cells following in vitro activation. STAT1-/- CD4+ T cells contained significantly less CXCR3 and T-bet mRNA than WT CD4+ T cells.

However, CXCR3 and T-bet mRNA levels were comparable in WT and STAT1-/-

CD8+ T cells. There were no significant differences between eosmesodermin mRNA levels in STAT1-/- and WT T cells. These findings show that IFN-γ and the STAT1 pathway is required for efficient induction of CXCR3 on CD4+ but not

CD8+ T cells.

81

Introduction:

The chemokine receptor CXCR3 is expressed on plasmacytoid dendritic cells

(pDCs), natural killer (NK) cells, and CD4+ and CD8+ T cells, and microvascular endothelial cells (163-165, 130). Three CXC chemokines, CXCL9 (Mig),

CXCL10 (IP-10), and CXCL11 (I-TAC) signal via CXCR3 (71, 165), and mediate biological functions such as cell migration and proliferation (127).

CXCR3 mediates immunity against pathogens by regulating recruitment and function of effector T cells (60, 166-169), however, CXCR3 also contributes to pathogenesis of allograft rejection and autoimmune diseases by promoting recruitment of pathogenic T cells (81, 170). Resting T cells express low levels of

CXCR3 but they up-regulate CXCR3 when activated thereby gaining responsiveness to its ligands (171).

Several studies suggest that Th1/Th2-associated cytokines regulate levels of CXCR3 on T cells (80, 172, 173). Interferon-γ (IFN-γ), which signals via STAT1, enhances CXCR3 expression on T cells (80), but its role in regulating CXCR3 on CD4+ versus CD8+ T cells is not clear. We therefore examined the role of IFN-γ and STAT1 in regulating CXCR3 on CD4+ and CD8+

T cells. Our findings show that both the IFN-γ and the STAT1 pathway are required for induction of CXCR3 on CD4+ T cells, but CD8+ T cells up-regulate

CXCR3 via IFN-γ/STAT1-independent mechanism.

82 Results and Discussion:

Although IFN-γ enhances CXCR3 expression on T cells (17), it is not clear whether this cytokine is essential for CXCR3 induction on CD4+ or CD8+ or both T cells. CD4+ Th1 cells preferentially express CXCR3 (130,133); however, CD4+ Th2 cells also express CXCR3 under certain conditions indicating that CXCR3 expression on CD4+ T cell may not be exclusively dependent on IFN-γ (8,134). In fact, a recent study showed that expression of transcription factor T-bet in IFN-γ-/-/T-bet-/-CD4+ T cells induces CXCR3, suggesting that T-bet can control CXCR3 expression in the absence of IFN-γ

(86). T-bet also regulates CXCR3 levels on CD8+ T cells (176), although it not clear whether IFN-γ and/or STAT1 are required for CXCR3 induction in CD8+ T cells.

The goal of our study was to determine whether IFN-γ and/or the STAT1 pathways control CXCR3 expression in CD4+ T cells, CD8+ T cells or both during activation. Therefore, we compared the effect of IFN-γ blockade or

STAT1 deficiency on CXCR3 expression in CD4+ and CD8+ T cells. CD4+ T cells activated in the presence of anti-IFN-γ neutralizing Ab (Pharmingen, clone# XMG1.2) expressed less CXCR3 than controls (Fig. 31A). However,

IFN-γ blockade had no effect on CXCR3 levels in CD8+ T cells (Fig. 31B).

Control CD4+ and CD8+ T cells produced comparable IFN-γ (data not shown) and efficiently up-regulated CXCR3 (Fig. 31). These findings show that IFN-γ is required for optimal induction of CXCR3 in CD4+, but not CD8+ T cells.

83 STAT-1 mediates signaling of cytokines, such as IFN-γ, IFN-α/β, and IL-

27 (177-180). STAT-1 also induces expression of T-bet, which controls CXCR3 levels in T cells. The effects of IFN-γ are mediated primarily by the STAT1 pathway, but several immunologically important genes are also up-regulated via

STAT-1-independent IFN-signaling (181, 182). We, therefore, asked whether

STAT1 is required for induction of CXCR3 on T cells. We isolated CD4+ and

CD8+ T cells from WT and STAT1-/- mice and compared CXCR3 surface expression and mRNA levels in them following in vitro stimulation with anti-

CD3/anti-CD28. Activated STAT1-/- CD4+ T cells failed to up-regulate CXCR3 as efficiently as WT CD4+ T cells (Fig. 31C). In contrast, STAT1-/- CD8+ T cells showed a significant induction of CXCR3 similar to WT CD8+ T cells (Fig. 31D).

Suspecting that another Th1-associated cytokine could be responsible for CXCR3 expression in the face of disrupted IFN-γ signaling, we tested the importance of TNF-alpha for CXCR3 induction. Unlike with the blockade of

IFN-γ, neither CD4+ nor CD8+ T cells were affected by neutralizing the Th1 cytokine TNF-alpha. This was seen for both STAT1-/- and STAT1+/+ derived T cells (Fig. 32).

Low CXCR3 expression on STAT1-/-CD4+ T cells also correlated with low levels of CXCR3 mRNA (Fig. 33 A, B). Both WT and STAT1-/- T cells produced significant IFN-γ upon activation, but IFN-γ levels were lower in STAT1-/- T cells

(data not shown). Blockade of IFN-γ did not inhibit expression of CXCR3 on

STAT1-/- CD8+ T cells (data not shown). These results indicate that STAT1 is

84 essential for efficient induction of CXCR3 in CD4+ but not CD8+ T cells and that

STAT1 controls CXCR3 levels on CD4+ T cells by regulating CXCR3 gene transcription. In addition, they suggest that IFN-γ is not involved in up-regulating

CXCR3 on STAT1-/- CD8+ T cells via STAT1-independent pathway.

T-bet and eomesodermin (eomes) are key transcription factors that regulate T cell function. T-bet modulates CD4+ and CD8+ T cell activity by regulating expression of several genes including CXCR3; whereas, eomes controls CD8 T cell function (183). Since T-bet and eomes can be induced in T cells via a STAT1-independent mechanism (184, 185), we hypothesized that increased expression of either or both these factors may be responsible for induction of CXCR3 in STAT1-/- CD8+ T cells. Hence, we compared T-bet and eomes mRNA levels in CD4+ and CD8+ T cells from both groups. STAT1-/-

CD4+ T cells showed decreased induction of T-bet mRNA compared to WT

CD4+ T cells (Fig. 33C). In contrast, STAT1-/- CD8+ T cells contained more T- bet mRNA than WT CD8+ T cells, but the difference was not significant (Fig.

33D). STAT1-/- CD4+ and CD8+ T cells also contained less eomes mRNA than

WT T cells but these differences were not significant (Fig. 33 E, F). Together, these findings show that STAT1 is critical for induction of T-bet in CD4+, but not

CD8+ T cells. Furthermore, they suggest that T-bet rather than eomes is likely to be involved in up-regulating CXCR3 on STAT1-/- CD8+ T cells.

85 In conclusion, both IFN-γ and STAT1 are critical for efficient induction of

CXCR3 on CD4+ T cells upon activation. However, neither IFN-γ nor STAT1 is required to up-regulate CXCR3 on CD8+ T cells. To the best of our knowledge this is the first study to demonstrate differential requirement for IFN-γ in up- regulation of CXCR3 on CD4+ versus CD8+ T cells. These findings are important in designing therapeutic strategies to control CXCR3 expression in T cells for treatment of autoimmune and inflammatory diseases.

Future Directions:

What induces Tbet expression in CD8+ T cells independently of the

STAT1/IFNγ pathway? It has been reported that for optimum Tbet-dependent

IFNγ production by CD8+ T cells in vivo, an IL-27Ralpha, but not an IFNγ- mediated signal is required (212). While it is known that IL-27 activates STAT1 and promotes Tbet expression in CD8+ T cells (213) the study mention above did not explore whether IL-27 was in fact acting through STAT1 to bring about

Tbet and IFNγ expression – leaving open the possibility that a STAT1- independent effect of IL-27 might be responsible for CXCR3 induction by CD8+

T cells.

Even though IL-27 is not reported to be made by T cells, we investigated the possibility that a contaminating cell type or hitherto unrecognized IL-27 source could be supplying our STAT1-deficient CD8+ T cells with the Tbet- inducing cytokine, we compared CXCR3 expression by in vitro activated WT

C57BL/6 T cells with those from TCCR-/- mice. As expected, we found no

86 significant deficiency in CXCR3 expression by TCCR-/- T cells (data not shown). Therefore, the Tbet induced in our CD8+ T cells is likely due to some other STAT1/IF gamma-independent source.

Another possibility is that Tbet is induced in CD8+ T cells, but not CD4+ via a STAT4-utilizing signal as described previously (214). However, since the

T cells in our model were stimulated in the absence of IL-12 producing cells (i.e. macrophages, DCs), this possibility seems less probable. It is also possible that Tbet and therefore CXCR3 is induced by an element of the TCR signaling pathway in CD8+ T cells.

One potential pathway involves the p38 MAPKinase. In addition to being involved in the TCR activation cascade, dysfunction of this kinase has been associated with decreased IFN-γ production by T cells (160) and phosphorylation of p38 occurs in response to numerous stimuli including Th1- promoting cytokines (161). Therefore we hypothesized that a STAT1- independent signaling pathway might utilize this molecule to bring about

CXCR3 induction in CD8+ T cells. A commercially available inhibitor,

SB203580 (Sigma) was used to assess the importance of the p38 pathways for

CXCR3 expression. If this pathway is important for CXCR3 expression, inhibitor treatment of STAT1-deficient CD8+ T cells should result in reduced

CXCR3 expression.

87 Interestingly we found that p38 inhibition resulted in reduced CXCR3 expression by CD4+ as well as CD8+ T cells (Fig. 34). This suggests that p38 may be important for Tbet induction in either cell type since it has been demonstrated that expression of this transcription is required for CXCR3 expression by both CD4+ and CD8+ T cells (86, 176). Indeed further work must be done to confirm the p38-Tbet link and also to elucidate which p38 utilizing pathway is involved here.

3.3 Materials and Methods:

Mice and Parasites: Sex, age matched wild type C57BL/6 and BALB/c mice were purchased from Harlan (Indianapolis, IN) and maintained at The Ohio

State University according to institutional regulations. IL-4Rα-/- mice where kindly provided by Alison Finnegan of Rheuma, Chicago, Illinois. STAT1- deficient C57BL/6 mice were a gift from Dr. Joan Durbin of The Ohio State

University. For intradermal infections 1 x 104 L. major promastigotes (LV39) were injected into the ears of C57BL/6 and BALB/c mice.

Leukocyte isolation and flow cytometry: At time-points post infection, 3-4 L. major-infected mice were sacrificed. dLN were excised and single-cell suspensions of LN cells were obtained by teasing through a 70µm mesh.

Lesion leukocytes were isolated as described previously (60). Cells were

88 stained with PE-conjugated anti-mouse CXCR3 (R and D systems, MN) and either FITC-labeled anti-CD4 or anti-CD8 antibodies (Biolegend, CA). Flow cytometric analysis of LN and lesion cells was performed with a BD

FACScalibur.

In vitro polyclonal stimulation and treatment of T cells: Cell suspensions were obtained from the excised LNs and spleens of uninfected mice. 90-95% pure

CD4+ and CD8+ T cells as well as mixed T cell populations were obtained via nylon wool columns and immunomagnetic isolation (Mitenyi Biotec USA, CA).

T cells were incubated 48 hours at 0.5-2.5 x106 cells/well in a 24-well plate pre- coated with 3 and 4 μg/ml anti-CD3 (clone 145-2C11) and anti-CD28 (clone

37.51) antibodies (Biolegend, CA), respectively. Following in vitro activation, cells were rested in their conditioned media for 24 hours prior to flow cytometry analysis similar to that described above.

Cytokine Elisa: Culture supernatants were sampled after 48 hours of incubation

o at 37 C, 5% CO2 and then analyzed for the presence of IFN- , IL-4, and IL-10 by sandwich ELISA as described previously (60).

Real Time PCR: Total RNA was extracted from T-cells using TRIZOL

Reagent (Invitrogen), and cDNA was generated and amplified in an Opticon2

DNA Engine (BioRad) using SYBR Green Taq polymerase. Fluorescence was

89 analyzed using Opticon Monitor 3 software. Primers and reaction conditions were found for CXCR3 using the PRIMER BANK website

(http://pga.mgh.harvard.edu/primerbank/index.html). Primers to T-bet and

Eomes were described elsewhere (174,175). Oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA). Data were normalized to

GAPDH and presented as fold induction over non-stimulated control cells from

WT and STAT1-/- mice.

Statistical analysis: Statistically significant differences were determined by an unpaired Student‟s t test. For real time PCR data, significant differences were determined by the Mann-Whitney Rank Sum test (SigmaStat). For either test, a

P value <0.05 was considered significant.

90

CHAPTER 4

THE ROLE OF CXCR3 IN VISCERAL LEISHMANIASIS.

Abstract:

We have shown previously that CXC chemokine receptor 3 (CXCR3) plays a critical role in the host defense against L. major by regulating recruitment of T cells to infected skin. Here, we demonstrate that although

CXCR3 regulates immune cell trafficking to liver during early phase of L. donovani infection, unlike L. major it is not essential for mediating immunity against L. donovani. CXCR3-/- C57BL/6 mice show delayed onset of hepatic inflammation and granuloma formation following L. donovani infection.

However, they mount an efficient Th1 response, recruit sufficient T cells into the liver, and control parasite growth as efficiently as CXCR3+/+ mice.

91 Introduction:

Leishmania donovani is an intracellular protozoan parasite, which causes visceral leishmaniasis (VL) (112,11). VL is characterized by the dissemination of parasites into the spleen, liver, lymph nodes and bone marrow resulting in hepatosplenomegaly, fever, abdominal pain, and weight loss. This disease is fatal if left untreated and complications such as secondary bacterial infection, anemia, and malnutrition due to hepatosplenomegaly are primarily responsible for VL- associated mortality (189).

The CXC chemokine receptor 3 (CXCR3) is expressed on plasmacytoid dendritic cells, neutrophils, NK cells, CD4+ Th1 cells, CD8+ T cells and B cells

(6,127,134). Three main ligands for CXCR3 include CXCL9 (MIG), CXCL10

(IP-10), and CXCL11 (I-TAC) (71,72). These chemokines mediate their biological activities by activating Ras/ERK, Src and the PI3K/Akt pathway (127) and their induction is closely associated with Th1 dominated responses (73).

CXCR3 and its ligands play a critical role in recruitment of NK cells, neutrophils and T cells to inflammatory sites in several disease models including allergic and autoimmune conditions (194, 195); graft rejection (81); and a variety of infectious agents (193,60). Additionally, some studies show that those CXCR3 ligands also regulate NK cell cytotoxicity as well as T cell activation and IFN-γ production (188).

92 Recent studies from our laboratory show that CXCR3 is required for the resolution of primary L. major infection (60). In the absence of CXCR3, L. major-resistant C57BL/6 mice mount a Th1 response but recruit fewer CD4+ and CD8+ T cells to infected skin, produce less IFN-γ locally, and develop lesions full of parasites. In contrast, the wild type C57BL/6 mice, which effectively control parasite growth, show a significant increase in CXCR3 expressing T cells in both the regional lymph nodes and the lesions. Taken together, these findings indicate that CXCR3 plays a non-redundant role in trafficking of IFN-γ producing effector T cells to the infected skin during cutaneous leishmaniasis. CXCR3 and its ligands also regulate recruitment of T cells into liver during infectious and non-infectious diseases (100,101).

Furthermore, patients suffering from VL show elevated levels of CXCL9 and

CXCL10 in their sera during active infection (102). Despite these observations it is not clear whether CXCR3 and its ligands contribute in host resistance against

VL. Therefore, we examined the in vivo role of CXCR3 in mediating host resistance against visceral leishmaniasis caused by L. donovani using CXCR3-

/- C57BL/6 mice. Our results demonstrate that CXCR3 may contribute to development of hepatic granuloma during early phase of visceral leishmaniasis but it is not essential for Th1 development, leukocyte recruitment and resolution of L. donovani infection.

93 Results and Discussion

We have previously demonstrated that CXCR3 is required for immunity against cutaneous leishmaniasis caused by L. major. Genetically resistant

C57BL/6 mice lacking CXCR3 develop chronic non-healing lesions full of parasites following L. major infection despite mounting a systemic Th1 response. In contrast, in the present study, CXCR3-/- C57BL/6 mice did not show increased susceptibility to L. donovani and controlled L. donovani growth in the liver and spleen as efficiently as resistant wild type C57BL/6 (Fig. 35).

Interestingly, 30 days post infection, CXCR3-/- mice showed more rapid resolution of parasite burden in their spleens than CXCR3+/+ mice (Fig 35B).

These findings indicate that CXCR3 is not required for the development of protective immunity against L. donovani in resistant C57BL/6 mice.

CXCR3 controls migration of CD4+ and CD8+ T cells to the site of inflammation in many diseases (60, 101,102). Therefore, it is perhaps not surprising that the development of chronic non-healing lesions in CXCR3-/-

C57BL/6 mice after L. major infection is associated with a significant impairment of T cell recruitment to infected skin (60). CD4+ and CD8+ T cells recruited to liver during L. donovani infection are critical for the host defense but they also contribute to formation of granuloma and induce liver pathology. We therefore analyzed pathology and enumerated granulomas in the livers of L. donovani- infected CXCR3+/+ and CXCR3-/- mice on days 15, 30 and 60 post-infection.

Onset of liver inflammation and granuloma formation was delayed in CXCR3-/-

94 mice and these mice contained significantly fewer foci of inflammation and granulomas in their liver as compared to CXCR3+/+ mice on days 15 and 30 after infection (Fig. 36). However, both groups developed similar liver inflammation and contained comparable number of granuloma by day 60 indicating that despite a delayed onset of cellular infiltration, CXCR3-/- mice ultimately recruit sufficient effector immune cells to the site of L. donovani infection (Fig. 36). Furthermore, no significant differences were noted in the proportion of CD11b+ macrophages, CD11chigh dendritic cells, NK1.1+ cells,

CD4+ T cells, and CD8+ T cells in infected livers and spleens from CXCR3+/+ and CXCR3-/- mice at all time points (data not shown). Our findings are similar to those reported in a previous study which reported that lack of CXCR3 impairs early granuloma formation during Mycobacterium tuberculosis infection in mice but has no effect on the final outcome of the infection. Furthermore, this study also found that neutrophils control granuloma formation via CXCR3-depdendent pathway (43). However, it is unlikely that lack of neutrophil recruitment to liver impaired early granuloma formation in CXCR3-/- mice during L. donovani infection because flow cytometry analysis showed that both CXCR3+/+ and

CXCR3-/- mice comparable proportions of Gr1+ neutrophils to their livers throughout the course of infection (data not shown). Nevertheless, our data show that although CXCR3 may be involved in controlling early liver inflammation and granuloma formation after L. donovani infection, it is not essential for the recruitment of immune cells to the liver during late phase of the

95 infection. Additionally, these findings also suggest that other T cell-associated

C-C and CXC chemokine receptors may be involved in migration of effector

CD4+ and CD8+ T cells to the liver and spleen.

There may be several reasons why a lack of CXCR3 impairs migration of

T cells to skin during L. major infection but has no effect on T cell trafficking to liver and spleen during L. donovani infection. First, L. major and L. donovani cause different diseases in humans and trigger complex immune responses that determine the outcome of the disease in the host. Second, it is becoming increasingly evident that chemokine receptors play organ-specific roles in regulating leukocyte trafficking. For example, selective recruitment of naïve lymphocytes to the secondary lymphoid organs is mediated largely by the action of CCR7 and its ligands (109,39) whereas CCR9 is known to mediate T cell development in the thymus (104). Similarly, among the so-called inflammatory chemokine receptors, CCR4 and CCR10 have been implicated in the selective recruitment of certain T cell subsets to the skin (105,106). Our findings in the present study together with our previous work demonstrate clearly that even though CXCR3 is necessary for optimal recruitment of T cells to skin, other mechanisms efficiently recruit T cells to the spleen and liver tissue during inflammation.

IL-12-driven CD4+ Th1-type lymphocytes play a critical role in immunity to L. donovani (95,96). However, the clear Th1-Th2 pattern of disease development shown for L. major is not observed in VL caused by L. donovani in

96 mice and humans because Th2 cytokines such as IL-4 and IL-13 do not determine susceptibility. In fact, IL-4-/- and IL-4Rα-/- mice are more susceptible to L. donovani, and respond poorly to anti-leishmanial drug therapy indicating that IL-4 may play a disease protective role during visceral leishmaniasis

(97,98). Previous studies have shown that CXCR3 plays a critical role in T cell activation. Similarly, CXCL9 and CXCL10 also enhance NK cell cytotoxicity, stimulate T cell proliferation and effector cytokine production (52,127). We therefore analyzed Th1 and Th2 responses in L. donovani-infected CXCR3+/+ and CXCR3-/- mice by determining titers of L. donovani Ag (LdAg)-specific Th1 associated IgG2a and Th2 associated IgG1, and measuring proliferation and cytokine production in spleen cells following in vitro stimulation with LdAg.

Additionally, we also compared levels of TNF-α, IL-12, IFN-γ, IL-4 and IL-10 mRNA in the infected livers by real-time RT-PCR. Throughout the course of infection, both groups displayed similar titers of L. donovani-specific Th1 associated IgG2a and Th2-associated IgG1 Ab (Fig. 37). On days 15, 30 and

60 post-infection, LdAg-stimulated spleen cells from L. donovani-infected

CXCR3+/+ and CXCR3-/- mice displayed comparable proliferation responses

(data not shown) and produced comparable amounts of IL-12, IFN-γ, IL-4 and

IL-10 (Fig. 38). At all time points, infected livers from both groups contained comparable amounts of TNF-α, IL-12, IFN-γ and IL-10 mRNA. Levels of IL-4 mRNA were also similar in CXCR3+/+ and CXCR3-/- mice on days 15 and 30, however, CXCR3-/- mice consistently contained significantly more IL-4 mRNA

97 in their livers on day 60 (Fig. 39). Additionally, since T cells can serve to amplify the recruitment of leukocytes by producing chemokines themselves

(99), we measured chemokine levels in the infected livers of CXCR3-/- and

CXCR3+/+ mice. No significant difference in CXCL9 and CXCL10 mRNA was found in the livers of CXCR3+/+ and CXCR3-/- mice at all time points assayed

(Fig. 40). These results demonstrate that lack of CXCR3 does not alter T cell activation, production of Th1 and Th2 cytokines and recruitment of IFN-γ producing cells to liver and spleen during L. donovani infection.

In sum, CXCR3-/- C57BL/6 mice show delayed formation of liver granuloma after L. donovani infection, but they eventually recruit sufficient T cells to liver and spleen and control L. donovani infection as efficiently as

CXCR3+/+ C57BL/6 mice. Furthermore, the resolution of L. donovani infection by CXCR3-/- mice is associated with the development of an efficient Th1 response. These results also suggest that although CXCR3 ligands, CXCL9 and CXCL10 are produced in high levels during L. donovani infection, they may play a minor role in the host defense against this parasite.

Future Directions:

While CXCR3-mediated recruitment of leukocytes to the L. donovani-infected liver is not crucial for long-term resistance to the parasite, other chemokine- chemokine receptor pairs will no doubt prove to be less dispensable for mediating immunity to L. donovani induced VL. The association of a

98 chemokine-chemokine receptor pair with a tissue-specific homing route is a well demonstrated trend in the field of chemokine biology (see discussion).

Pertinent for VL disease, would be the liver-directing chemokine- chemokine receptor pairs. Studies of the inflamed/diseased liver in diverse models suggest a key role for CXCR6 and its ligand, CXCL16 in leukocyte migration to the organ (108). It stands to reason that during VL, which greatly involves the liver, CXCR6-signaling would efficiently mediate recruitment of leukocytes to the infected liver even in the absence of irrelevant/redundant/or complementary CXCR3-signaling. Therefore we hypothesize that blocking

CXCR6-CXCL16 will reduce leukocyte recruitment to the L. donovani infected liver, rendering mice highly permissive to parasite growth and susceptible to disease. Since no CXCR6 knockout mice are readily available on the C57BL/6 background, blockade of CXCL16 with neutralizing antibody will allow us to manipulate this chemokine-chemokine receptor interaction. Experiments in our lab are currently underway to explore the potential contributions of CXCR6- siganling in L. donovani immunity.

Materials and Methods:

Eight to ten week-old, sex-matched CXCR3-/- C57BL/6 and WT C57BL/6 mice

(purchased from Harlan) were infected i.v. with 1x107 L. donovani (1 Sudan strain) amastigotes harvested from the spleens of infected hamsters. Animals were housed and maintained under institutional guidelines for animal research

99 at The Ohio State University. Disease progression was monitored by measuring parasite loads in livers and spleens 15, 30, and 60 days post- infection as described elsewhere (189). Also, at these time points, hematoxylin/eosin stained tissue sections from the livers were examined to enumerate granulomas as described previously (189). Furthermore, spleens were removed from these mice and parasite-specific T cell proliferation was assessed by Alamar Blue Assay (Biosource International). Levels of IFN- , IL-

12p70, IL-4, and IL-10 in the above supernatants were measured by ELISA as described previously (60).

For flow cytometric analysis, single cell suspensions of liver leukocytes were obtained as described previously (189). 1-2x106 cells were stained with

FITC- and PE-conjugated antibodies purchased from Biolegend and BD

PharMingen (San Diego, CA). Single cell suspensions of splenocytes were obtained as described for the proliferation assay and stained in a manner similar to the liver cells. Flow cytometry analysis was then performed using a

FACSCalibur flow cytometer and CellQuestPro software (Becton Dickinson).

Total RNA was extracted from liver sections using the SV Total RNA Isolation

System (Promega). mRNA was reversed transcribed, and cDNA was amplified in an Opticon2 DNA Engine (BioRad) using SYBR Green Taq polymerase. The primers used to amplify the cDNA in this semi-quantitative RT-PCR were found

100 using the PRIMER BANK website:

(http://pga.mgh.harvard.edu/primerbank/index.html).

In the aforementioned experiments, a Student‟s unpaired t-test was used to determine statistical significance of differences in the values observed. A value of p < 0.05 was considered significant.

101

CHAPTER 5

FUTURE DIRECTIONS AND CONCLUDING REMARKS

Abstract:

In this section we present the results of our additional investigations into the factors controlling CXCR3 expression by T cells. While it is known that TCR- activation is important for CXCR3 up-regulation by T cells, it is unclear precisely what elements of the TCR-signaling cascade are required. Also since the requisite TCR-stimulus must cease for effective CXCR3 up-regulation, it is possible that some element of T cell activation actually suppresses CXCR3 expression during initial activation. We also recognize the potential importance of co-stimulatory molecules in CXCR3 induction – a topic not well explored thus far. Also, we report on the preferential expression of CXCR3 by certain memory

T cell subsets generated during L. major infection. It is our hope that in the future, our work will form the basis for further inquiry as to the finer points of

CXCR3 regulation. We have focused on the importance of several TCR- signaling cascade participants.

102 5.1 PI3Kinase gamma as a regulator of CXCR3 expression in T cells.

The gamma isoform of PI3Kinase (PI3Kγ) controls leukocyte chemotaxis by participating in GPCR signaling, as well as regulating cellular polarization. Here we show that PI3Kγ is required for efficient induction of CXC chemokine receptor 3 (CXCR3) on T cells upon activation. Inhibition of PI3Kinases using wortmannin or blockade of PI3Kγ using a selective inhibitor suppressed induction of CXCR3 on T cells following activation. Levels of CXCR3 and T-bet mRNA were significantly lower in PI3Kγ inhibitor-treated T cells, indicating that

PI3Kγ controls CXCR3 expression by regulating gene transcription at least in part through induction of T-bet. Finally, T cells from PI3Kγ-/- mice up-regulated

CXCR3 less efficiently than wild type controls both upon in vitro activation as well as during Leishmania mexicana infection. These results reveal a novel role for PI3Kγ in the induction of CXCR3 on T-cells and suggest that PI3Kγ may regulate leukocyte chemotaxis by controlling expression of chemokine receptors. Also in this chapter we discuss some other interesting results regarding CXCR3 induction in T cells that should provide a basis for further study.

Introduction:

The chemokine receptor CXCR3 is expressed on plasmacytoid dendritic cells (pDCs), natural killer (NK) cells, CD4+ and CD8+ T cells, and microvascular endothelial cells (69). Three CXC chemokines, CXCL9, CXCL10,

103 and CXCL11 signal via CXCR3 (73) and mediate biological functions such as cell migration and proliferation (127).

CXCR3 mediates immunity against pathogens by regulating recruitment and function of effector T cells and other leukocytes (191,193, 60,77), however,

CXCR3 also contributes to pathogenesis of allograft rejection and autoimmune diseases by promoting recruitment of pathogenic T cells (81,194, 76, 160).

Resting T cells express low levels of CXCR3 but they up-regulate CXCR3 when activated thereby gaining responsiveness to its ligands (80,138).

The phosphoinositide-3-kinases (PI3Ks) are lipid signaling kinases which are required for generation of PIP3. Class I PI3Ks are dual-specificity lipid and protein kinases, which control cell growth, proliferation, survival, adhesion and motility. These kinases include class IA (consisting of PI3Kα, PI3Kβ and PI3Kδ) and class IB (PI3Kγ) (195). Of these, PI3Kγ is involved in leukocyte chemotaxis, mast cell degranulation, neutrophil respiratory burst and TCR- induced T cell activation (195-197).

Several studies have shown that TCR-induced activation is essential for up-regulation of CXCR3 on T cell suggesting that PI3Kγ may regulate CXCR3 expression on activated T cells (80,138). Therefore, we examined the role of

PI3Kγ in regulation of CXCR3 on T cells. Our findings show that PI3Kγ is critical for efficient induction of CXCR3 on activated T cells.

104 Results and Discussion:

T cells up-regulate CXCR3 upon activation, but the TCR-mediated signaling mechanisms which control CXCR3 expression are not clear. Class I

PI3Ks are dual-specificity lipid and protein kinases which participate in numerous intracellular signaling pathways. PI3Kγ is a class I PI3K which plays a critical role in leukocyte migration, and its expression is mainly restricted to hematopoietic cells (195,197). PI3Kγ is mainly activated by GPCRs which include chemokine receptors (195). However, a recent study has found that

PI3Kγ in T cells also participates in T cell activation induced by TCR engagement (196).

The goal of our study was to determine whether the PI3K pathway, and specifically PI3Kγ control CXCR3 expression in T cells following TCR-induced activation. To investigate the role of PI3K pathway in CXCR3 induction by T cells, we analyzed the expression of CXCR3 on C57BL/6 mouse T cells which were stimulated with anti-CD3/anti-CD28 in vitro and subsequently rested in the presence of PI3K inhibitor wortmannin or DMSO (control) for 24 hrs, as described previously (80). Activated T cells incubated with 100nM wortmannin during resting phase failed to efficiently up-regulate CXCR3 (Fig. 41A). Similar results were observed when T cells were treated with the PI3K inhibitor

LY294002 (data not shown). These findings indicate that the PI3K pathway is critical for efficient induction of CXCR3 on activated T cells.

105 TCR-induced T cell activation is essential for up-regulation of CXCR3, which mediates T cell chemotaxis, likely by activating PI3Kγ. Because PI3Kγ also participates in TCR-induced T cell activation (196), we determined whether

PI3Kγ is required for induction of CXCR3. We stimulated T cells from naïve WT and PI3Kγ-/- C57BL/6 mice in vitro with anti-CD3/anti-CD28 as described above and compared levels of CXCR3 using flow cytometry. In addition, we analyzed

CXCR3 expressing T cells in the lymph nodes of WT and PI3Kγ-/- mice infected with Leishmania mexicana. PI3Kγ-/- T cells failed to up-regulate CXCR3 as efficiently as WT T cells following in vitro stimulation (Fig. 41B). Furthermore, the lymph nodes of L. mexicana-infected PI3Kγ-/- mice contained fewer CXCR3 expressing CD4+ and CD8+T cells than WT mice (Fig. 41C) and the average intensity of CXCR3-PE staining was reduced in the knockout T cells (Fig. 41D).

Although these results suggest that PI3Kγ may control CXCR3 on T cells they do not exclude the possibility that the failure of PI3Kγ-/- T cells to up- regulate CXCR3 may due to an inherent defect in these T cells and/or impaired

TCR-induced activation. Therefore, we determined whether blockade of PI3Kγ using an isoform-selective small-molecule PI3Kγ inhibitor (AS-605240) after T cell activation prevents induction of CXCR3. The AS-605240 molecule competes with ATP for its binding pocket on PI3Kγ effectively inhibiting the enzyme (197). CD4+ and CD8+ T cells were isolated from naïve C57BL/6 mice, activated in vitro with anti-CD3/anti-CD28 for 48 hrs, then treated with 1.25 μM

AS-605240 for 24 hrs and surface expression and mRNA levels of CXCR3 were

106 measured by flow cytometry and real time RT-PCR respectively. Activated T cells treated with AS-605240 failed to up-regulate CXCR3 as efficiently as controls (Fig. 42A). Low CXCR3 expression on AS-605240-treated T cells also correlated with low levels of CXCR3 mRNA (Fig. 42B). Levels of IFN-γ, IL-4 and

IL-2 were comparable in culture supernatants from AS-604850-treated and control T cells (data not shown). AS605240 treatment had no effect on mRNA levels of CCR1, CCR5, CCR4, CCR7, CXCR6, and CCR10 (Fig. 42C). These results demonstrate that PI3Kγ is required for efficient induction of CXCR3 on activated T cells and that PI3Kγ controls CXCR3 levels on T cells by regulating

CXCR3 gene transcription. In addition, they suggest that PI3Kγ up-regulates

CXCR3 via IFN-γ-independent mechanism.

Expression of CXCR3 by T cells requires the transcription factor T-bet

(86,176). It has been also reported that SHP-1, which suppresses PI3K activity

(198) also inhibits T-bet expression in T cells (199). We therefore measured T- bet mRNA levels in AS-605240-treated and control T cells. AS-605240-treated

T cells showed decreased induction of T-bet mRNA than controls (Fig. 42D).

These findings indicate that PI3Kγ controls T-bet induction in TCR-activated T cells and suggest that T-bet may be involved in PI3Kγ-mediated up-regulation of CXCR3.

In conclusion, our study has revealed a previously unknown function of

PI3Kγ in induction of CXCR3 on activated T cells. The findings also suggest that since PI3Kγ may regulate T cell chemotaxis by modulating CXCR3 levels,

107 PI3Kγ-selective inhibitors could be used for manipulating CXCR3 levels on pathogenic T cells to inhibit their chemotaxis in chronic inflammatory diseases.

5.2 CXCR3 induction and other aspects of the TCR signaling cascade:

Besides the PI3Kinases, other elements of the TCR-signaling pathways may be involved in the up-regulation of CXCR3 in T cells. We have found that inhibiting the calcium flux associated with TCR signaling and inhibition of ERK Kinases resulted in either no effect or slight decrease in CXCR3 induction, respectively

(data not shown). However, we have uncovered an interesting role for another

TCR cascade participant in CXCR3 induction.

PLC- is an important component of the TCR-signaling cascade as well as GPCR signaling. Therefore we tested how this molecule affects CXCR3 expression by T cells by incubating in vitro stimulated T cells with the inhibitor

U73122. We hypothesized that PLC inhibition might decrease CXCR3 expression since both this enzyme and PI3Kinase are involved in TCR signaling and since inhibition resulted in reduced receptor induction. In fact, we found the opposite to be true. Through flow cytometric staining of U73122 and DMSO treated activated T cells we found that both C57BL/6 and BALB/c derived T cells expressed higher levels of CXCR3 when they were treated with the PLC inhibitor (Fig. 43A). Curiously, these treated cells also produced lower levels of

IL-10 – a factor we have shown to be important for suppressing CXCR3

108 expression. In fact we found a dose dependent reduction of IL-10 production in response to PLC inhibition (Fig. 43B). It is tempting to hypothesize that PLC inhibits CXCR3 expression by IL-10 production. Indeed more investigation is needed to properly test this notion.

These results shed some light on an under-explored aspect of CXCR3 regulation. While the cytokines and transcription factors required for CXCR3 expression have been studied (80,86), the need for TCR activation has not been explained experimentally. Particularly, it is not known exactly what aspects of this signaling cascade are needed for CXCR3 induction. With these preliminary findings, we have provided the framework for further study.

5.3 CXCR3 expression on memory T cells:

In addition to being highly expressed by activated effector T cells, CXCR3 has also been found to be expressed by memory T cells. Of the memory T cells, central memory T cells (Tcm) have been shown to be important for immunity to re-infection by leishmania parasites. To determine if CXCR3 is expressed on the memory T cells generated in response to parasitic infection, L. major- resistant C57BL/6 mice were infected with L. major and allowed to resolve the infection (this was accomplished approximately 12 weeks after infection). Cells were isolated from the lymph node draining the site of infection and their surface markers were analyzed by multi-color flow cytometry. We found that, in agreement with previous reports, naïve T cells (defined as CD3+/CD44-

109 /CD62L+) expressed very little CXCR3 while many effector T cells (Teff =

CD3+/CD44+/CD62L-) and Tcm cells (CD3+/CD44+CD62L+) expressed CXCR3

(Fig. 44).

Based on these results, it would appear that CXCR3 is expressed by T cells of the central memory phenotype. Therefore CXCR3 may play an important role, not only in the initail primary response to L. major, but also in the memory response mounted against re-exposure to the parasite. This observation demands further study of CXCR3 in the memory response. If indeed CXCR3 is important for marshalling an effective memory response, vaccine strategies should be explored that optimize CXCR3 induction and/or function.

Remaining questions:

The molecular reasons behind the need for a release from TCR stimulation to date remain unknown. Intuitively, it makes sense to have CXCR3 induction by naïve T cells delayed until after cessation of TCR-APC interaction. Up- regulation of CXCR3 by these cells in the dLN could result in their retention there instead of the homing to peripheral sites of inflammation (since the

CXCR3 ligands are expressed in the LN). CXCR3 ligand expression in the dLN has been shown to promote T cell retention in these secondary lymphoid organs during Leishmania infection. On the other hand, if the newly activated T

110 cells are permitted to exit the LN prior to CXCR3 up-regulation, they might more efficiently home to sites of inflammation –probable sources of CXC3 ligands.

While many elements of TCR signaling are requisite for CXCR3 induction on naïve T cells (80), it may be that certain elements of the signal- cascade may suppress CXCR3 up-regulation and only after their activity wanes can CXCR3 expression be increased. Another possibility is that sufficient and appropriate transcription factors must be given time to accumulate in the activated T cells. It may be that such an accumulation requires time to affect

CXCR3 expression, however, we and others have found that given extended periods of stimulation with no subsequent resting of T cells results in little-to-no

CXCR3 up-regulation. It may yet be that transcription factors must first be up- regulated or accumulated for optimal CXCR3 expression, but perhaps ongoing

TCR stimulation impedes this, or sequesters transcription factors and their accessory molecules in more immediately pertinent activities. Clearly much experimentation must be done to test these notions. Also a related question would be: is this a CXCR3-specific phenomenon? Or do other “inflammatory” chemokine receptors require such a sequence of events for optimal expression?

The enigmatic up-and down-regulation of CXCR3 that occurs upon re- stimulation or upon entry of cells into peripheral sites (138) also remains to be investigated. Could IL-10, a proven CXCR3-suppressive factor be responsible for a loss of CXCR3 in peripheral tissues? The fact that T cells re-stimulated ex

111 vivo with by antigen presenting cells also down-regulate CXCR3 suggests that something else is involved in this up-and-down regulatory cycle. It may be that cell-cell interactions also play a role.

Another unexplored aspect of CXCR3 regulation is how expression of this chemokine receptor is influenced by co-stimulatory molecules. The delivery of co-stimulatory signal to T cells during their activation can greatly impact their later function and capacity for activation. It is possible that the cumulative interactions of a T cell with co-stimulatory and co-inhibitory molecules at the

APC-interface might determine the expression of inflammatory chemokine receptors such as CXCR3. While we have only begun to investigate the impact of co-stimulatory (CD80/CD86) and co-inhibitory (PDL1/2) deficiency, much work remains to be done to fully understand how this accessory signal affects

CXCR3 expression.

Investigation of these lingering questions will no doubt bring to light numerous intricacies of T cell activation that certainly involve the enigmatic events that occur within the secondary lymphoid organ. As advances in vivo imagery have revealed, the interactions of T cells with their lymph node neighbors (antigen presenting, and other support cells) are both dynamic in nature and on the verge of being understood. Such interactions seem to have an impact on the activation state of T cells and therefore are likely to influence

CXCR3 up-regulation.

112

5.4 Materials and Methods:

Mice: PI3Kγ-/- C57BL/6 mice were maintained at The Ohio State University.

Wild type (WT) C57BL/6 mice were purchased from Harlan (Indianapolis, USA).

Age (6-8 wks) and sex matched animals were used for experiments.

In vitro stimulation of T cells: Single cell suspensions were prepared from 3-

5 spleens of WT or PI3Kγ-/- mice and either CD4+ or CD8+, or mixed T-cells were isolated by immunomagnetic separation (Myltenyi Biotech). Briefly, cells

(90-94% pure) were plated in 24-well plates at 1x106/ml and stimulated with plate-bound anti-CD3e (3μg/ml) and anti-CD28 (4μg/ml) at 37oC for 48 hr.

Subsequently, cells and their conditioned media were incubated for 24 hours without stimulation as described previously (Chapter 3) either in the presence of a specified chemical inhibitor or a DMSO vehicle.

Flow Cytometry: 1-2x105 cells in PBS were stained with PE-conjugated anti-

CXCR3 antibodies (R&D systems) and analyzed by flow cytometry.

Real Time-PCR: Total RNA was extracted from T-cells using TRIZOL Reagent

(Invitrogen), and cDNA was generated and amplified as described previously

(103). Primers and reaction conditions were found using the PRIMER BANK website.

113 Leishmania mexicana parasites and infections: Five age, sex matched (8-12 wks old) WT and PI3Kγ-/- C57BL/6 mice were infected by inoculating 1 x 106 metacyclic Leishmania mexicana promastigotes into the footpads. Three weeks post-infection, mice were sacrificed and CXCR3 expressing T cells in the draining LNs were analyzed by flow cytometry.

Statistical Analysis: Significance of differences was determined by an unpaired Student‟s t Test. A value of p < 0.05 was considered significant.

5.5 Concluding Statement:

While much remains to be explored in regards to both chemokine receptor regulation as well as the role played by such molecules during leishmaniasis, we have demonstrated the importance and irrelevance of several

“inflammatory” chemokine receptors in the ant-leishmania response and partially we have characterized the consequences of CXCR3-deficiency for this response in mice. We have taken the initial steps towards uncovering the mechanism by which CXCR3 mediates resistance to L. major. Additionally we elucidated several hitherto unrecognized regulatory factors governing CXCR3 expression by T cells.

114

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136

APPENDICES:

137

APPENDIX A:

FIGURES 1-23

138

Fig. 1. Course of L. major infection in CXCR3+/+ and CXCR3–/– mice. (A) Mice were infected by inoculating 2x106 L. major SPP into the footpad. Lesion growth was monitored by measuring the increase in thickness of the infected footpad and comparing this to the thickness of the contra-lateral uninfected footpad.

Significant differences in footpad lesions were found after four weeks of infection. Data are representative of two independent experiments. Note that footpads from CXCR3–/– mice contained 4–5 log more parasites than those from CXCR3+/+ mice at week 8 post-infection. (B) Footpad and (C) lymph node parasites burdens were determined by limiting-dilution analysis at weeks 3 and

8 post-infection. Data are expressed as mean log titer +/- SEM. BALB/c mice displayed the highest parasite loads (data not shown). Similar results were observed in three independent experiments.

139

*

140

Fig. 2. Infection of CXCR3-/- and CXCR3+/+ mice with an intermediate dose of

L. major promastigotes in the ear pinna resembles disease caused by LFP infection and CXCR3-/- do not resolve L. major infection. Mice were infected with 1x104 L. major promastigotes intradermally and the lesion growth was measured throughout the infection course (A). Then mice were euthanized and parasite burdens were found by limiting dilution assay (B). Significant differences in lesion diameter were found after 6 weeks of infection and

CXCR3-/- lesions harbored significantly more parasite at week 15 than

CXCR3+/+ mice.

141

A.

B.

*

142

Fig. 3. Analysis of the histopathology of infected footpads from L. major infected CXCR3+/+ and CXCR–/– mice at week 8 post-infection. (A,C)

Hematoxylin/eosin-stained lesions from CXCR3+/+ mice showed inflammatory infiltrates comprised of macrophages with few parasites and lymphocytes.

(B,D) Lesions from L. major-infectedCXCR3–/– mice showed tissue destruction associated with inflammatory infiltrate comprised of heavily parasitized macrophages, neutrophils, eosinophils, and lymphocytes. Shown are representative images from one of three independent experiments (n=5).

143

144

Fig. 4. Antibody responses in L. major-infected CXCR3–/– and CXCR3+/+ mice at weeks 3, 6, and 8 post-infection. (A) LmAg-specific IgG1and (B) LmAg- specific IgG2a measured in sera. Serum taken from both groups at the time points specified contained similar levels of IgG2a and IgG1. Data are from two independent experiments and presented as mean of endpoint titers on log scale. Eight to ten mice were analyzed in each group.

145

146

Fig. 5. Analysis of in vitro proliferation (A) and enumeration of CD4+ (B) and

CD8+ T cells (C) in the draining lymph nodes cells from L. major-infected

CXCR3–/– and CXCR3+/+ mice at weeks 3 and 8 post-infection (eight mice per group). (A) At weeks 3 and 8 post-infection, the draining lymph node cells from

CXCR3+/+ and CXCR3–/– mice were stimulated in vitro with 20 µg/ml of LmAg and their proliferation was measured using Alamar blue as per manufacturer's instructions. Data are expressed as the mean % proliferation +/- SEM. (B, C) At these time points, lymphocytes from the lymph nodes of L. major infected

CXCR3+/+ and CXCR3–/– mice were enumerated and analyzed by flow cytometry. Data are expressed as the number of CD3+CD4+ (B) and

CD3+CD8+ (C) T cells per lymph node. Four to five mice were used per group at each time point. These data are means of two independent experiments

(n=8)..

147

148

Fig. 6. Analysis of in vitro cytokine production by LmAg stimulated lymph node cells from L. major-infected CXCR3+/+ and CXCR3–/– mice. The levels of IL-12, IFN-γ, and IL-4 were measured in culture supernatants by sandwich ELISA. The data are means of two independent experiments at each time point. Asterisks indicate statistically significant (p<0.05) differences between groups (n=8).

149

150

Fig. 7. Quantification of IFN-γ and IL-4 gene transcript levels in lesions of L. major-infected CXCR3+/+ and CXCR3–/– mice by real-time RT-PCR. Total

RNA was isolated from lesions of L. major-infected CXCR3+/+ and CXCR3–/– mice at weeks 3 and 8 post-infection, reverse-transcribed, and levels of cytokine gene transcripts were measured by SYBR green incorporation by real- time PCR. Data are shown as mean fg/ml +/-SEM. Asterisks indicate statistically significant differences between the groups (p<0.05).

151

152

Fig. 8. Analysis of CXCR3-expressing CD4+ and CD8+ T cells in lesions from

L. major-infected CXCR3+/+ mice at week 3 post-infection. Lesion-derived cells from CXCR3+/+ and CXCR3–/– mice were stained with PE-conjugated anti-

CXCR3 and FITC-conjugated anti-CD4 or anti-CD8 Ab, and analyzed by flow cytometry. Note that the lesions from L. major infected CXCR3+/+ mice contained CXCR3-expressing CD4+ and CD8+ T cells. Similar results were also observed at week 8 post-infection (data not shown). The percentages indicated in all the quadrants are for the gated lymphocytes. The percentages of demarcated CXCR3+CD4+ and CXCR3+CD8+ cells in wild-type mice are shown in bold outside the square (upper right side).

153

154

A. B.

* * * *

Fig. 9. Enumeration of CD4+ and CD8+ T cells in lesions from L. major-infected

CXCR3+/+ and CXCR3–/– mice. At weeks 3 and 8 after infection, lesion- derived lymphocytes from the ear lesions of L. major-infected CXCR3+/+ and

CXCR3–/– mice were enumerated and analyzed by flow cytometry. Data are expressed as the number of CD3+CD4+ (A) and CD3+CD8+ (B) T cells per ear lesion. Four to five mice were used per group at each time point. Data are shown as mean cell numbers +/-SEM. Asterisks indicate statistically significant differences between the groups (p<0.05).

155

Fig. 10. IFNγ production by T cells from CXCR3+/+ and CXCR3-/- mice infected with L. major. CD4+ or CD8+ T cells were isolated from CXCR3+/+ or

CXCR-/- dLN (A,B) or ear lesions (C,D). These cells were incubated with

3ug/ml anti-CD3e and 5ug/ml anti-CD28 antibodies and the cytokines released into the supernatants were quantified by ELISA. Data are shown as mean concentrations +/-SEM. Asterisks indicate statistically significant differences between the groups (p<0.05).

156

A. B. A. B.

C. D.

C. D.

*

157

Fig. 11. IFNγ production by T cells of the CXCR3+/+ and CXCR3-/- dLN and lesion during an in vitro APC assay. T cells were purified from either the LN

(A,B) or lesions (C,D) of L. major-infected CXCR3+/+ and CXCR3-/- mice and co-cultured with BMDM pulsed with either L. major promstigotes (at a 1:10 cell to parasite ratio) or 20 ug/ml L. major antigen extract. IFNγ production by these

T cells was quantified by ELISA. Data are shown as mean concentrations +/-

SEM. Asterisks indicate statistically significant differences between the groups

(p<0.05). Shown are the results of two to three independent experiments.

158

A.

B.

* *

C. * *

159

*

Fig. 12. CXCL10 responsiveness of CD4 and CD8 T cells from C57BL/6 mice infected with L. major. FACS purified T cells were used in a Transwell migration assay determining the responsiveness of CXCR3+/+ CD4+ and CD8+

T cells to rCXCL10. Results are presented as the percentage of cells that migrating to chemokines over those responding to media alone. Data are shown as mean percentages +/-SEM. Asterisks indicate statistically significant differences between the groups (p<0.05). Shown are the results of two to three independent experiments.

160

Fig. 13. Flow cytometric analysis of non-T cell populations in CXCR3+/+ and

CXCR3-/- lesions following L. major infection. A. The lesions of CXCR3-/- mice contain higher numbers of NK cells (NK1,1+), B. neutrophils (GR1+), and C. monocytes/macrophages (CD11b+) than CXCR3+/+ mice. D. CXCR3 -/- mice also recruit comparable numbers of neutrophils and monocyte/macrophages to the peritoneal cavity to CXCR3+/+ mice in a thioglycollate induced peritonitis model. The data are a representative result from one of 2-3 independent experiments at each time point.

161

A. Total NK1.1+ Cells/Lesion B. Total CD11b+ Cells/Lesion

10 20 8 15 CXCR3 +/+ 6 CXCR3 +/+ 10 CXCR3 -/- 4 CXCR3 -/-

5 2

Number Number of Cells

Number Number of Cells recovered (10^3) recovered 0 (10^4) Recovered 0 WEEK 3 WEEK 8 WEEK 3 WEEK 8

C. Total GR1+ Cells/Lesion

12 10 8 CXCR3 +/+ 6 CXCR3 -/- 4

2 Number Number of Cells Recovered (10^3) Recovered 0

D.

162

Fig. 14. Determining the role of CXCR3 in DC migration to LN tissues. (A)

Fluorescence microscopy analysis of dendritic cell recruitment to the lesion- draining LN of CXCR3-/- and CXCR3+/+ mice. White arrows indicate FITChigh langerhans cells. (B) Flow cytometric analysis of CD11c+ cell presence in the dLN tissue of CXCR3+/+ and CXCR3-/- L. major-infected mice 6 weeks post infection. Numbers shown represent the percentage of leukocytes expressing

CD11c. The data shown here are representative of two independent experiments.

163

A. 40X

CXCR3-/-

CXCR3+/+ CXCR3-/-

B. CXCR3+/+ Data.023 4.7 CXCR3Data.024-/- 4.3

R2 R2

100 101 102 103 104 100 101 102 103 104 CD11c FITC CD11c FITC

CD11c-FITC

164

Fig. 15. In vitro infection rate and infectivity of BMDM from wild type, CXCL9-/- and CXCL10-/- and CXCR3-/- C57BL/6 mice. Depicted are the average percent of macrophages infected after co-culture with 10 promastigotes per cell followed by 36 or 48 hours of either no stimulation (A), stimulation with 100U/ml rIFNγ (B), or stimulation with rIFNγ and 1ug/ml E. coli LPS (C). Shown are the mean results (+/-SEM) of 2-3 independent experiments. Asterisks indicate statistically significant (p<0.05) differences between groups.

165

A.

B.

* * * *

C.

* * * *

*

166

10 mig 8 ip10 6

4 cxcr3 2 wt 0 (uM) concentration NO 12 24 36 48 Time (hours)

Fig. 16. Indirect assessment of NO production by in vitro infected BMDM from

C57BL/6 WT, CXCL9-/- and CXCL10-/- and CXCR3-/- mice. Cells were infected with L. major promastigotes (10:1 parasite-to-macrophage ratio), stimulated with rIFNγ and LPS as above, and at indicated time points culture supernatants were collected and later subjected to the Greiss reaction in triplicate. Shown are the mean results of 3 independent trials +/-SEM.

Asterisks indicate statistically significant (p<0.05) differences between groups.

167

CCR3-FITC

Fig. 17. CCR3 is expressed by the BMDM of C57BL/6 mice. Macrophages were derived from bone marrow precursors in vitro and then cultured with and without stimulus for 48 hours. Cells were detached and stained with anti-

CCR3-FITC monoclonal antibody (solid black peak). The gray hollow peak represents isotype control treated cells. Shown are representative results from both stimulated and non-stimulated from two independent experiments.

168

Fig. 18. Analysis of the L. major-induced lesion growth and parasite burdens of infected C57BL/6 WT, CXCL9-/-, and CXCL10-/- mice. After injection of footpads with 2x106 promastigotes, lesions were measured weekly throughout the infection course relative to the contra-lateral, uninfected footpad (A).

Parasite burdens were determined by limiting dilution assay at weeks 6 and 9 post infection (B). Shown are the mean results of 3 independent trials +/-SEM.

Asterisks indicate statistically significant (p<0.05) differences between groups.

169

A. Lesion Diameter 2 WT CXCR3KO 1.5 MIGKO IP10KO * * * * * 1

0.5

0 1 2 3 4 6 7 8 9 Weeks B. WEEK 6

20 * 15

10

5

Log parasite dilution 0 WT CXCR3CXCR3-/- CXCL9MIG -/- CXCL10IP10 -/-

WEEK 9

6 *

4

2

0 Logdilution parasite WTWT CXCR3CXCR3 -/- CXCL9MIG -/- CXCL10IP10 -/-

170

Fig. 19. Analysis of the cytokines made by the dLN cells from L. major-infected

C57BL/6 WT, CXCL9-/-, and CXCL10-/- mice ex vivo upon re-stimulation by L. major antigen. dLN cells were isolated from euthanized mice after 6 or 9 weeks of infection. Levels of IL-10 (A), IL-12 (B), IFNγ (C), and IL-4 (not shown) in culture supernatants were determined by sandwich ELISA. Shown are the mean results of 3 independent trials +/-SEM. Asterisks indicate statistically significant (p<0.05) differences between groups.

171

A. IL-10 WEEK 6 IL-10 WEEK 9

3500 2500 3000 2000 2500 * 2000 1500 1500 1000 1000 500 500 [cytokine] [cytokine] pg/ml 0 0 [cytokine] pg/ml

B. IL-12 WEEK 6 IL-12 WEEK 9

2500 4500 2000 4000 3500 1500 3000 2500 1000 2000 1500 500 1000 0 500

0

[cytokine] [cytokine] pg/ml [cytokine] [cytokine] pg/ml

C. IFNg WEEK 6 IFNg WEEK 9 100000 160000 90000 80000 140000 70000 120000 60000 100000 50000 80000 40000 60000 * * 30000 40000 20000 10000 20000

0 0

[cytokine] [cytokine] pg/ml [cytokine] [cytokine] pg/ml

172

Fig. 20. Course of L. major infection in CCR5+/+ and CCR5-/- mice. (A) Mice were infected by inoculating 2x106 L. major SPP into the footpad. Lesion growth was monitored by measuring the increase in thickness of the infected footpad and comparing this to the thickness of the contra-lateral uninfected footpad.

Data are representative of two independent experiments. (B) Footpad parasites burdens were determined by limiting-dilution analysis at weeks 3 and 8 post- infection. Data are expressed as mean log titer +/-SEM. Similar results were observed in three independent experiments.

173

AA..

Lesion diameter (mm)

B.

174

Fig. 21. Analysis of ex vivo cytokine production by LmAg stimulated lymph node cells from L. major-infected CCR5+/+ and CCR5–/– mice. The levels of IL-12

(A), IFN-γ (B), IL-10 (C) and IL-4 (D) were measured in culture supernatants by

ELISA. Shown here are the mean results (+/-SEM) of three independent experiments. Asterisks indicate statistically significant (p<0.05) differences between groups (n=8).

175

A.

700 600 500 400 300 200

[IL-12] (pg/ml) [IL-12] 100 0 CCR5+/+ CCR5-/- CCR5+/+ CCR5-/- LN LN SPL SPL

B. 8000 7000 6000 5000 4000 3000 2000 1000

0 [IFNgamma] (pg/ml) [IFNgamma] CCR5+/+ CCR5-/- CCR5+/+ CCR5-/- LN LN SPL SPL

C. 70 60 50 40 30 20

[IL-10] (pg/m)l [IL-10] 10 0 CCR5+/+ CCR5-/- CCR5+/+ CCR5-/- LN LN SPL SPL

D. 50 40 30

20

10 [IL-4] (pg/ml) [IL-4] 0 CCR5+/+ CCR5-/- CCR5+/+ CCR5-/- LN LN SPL SPL

176

Fig. 22. Course of L. major infection in CCR3+/+ and CCR3-/- Mice on a

BALB/c background. (A) Mice were infected by inoculating 2x106 L. major SPP into the footpad. Lesion growth was monitored by measuring the increase in thickness of the infected footpad and comparing this to the thickness of the contra-lateral uninfected footpad. Data are representative of two independent experiments. (B) Footpad parasites burdens were determined by limiting- dilution analysis at weeks 3 and 8 post-infection. Data are expressed as mean log titer +/- SEM. Similar results were observed in three independent experiments.

177

A. Lesion Diameter

6 5 CCR3+/+ 4 CCR3-/- 3

Lesionsize (mm) 2 1 0 1 2 3 4 5 6 6.5

Weeks post infection

B. Parasite Burden

16 14 12 * 10 CCR3+/+ 8 6 CCR3-/-

Log Parasite Log Titer 4 2 0

178

Fig. 23. Analysis of ex vivo cytokine production by LmAg stimulated lymph node cells from L. major-infected CCR3+/+ and CCR3–/– mice. The levels of IL-12

(A), IFN-γ (B), IL-10 (C) and IL-4 (D) were measured in culture supernatants by

ELISA. The data are means of two independent experiments (+/-SEM). No statistically significant (p<0.05) differences were found between the groups

(n=6).

179

A. 250 200 150 100

50 [IL-12] (pg/ml) [IL-12] 0 CCR3+/+ CCR3-/-

B. 12000 10000 8000 6000 4000 2000 0 [IFNgamma] (pg/ml) [IFNgamma] CCR3+/+ CCR3-/-

C. 700 600 500 400 300 200

100 [IL-10] (pg/ml) [IL-10] 0 CCR3+/+ CCR3-/-

D.

700 600 500 400 300 200

[IL-4] (pg/ml) [IL-4] 100 0 CCR3+/+ CCR3-/-

180

APPENDIX B:

FIGURES 24-33

181

Fig. 24. C57BL/6 T cells express higher levels of CXCR3 than those of BALB/c mice both in vivo and in vitro. The draining LN (A) and (B) lesions of C57BL/6 mice infected with L. major contain higher percentages of CXCR3+/CD4+ and

CXCR3+/CD8+ lymphocytes than those of infected BALB/c mice after 3 weeks of L. major infection. Also CXCR3+ cells comprised a larger percentage of the

CD4+ and CD8+ T cell compartments in the C57BL/6 dLN throughout the observed course of infection (C). Upon in vitro mitogenic stimulation, C57BL/6

T cells dramatically up-regulated CXCR3 (solid peak) while BALB/c T cells did not (hollow gray peak). Isotype controls were denoted by dotted hollow peaks.

Real Time-PCR measurement of CXCR3 mRNA showed that this trend in expression was observed at the transcript level (D). In panels A, B and D, representative results from one of five trials are shown. Panels C and E represent the averaged result of three independent trials (+/-SEM). *= a P value < 0.05.

182 C57BL/6 BALB/c C57BL/6 BALB/c A. B. 19.0 3.0 4.1 <1 4.8 6.8 2.4 1.9

19.8 15.4 11.1 35.7

0 1 2 3 4 0 1 2 3 4

100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 10 10 10 10 10

PE

PE

- - CD4-FITC CD4-FITC

9.7 7.2 4.8 <1 9.0 4.2 4.0 1.4

CXCR3 CXCR3

2.0 2.6 7.3 9.0

0 1 2 3 4 10 10 10 10 10 100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 CD8-FITC CD8-FITC C. D.

20 CD4+ T cells 60 CD8+ T cells 50 15 40 C57BL/6 10 * 30 BALB/c * * 20 5 *

10 * D.E. cxcr3 mRNA

% cellsT CD4+ of %

% cellsT CD8+ of % expressing CXCR3 expressing 0 CXCR3 expressing 0 20 C57BL/6 1 3 7 1 3 7 15 BALB/c Weeks post-infection Weeks post-infection 10 5 *

Fold Induction Fold 0

183

Fig. 25. BALB/c and C57BL/6 derived T cells produce comparable levels of

IFNγ, and despite the greater amounts of Th2 cytokine produced by BALB/c T cells, blocking IL-4 signaling did not alter CXCR3 expression by BALB/c. (A)

BALB/c T cells activated in vitro with anti-CD3/anti-CD28 make more Th2 cytokines (IL-4 and IL-10) than C57BL/6 T cells and comparable levels of IFNγ and (B) Tbet. (C) Neutralizing IL-4 (11B.11, 10μg/ml) during and post stimulation did not affect CXCR3 surface expression by C57BL/6 or BALB/c derived T cells as measured by flow cytometry. Solid black peaks represent control antibody treated cells, while gray hollow peaks and dotted lines represent anti-IL-4 treated cells and straining isotype controls, respectively. (D)

Likewise IL-4Rα-/- BALB/c T cells (black hollow peak) did not up-regulate

CXCR3 more efficiently than WT BALB/c T cells (gray hollow peak). Isotype controls are represented by a solid black peak. In panels A and B, the averaged results of three to five independent trials (+/-SEM) are shown (*= a P value < 0.05). Panels C and D show representative results from one of three independent trials.

184 300 4500 25000 C57BL/6 4000 BALB/c 250 A. 3500 20000 200 3000 15000 2500 150 2000

10000

4] pg/ml 4]

10] pg/ml 10] - 100 - 1500

* * pg/ml [IFNg] [IL [IL 1000 50 5000 500 0 0 0

B. Tbet mRNA C. C57BL/6 BALB/c 3 2.5 C57BL/6 2 1.5 BALB/c 1

Fold Induction Fold 0.5 0

D.

185

Fig. 26. IL-10 suppresses CXCR3 expression by BALB/c T cells, but not those of C57BL/6. (A) Blocking IL-10R (1B1.3A, 10μg/ml) allowed moderate surface

CXCR3 expression by BALB/c T cells activated in vitro (solid black peak).

Control Ig-treated cells are represented by a hollow gray peak. (B) Likewise, IL-

10-/- BALB/c T cells (black hollow peak) induce CXCR3 more readily than WT controls (gray hollow peak). A solid black peak represents isotype controls. (C)

Treatment of C57BL/6 T cells with rIL-10 (2ng/ml) (hollow gray peak) did not suppress CXCR3 expression compared to untreated controls (solid black peak).

(E) Real-time PCR and (F) flow cytometric analysis of IL-10R revealed that

BALB/c derived T cells (white bars and hallow gray peaks)express more IL-10R mRNA and surface protein than C57BL/6 T cells (black bars and solid black peaks). In panels A-C and E, representative results from one of three independent trials are shown. Panel D represents the averaged results of three independent trials (+/-SEM). *= a P value < 0.05.

186 A. B.

Il-10r mRNA 5 8 C. D. * C57BL/6 4 * 6 BALB/c 3 4 2 2

Fold InductionFold 1 0 0 100 101 102 103 104 FL2-H CD4+ T cells CD8+ T cells E.

187

Fig. 27. BALB/c and C57BL/6 derived T cells express comparable levels of

IFNγ receptor upon in vitro stimulation. Either CD4+ (A) or CD8+ (B) T cells were isolated from the spleens of C57BL/6 (black bars) or BALB/c mice (empty bars). T cells were activated in vitro with ant-CD3/CD28 antibodies for 48 hours and RNA was extracted and used to generate cDNA for RT-PCR analysis.

Shown are the mean results (+/-SEM) of three independent experiments.

188

CD4: IFNgamma Receptor RNA B. CD8: IFNgamma Receptor RNA A. 6 9 5 8 4 7 C57BL/6 C57BL/6 6 5 BALB/c 3 BALB/c 4

2 3 Fold Induction Fold Induction 2 1 1 0 0

189

Fig. 28. Transgenic BALB/c T cells do not up-regulate CXCR3 upon antigen- specific stimulation unless removed from stimulus and endogenous cytokines.

DO11 T CD4+ T cells were co-cultured with BMDM pulsed with OVA peptide

(1000ug/ml). Cells were removed from co-culture and rested 24 hours either with or without washing the cells to remove cytokines secreted during stimulation. RNA was extracted and RT-PCR determined the level of CXCR3 mRNA in these cells. Data is presented as mean fold induction (+/-SEM) over that of non-stimulated cells. These results were also normalized to the housekeeping gene, GAPDH. Shown is a representative of two independent experiments with similar results.

190

DO11 in vitro APC assay CXCR3 mRNA Levels 15

10 washed

5 unwashed Fold induction Fold 0

191

Fig. 29. Flow cytometric analysis of CD49b+ (DX5+) NK cells in the lesion and

LN of C57BL/6 and BALB/c mice during infection by L. major: Leukocytes were isolated from the designated tissues and stained for flow cytometric analysis of surface markers. (A) CXCR3 expression by NK cells of C57BL/6 and BALB/c infected with L. major was determined by flow cytometry analysis of LN cells.

Also, CXCR3 expression by NK1.1+ lesion cells of C57BL/6 mice was similarly determined (B). Shown are the representative findings of one of three independent experiments with similar results. Numbers represent the percentage of leukocytes positive for the designated marker.

192 A. C57BL/6 BALB/c

17.2 1.8 15.2 0.4

WEEK 3

1.1 0.1

18.9 6.1 11.3 4.0

WEEK 4

PE - 6.6 8.2

CXCR3

16.5 4.1 11.0 3.0

WEEK 6

6.0 12.5

CD49b - FITC

B. Week1 Week2 Week3 % % Data.009 7.3% Data.007 7.2% Data.007 6.8%

CXCR3

-PE 9.3% 20% 8.9%

0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 100 101 102 103 104 NK1.1 FITC NK1.1 FITC NK1.1 FITC

NK1.1-FITC

193

Fig. 30. IFN- and STAT1 are required for efficient induction of CXCR3 on

CD4+ but not CD8+ T cells. After stimulation with anti-CD3 and anti-CD28 antibody, cells were rested 24 hours without stimulation. CXCR3 surface protein expression was measured by flow cytometric staining of wild-type CD4

(A) and CD8 (B) cells in the presence of IFN-γ-neutralizing antibody or an isotype control. Stained cells treated with anti-IFN-γ are represented by hollow gray peaks; cells stimulated in the presence of control antibody are depicted by solid peaks; and dotted lines represent a PE-labeled isotype control. Surface

CXCR3 was also analyzed on STAT1-/- CD4 (C) and CD8 T-cells (D). STAT1+/+ cells are represented by solid peaks; STAT1-/- cells are represented by hollow grey peaks; and isotype control staining is repressed by a dotted line. Numbers represent the mean fluorescence intensity (MFI, +/-SEM). Panels A and B are representative of three independent experiments, while panels C and D represent one of five independent experiments.

194

A. B.

MFI MFI

Control Ab 113.5 Control Ab 102.5 Anti-IFNγ 40.5 Anti-IFNγ 124.6

C. CXCR3 -PE D.

MFI MFI

STAT1+/+ 118.6 STAT1+/+ 105.2 STAT1-/- 24.8 STAT1-/- 111.0

CXCR3-PE

195

Fig. 31. TNF-alpha is not required for CXCR3 expression by CD8 or CD4 T cells. Purified CD4+ and CD8+ T cells from STAT1+/+ and STAT1-/- mice were stimulated in vitro with anti-CD3/CD28 antibodies as described above with a

TNF-alpha neutralizing antibody (gray hollow peaks) or an inert isotype control antibody (black peaks). Staining controls are depicted by dotted hollow peaks.

CXCR3 expression was then analyzed by flow cytometric analysis. Shown are the results of two independent experiments with like results.

196

STAT1+/+ STAT1-/-

CD4+ T cells

100 101 102 103 104 100 101 102 103 104 FL2-H FL2-H

CD8+ T cells

100 101 102 103 104 100 101 102 103 104 FL2-H FL2-H CXCR3-PE

197

Fig. 32. STAT1 controls CXCR3 and T-bet gene transcription in CD4+ but not

CD8+ T-cells. Levels of CXCR3 (A, B), T-bet (C, D), and Eomes (E, F) mRNA in activated CD4 and CD8 T-cells from WT as well as STAT1-/- mice were measured by real time RT-PCR. Data were normalized to the housekeeping gene GAPDH and the results are presented as fold-induction of gene expression over non-activated cells. Empty bars represent STAT1+/+ derived

CD4+ or CD8+ T cells while black bars represent the T cells of STAT1-/- mice.

Shown are the averaged results (+ SEM) of five independent experiments

(*P<0.05)

198

A B

CD4 CXCR3 CD8 CXCR3

40 * 8 30 6

20 4

10 2

Fold Induction Fold Induction 0 0

C D

CD4 Tbet CD8 Tbet

15 * 15

10 10

5 5

Fold Induction Fold Induction 0 0

E F

CD4 Eomes CD8 Eomes

8 8

6 6

4 4

2 2

Fold Induction Fold Induction 0 0

199

STAT1+/+ STAT1-/- Data.007 Data.006 Data.007 Data.008

R6 R6 R6 R6

12.2 8.6 19.7 7.0

0 200 400 600 800 1000 FSC-H 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 Vehicle InhibitorFSC-H FSC-H FSC-H Vehicle Inhibitor

Fig. 33. STAT1-independent mechanisms for CXCR3 induction by CD8+ T cells. CD8+ T cells were activated in vitro by anti-CD3/CD28 antibody either in the presence of an p38 inhibitor (100nM) or a DMSO vehicle control. Numbers represent the percentage of CD8+ T cells staining positive for CXCR3 post- stimulation and rest. Shown are the results of one of two experiments with similar results.

200

APPENDIX C:

FIGURES 34-39

201

Fig. 34. CXCR3-/- mice and wild type C57BL/6 control parasite burdens comparably and are both resistant to L. donovani infection. Liver and spleen (A and B) parasite loads were determined 15, 30, and 60 days post- i.v. inoculation with 1 X 107 L. donovani amastigotes. Parasite burdens in liver (A) and spleen

(B) are expressed as the mean LDU + SEM. The data shown are mean values of eleven to twelve mice per group at each time point in three independent experiments with like results. Statistical significance was determined using an unpaired Student‟s t-test, *(p < 0.05).

202 A. LIVER

1600 CXCR3+/+ 1400

1200 CXCR3-/- 1000

800 LDU 600

400

200

0 Days after infection 0 20 40 60

B. SPLEEN

45 CXCR3+/+ 40 35 * 30 CXCR3-/-

LDU 25 20 15 * 10 5 0 0 15 Days after30 infection 45 60

203 Fig. 35. CXCR3-/- mice show delayed liver granuloma formation, but eventually develop granuloma counts comparable to CXCR3+/+ mice. Liver and spleen (A and B) parasite loads were determined 15, 30, and 60 days post- i.v. inoculation with 1 X 107 L. donovani amastigotes. At such time points, routine H/E staining of histological liver sections were examined and granulomas were enumerated

(A). Granuloma counts represent the mean numbers of granuloma per 10 high power field (hpf) + SEM from 4 mice per group, for each time point.

Histological images from a representative experiment are depicted for CXCR3-/- and CXCR3+/+ mice at relevant time points (B). Parasite load and granuloma data are mean values of data from twelve individual mice per group at each time point collected from three independent experiments with like results.

Statistical significance was determined using an unpaired Student‟s t-test, *(p <

0.05).

204 A.

100 * 90 * 80 70 CXCR3+/+ 60 50 CXCR3-/- 40 30 20 Granulomas/10 hpf Granulomas/10 10 0 15 30 60 Days after infection

B.

Day 15 Day 30 Day 60

CXCR3+/+

X40 X40 X40

CXCR3-/-

X40 X40 X40

205 . A. B. CXCR3+/+ 100000 CXCR3-/- 100000 10000 10000

1000 1000 100 100 10 10

1 1

15 30 45 60 15 30 45 60 IgG1 titers titers (log)IgG1 Days after infection IgG2a titers (log) Days after infection

Fig. 36. Kinetics of the antibody response in L. donovani-infected CXCR3-/- and CXCR3+/+ mice at 15, 30, 45, and 60 days after infection. LdAg-specific

IgG1 (A) and LdAg-specific IgG2a (B) measured in the sera of infected mice.

Data from three like experiments are presented as the mean of endpoint titers on a log scale. Twelve mice were analyzed in each group.

206

Fig. 37. Kinetics of the cytokine response of LdAg-stimulated splenocytes from infected CXCR3+/+ and CXCR3-/- mice. Spleen cells were stimulated during a

72-hour incubation with 20 g/ml of LdAg, and levels of cytokines were measured by ELISA. IL-12p70 (A), IFN- (B), IL-10 (C), and IL-4 (D) production by spleen cells from L. donovani-infected CXCR3+/+ and CXCR3-/- mice are show. These results are the means +/- SEM of triplicate samples from eleven to twelve mice per group from three independent experiments.

207

B 50000 40000 30000

20000 IFNg (pg/ml) 10000 0 0 15 30 45 60 Days after infection

C D 8000 400 7000 6000 300 5000 4000 200

3000

IL-10 (pg/ml)IL-10 IL-4(pg/ml) 2000 100 1000 0 0 0 15 30 45 60 0 15 30 45 60 Days after infection Days after infection

208

Fig. 38. The liver infiltrate of CXCR3-/- mice induce cytokine mRNA as efficiently as that of CXCR3+/+ mice. Fold induction of the mRNA encoding IL-

12 (A), IFN (B), TNF (C), IL-10 (D), and IL-4 (E) are shown as mean fold induction + SE. Shown are the mean results of two independent experiments with similar results.

209

IL-12 IFN-gamma A. CXCR3+/+ B. CXCR3-/- 100 250 90 80 200 70 60 induction Fold 150 Fold induction Fold 50 40 100 30 20 50 10 0 0 0 15 30 60 0 15 30 60 Days after infection Days after infection

IL-10 C. TNF-alpha D.

200 35 180 30 160 25 140 induction Fold Fold induction Fold 120 20 100 80 15 60 10 40 20 5 0 0 0 15 30 60 0 15 30 60 Days after infection Days after infection

IL-4 E.

400 350

300 Fold induction Fold 250 200 150 100 50 0

0 Days after15 infection30 60

210

Fig. 39. The CXCR3-ligands are not significantly reduced in CXCR3-/- mice infected with L. donovani. Mean fold induction (+/- SEM) of the mRNA encoding CXCL10 (A), and CXCL9 (B) are shown are representative results from one of two independent trials.

211

A.

B.

212

APPENDIX D:

FIGURES 40-43

213

Fig. 40. Inhibiting PI3Kgamma suppresses CXCR3 induction following in vitro T cell activation. (A) General PI3Kinase inhibition by incubating activated T cells with 100nM wortmannin (gray hollow peaks) suppressed CXCR3 surface expression by these cells as detected by flow cytometry. Vehicle (DMSO) treated controls and isotype control stained cells are represented by black hollow and solid peaks, respectively. (B) Treatment of activated T cells with the PI3Kgamma-specific inhibitor, AS605240 suppressed CXCR3 expression.

Numbers given are the percentage of cells positive for CXCR3 and CD3. (C)

Semi-quantitative real time-PCR analysis of CXCR3 transcript induction.

AS605240 treated T cells (white bars) induce significantly less CXCR3 mRNA than vehicle treated cells (black bars). (D) AS605240 treated T cells (white bars) had suppressed Tbet induction during in vitro T cell activation compared to vehicle controls (black bars). Real time-PCR data for each group was normalized to the house keeping gene gapdh and is expressed as fold induction over non-stimulated cells. Shown in panels A and B are representative results of three or more independent experiments. Panels C-D represent the mean result (+/-SEM) of 3 or more independent experiments. A P value from an unpaired Students t-test < 0.05 (*) was considered significant.

214

DMSO INHIBITOR A. B. 32 17

CD4+ T cells

PE -

CXCR3 45 35

CXCR3 -PE CD8+ T cells Inhibitor DMSO Isotype ctrl CD3-FITC

C. CXCR3 mRNA D. Tbet mRNA

25 15 DMSO DMSO 20 INHIBITOR INIHIBITOR 15 10 10 * 5 *

5 * FOLD INDUCTION FOLD 0 INDUCTION FOLD 0 CD4+ T cells CD8+ T cells

215

Fig. 41. CXCR3 uniquely requires PI3Kgamma for expression by T cells. (A)

Other chemokine receptors (CCR4, CCR5, CCR1, CCR10, CXCR6, CCR7) were not similarly affected by PI3Kgamma inhibition. However, T cells genetically deficient in functional PI3Kgamma express less CXCR3 than wild type cells. (B) Flow cytometric analysis of CXCR3 surface protein on wild type

(gray hollow peak) and PI3Kγ-/- derived T cells (solid black peak). Also shown is the flow cytometric analysis of lesion-draining LN cells excised from L. mexicana-infected wild type and PI3Kγ-/- mice (C). Shown are representative results of three or more independent experiments.

216

10 CD4+ T cells 2 CD8+ T cells A.

5 1

FOLD INDUCTION

0 0

DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO DMSO

INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR INHIBITOR CCR4 CCR5 CCR7 CXCR6 CCR10 CCR1 CCR4 CCR5 CCR7 CXCR6 CCR10 CCR1

C. WT PI3Kγ-/-

B. WT 3 1

PI3Kγ-/-

PE -

CD4-FITC

CXCR3 13 2

CXCR3 -PE

CD8-FITC

217

Fig. 42. PLC and CXCR3 regulation. T cells of C57BL/6 (A) and BALB/c (B) mice were stimulated with anti-CD3/CD28 antibodies in vitro. Cells were rested in the presence of the PLC inhibitor U73122 or a DMSO vehicle control. After

24 hours of resting, the cells were stained for surface CXCR3 expression and analyzed by flow cytometry. Shown are the representative results of three independent experiments yielding similar results. The solid black peaks represent isotype control treated cells, hollow black peaks depict inhibitor treated cells and gray hollow peaks represent vehicle treated cells. The supernatants of these cells were subjected to cytokine Elisa (C). The mean concentrations of IL-10 in the culture supernatants of activated cells rested in the presence or absence of U73122 are shown (+/- SEM).

218

C57BL/6

A.

CXCR3-PE

B. BALB/c

C. CXCR3-PE

219

Fig. 43. Expression of CXCR3 on memory T cells generated during L. major infection. To determine if CXCR3 is expressed on the memory T cells generated in response to parasitic infection, L. major-resistant C57BL/6 mice were infected with L. major and allowed to resolve the infection (this was accomplished approximately 12 weeks after infection). Cells were isolated from the lymph node draining the site of infection and their surface markers were analyzed by multi-color flow cytometry. Shown below are histograms representing the proportion of cells expressing naïve T cell markers

(CD3+/CD62L+), the central memory profile(CD3+/CD44+/CD62L+), and the markers of effector/memory T cells (CD3+/CD44+). Numbers represent the percentage of T cells expressing the specified markers while numbers in parathsises denote the percentage of total LN leukocytes.

220

CXCR3 and Memory T cells:

C57BL/6 LN cells CD3+/CD62L+ CD3+/CD62L+/CD44+ CD3+/CD44+ 2.45 (0.34) 27.61 (16.15) 54.29 (12.09)

M1 M1 M1

M2 M2 M2

0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 FL2-H 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL2-H FL2-H CXCR3

Naïve T Cells TCM Cells TEFF Cells

221