The Role of the Costimulatory TNFR Family Member GITR in T Cell Immunity During an Acute Respiratory Infection

Kuan-Lun Chu

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Immunology University of Toronto

Abstract

The Role of the Costimulatory TNFR Family Member GITR in T Cell Immunity During an Acute

Respiratory Infection.

Doctor of Philosophy (2020)

Kuan-Lun Chu, Department of Immunology, University of Toronto

T cells play a critical role in control of influenza virus, a major human pathogen.

Glucocorticoid-induced TNFR-related Protein (GITR), a prosurvival member of the TNFR family, contributes to maximal CD8 T cell responses and protects mice from death upon severe influenza infection. In this thesis, I demonstrate that during influenza infection, GITR ligand

(GITRL) is mainly expressed by a group of monocyte-derived tissue infiltrating cells known as inflammatory antigen presenting cells (InfAPC). I show that both GITR and GITRL are detected early in the mediastinal lymph node (mLN) and in the lung tissue when T cells start to accumulate after influenza infection. By detecting phospho-signals downstream of GITR, I show that InfAPC likely provide crucial signals through GITR on T cells in the mLN and afterwards in the lung. This signal is critical for the accumulation of lung effector CD4 and CD8

T cells as well as optimal formation of lung CD4 and CD8 tissue-resident memory T cells (Trm) after influenza infection.

The signals that GITR delivers for effector T cell and Trm accumulation most likely occur at the effector phase of the response since GITRL expression is mainly confined to the

first week of influenza infection. Thus, I investigated how GITR affects effector T cell subpopulations during influenza infection. Ly6C expression defines two effector CD4 T cell subsets while Ly6C and CX3CR1 expression defines three effector CD8 T cell subpopulations.

In CD8 T cells, GITR affects the accumulation of all three subsets, with a predominant effect on the least differentiated subset. GITR also selectively upregulates CXCR6 on the less differentiated CX3CR1lo CD8 T cell subsets. GITR affects the accumulation of both CD4 T cell subsets, but selectively upregulates CD127 in the least differentiated CD4 T cell subpopulation which shows a preference for entry into the lung parenchyma. Together, this thesis demonstrates that during influenza infection GITR costimulation in the lung plays an important role in sustaining both highly differentiated effector T cells and Trm precursors, but with differential effects on effector T cell subpopulations. This signal is also critical for optimal formation of lung CD4 and CD8 Trm.

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Acknowledgments

I would first like to thank my supervisor, Dr. Tania Watts. Tania, I am so grateful for the support and guidance you provided during my graduate studies. Thank you for allowing me to just walk into your office anytime (even lunch time) to discuss my project. I also want to thank you for letting me purse my research ideas. Some of these ideas were bad ideas that led to nowhere.

I would like to thank all the members of the Watts Lab. Ali Abdul-Sater, thank you for providing assistance on molecular biology aspects as well as your computer expertise. Samia Afzal, for answering my random research questions. Nathalia Batista, for hunting free food and free drinks with me. Frank Chang, for making funny jokes that are actually funny. Derek Clouthier, for training me when I first joined the lab. Maria Edilova, for helping me out on that course. Birinder Ghumman, for providing technical assistance and bringing tandoori chicken to lab parties. Melanie Girard, for laughing at my jokes. Jaclyn Law, for listening to my poorly spoken Cantonese. Achire Mbanwi, for the April Fool’s Day joke which I did not fall for. Anh Tran, for telling me where to get good food and bubble tea. Johnny Wang, for making jokes and making strange noise while doing experiments. Angela Zhou, for teaching me lab techniques. I would also like to thank the summer and 4th year undergraduate project students of the Watts Lab: Wenting Gao, Gloria Hou, Ruty Khanolkar, Adam Komorowski, Juliana Lee, Kymberly Litman, Miguel Torres Perez, Kenneth Ting, Rebekah Yuan.

I would like to thank my supervisory committee members, Dr. Clinton Robbins and Dr. Jennifer Gommerman, for very helpful comments and discussion during committee meetings. I thank Dionne White and Joanna for flow training and cell sorting. Stacy Nichol and Janice Suarez for taking good care of the animal colony. Carlo Riccardi and Pier Paolo Pandolfi for provision of GITR-/- mice. Ethan Shevach for provision of GITRLexon2fl/fl mice. David Brooks for provision of CD45.1 OT-II mice. I would like to thank my 4th year research project supervisor, Dr. James Carlyle, for giving me the opportunity to do research in his lab as an undergraduate student. As well as my undergraduate project mentor, Tina Kirkham. Tina, I am grateful to have you as my mentor. You not only taught me basic research skills, but also how to deal with failed experiments (technically and emotionally).

In particular, I would like to thank my parents and my brother for their endless love and support. Especially, when they flew from the other side of the world to Toronto just to celebrate my birthday with me. Last but not least, I would like to thank Minnie Hung for her unconditional love and support during my graduate studies. Minnie, thank you for teaching me all those functions in that word processor which made formatting this thesis a lot easier.

Table of Contents

Abstract ...... ii

Acknowledgments ...... iv

Table of Contents ...... vi

List of Tables ...... xi

List of Figures ...... xii

List of Publications ...... xv

Abbreviations ...... xvi

Chapter 1 Introduction ...... 1

1 Introduction ...... 2

1.1 Influenza virus ...... 2

1.1.1 Influenza ...... 2

1.1.2 Influenza structure and function ...... 2

1.1.3 Influenza life cycle ...... 4

1.2 Immune responses against influenza virus ...... 6

1.2.1 Innate immune responses ...... 6

1.2.2 APC subsets and priming of T cells ...... 7

1.2.3 CD8 T cell responses ...... 9

1.2.4 CD4 T cell responses ...... 10

1.2.5 Central memory and effector memory T cells ...... 11

1.2.6 Tissue resident memory T cells ...... 11

1.2.7 B cell responses ...... 14

1.3 The TNFR superfamily ...... 16

1.3.1 Classifications and functions of the TNFR superfamily...... 16

1.3.2 TNFR signaling ...... 18

1.3.3 TNFRs and viral infections ...... 21

1.3.4 TNFRs and Trm formation ...... 23

1.4 GITR and GITRL ...... 26

1.4.1 GITR structure and expression ...... 26

1.4.2 GITR signaling ...... 28

1.4.3 GITRL structure and expression ...... 31

1.4.4 GITRL reverse signaling ...... 32

1.4.5 Effect of GITR on DC and macrophages ...... 33

1.4.6 Effect of GITR on ILC ...... 34

1.4.7 Effect of GITR on NK and NKT cells ...... 35

1.4.8 Effect of GITR on T cells ...... 35

1.4.9 Effect of GITR on B cells ...... 37

1.4.10 GITR and viral infections ...... 37

1.4.11 GITR and signal 4 ...... 40

1.5 Thesis synopsis ...... 43

Chapter 2 GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T cell accumulation and tissue-resident memory T cell formation ...... 45

2 GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T cell accumulation and tissue-resident memory T cell formation ...... 46

2.1 Summary ...... 46

2.2 Introduction...... 47

2.3 Materials and Methods ...... 48

2.3.1 Mice ...... 48

2.3.2 Reagents and antibodies ...... 48

2.3.3 Influenza virus infection ...... 51

2.3.4 Tissue harvest and processing ...... 51

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2.3.5 Flow cytometry ...... 51

2.3.6 T cell isolation and adoptive transfers ...... 52

2.3.7 Phosphoflow assays ...... 52

2.3.8 Mixed bone marrow chimeras ...... 53

2.3.9 In vivo IFNAR-1 blockade...... 53

2.3.10 Data analysis and statistics ...... 53

2.4 Results ...... 54

2.4.1 GITR is required for optimal effector CD4 and CD8 T cell accumulation in a competitive influenza model...... 54

b 2.4.2 GITR signaling rescues low affinity D /NP366-374-specific CD8 T cells ...... 60

2.4.3 GITR is required for optimal lung CD4 and CD8 Trm formation following influenza infection ...... 64

2.4.4 Preferential effect of GITR on CD8 over CD4 T cell responses in a non- competitive influenza model...... 68

2.4.5 GITR is transiently upregulated on Th1, Treg, and influenza-specific CD8 T cells in the lung and mLN ...... 71

2.4.6 GITRL expression is highest on inflammatory APC subsets after influenza infection ...... 73

2.4.7 GITRL expression in the lung is partially mediated by IFN-I during influenza infection ...... 78

2.4.8 GITR-dependent signals in T cells take place in the mLN and then in the lung tissue ...... 79

2.5 Discussion ...... 85

Chapter 3 GITR differentially affects lung effector T cell subpopulations during influenza virus infection ...... 90

3 GITR differentially affects lung effector T cell subpopulations during influenza virus infection ...... 91

3.1 Summary ...... 91

3.2 Introduction...... 92

3.3 Materials and Methods ...... 94 viii

3.3.1 Mice ...... 94

3.3.2 Reagents and antibodies ...... 94

3.3.3 Influenza virus infection ...... 95

3.3.4 Tissue harvest and processing ...... 95

3.3.5 Flow cytometry ...... 95

3.3.6 T cell isolation and adoptive transfers ...... 96

3.3.7 Effector T cell transfers ...... 96

3.3.8 Mixed bone marrow chimeras ...... 97

3.3.9 Data analysis and statistics ...... 97

3.4 Results ...... 98

3.4.1 During influenza infection, CD4 T subsets can be divided into Ly6Chi and Ly6Clo subpopulations ...... 98

3.4.2 Ly6Chi or Ly6Clo CD4 subsets are similarly dependent on GITR costimulation for their accumulation ...... 101

3.4.3 Cytokine producing Ly6Chi CD4 T cells are more dependent on GITR costimulation than cytokine producing Ly6Clo CD4 T cells ...... 104

3.4.4 GITR differentially regulates CD127 (IL-7Rα) in CD4 effector T cell subsets .. 107

3.4.5 Effect of GITR signaling on CXCR6 expression on CD4 T cells during influenza infection ...... 108

3.4.6 Upon adoptive transfer, the Ly6Clo CD4 T cells preferentially enter the lung parenchyma ...... 114

3.4.7 Ly6C and CX3CR1 expression defines three subsets of effector CD8 T cells in the lung ...... 116

3.4.8 The Ly6Clo CX3CR1lo CD8 T cell subset is the most dependent on GITR costimulation...... 118

3.4.9 Effect of GITR on OX40 induction on CD8 T cells during influenza infection . 121

3.4.10 GITR regulates CXCR6 expression in Ly6Chi CX3CR1lo and Ly6Clo CX3CR1lo effector CD8 T cells during influenza infection ...... 121

3.5 Discussion ...... 125

Chapter 4 Summary and Future Directions ...... 129

4 Summary and Future Directions ...... 130

Chapter 5 References ...... 138

5 References ...... 139

List of Tables

Table 1.1 Murine GITR expression ...... 27

Table 2.1 Anti-mouse antibodies for flow cytometry ...... 49

List of Figures

Figure 1.1. Structure of influenza virion...... 3

Figure 1.2. Life cycle of influenza...... 5

Figure 1.3. The TNF/TNFR superfamily...... 17

Figure 1.4. The NF-κB pathways downstream of TNFRs...... 20

Figure 1.5. GITR signaling pathways...... 30

Figure 1.6. Role of InfAPC in providing signal 4 for T cells...... 42

Figure 2.1. GITR is required for effector CD4 and CD8 T cell accumulation in a competitive influenza model...... 57

Figure 2.2. Reconstitution ratio of GITR+/+ to GITR-/- CD4 and CD8 T cells in the blood, mLN, and spleen...... 58

Figure 2.3. GITR is required for accumulation of influenza-specific IFNγ-producing CD4 and CD8 T cells...... 59

b Figure 2.4. GITR rescues low affinity D /NP366-374-specific CD8 T cells...... 63

Figure 2.5. GITR is required for optimal lung CD4 and CD8 Trm formation after influenza infection...... 66

Figure 2.6. GITR is not required for the accumulation of splenic CD4 and CD8 Trm...... 67

Figure 2.7. Preferential effect of GITR on CD8 over CD4 T cells in a non-competitive influenza model...... 69

Figure 2.8. Influenza-specific T cell responses in secondary lymphoid organs in GITR-deficient mice...... 70

Figure 2.9. GITR upregulation on activated CD4 and CD8 T cells in the mLN and lung following influenza infection...... 72

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Figure 2.10. Gating strategy for APC subsets in the lung and mLN...... 74

Figure 2.11. CCR2 expression in the lung and mLN during influenza infection...... 75

Figure 2.12. GITRL expression is highest on inflammatory APCs after influenza X31 infection.76

Figure 2.13. GITRL expression is highest on inflammatory APCs after influenza PR8 infection.77

Figure 2.14. Lung GITRL expression is mediated in part by IFN-I during influenza infection. .. 78

Figure 2.15. GITR signaling increases pS6 levels in antigen-specific OT-II and OT-I T cells. ... 82

Figure 2.16. GITR is required for optimal IL-2 producing OT-II accumulation...... 83

Figure 2.17. GITR signaling increases pS6 levels in antigen-specific OT-II T cells in the lung parenchyma...... 84

Figure 3.1. Effector CD4 T cells in the lung can be divided into Ly6Chi and Ly6Clo subpopulations...... 100

Figure 3.2. Similar effect of GITR on accumulation of Ly6Chi or Ly6Clo OT-II CD4 T cells in the lung during influenza infection...... 102

Figure 3.3. Similar effect of GITR on accumulation of endogenous Ly6Chi or Ly6Clo effector CD4 T cells in the lung during influenza infection...... 103

Figure 3.4. IFNγ producing or IL-2 producing Ly6Chi OT-II CD4 T cells are more dependent on GITR costimulation than their Ly6Clo counterparts...... 106

Figure 3.5. OX40, CD127, and CXCR6 expression on naïve OT-II CD4 T cells...... 109

Figure 3.6. Regulation of OX40 and CD127 expression by GITR in antigen specific OT-II CD4 T cells during influenza virus infection...... 110

Figure 3.7. Regulation of OX40, CD127, and CXCR6 expression by GITR in endogenous effector CD4 T cells during influenza infection...... 112

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Figure 3.8. Similar CX3CR1 expression between GITR+/+ and GITR-/- OT-II cells following influenza infection...... 113

Figure 3.9. Compared to Ly6Chi effector CD4 T cells, Ly6Clo effector CD4 T cells preferentially home to the lung parenchyma and have a higher potential to form CD4 Trm...... 115

Figure 3.10. Lung effector CD8 T cells can be divided into three subsets by Ly6C and CX3CR1.117

Figure 3.11. In the absence of GITR, there is a greater loss of Ly6Clo CX3CR1lo OT-I CD8 T cells in the lung during influenza infection...... 119

Figure 3.12. In the absence of GITR, there is a greater loss of endogenous Ly6Clo CX3CR1lo effector CD8 T cells in the lung during influenza infection...... 120

Figure 3.13. Regulation of OX40 and CXCR6 expression by GITR in endogenous effector CD8 T cells during influenza infection...... 123

Figure 3.14. OX40 and CXCR6 expression on naïve OT-I CD8 T cells...... 124

Figure 4.1. Model of GITR costimulation...... 131

Figure 4.2. Model of effect of GITR on effector T cell subpopulations...... 132

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List of Publications

1. Chu, K. L., Batista, N. V., Wang, K. C., Zhou, A. C., & Watts, T. H. (2019). GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T-cell accumulation and tissue-resident memory T-cell formation. Mucosal Immunol, 12(2), 363-377. doi:10.1038/s41385-018-0105-5

2. Wang, K. C., Chu, K.-L., Batista, N. V., & Watts, T. H. (2018). Conserved and Differential Features of TNF Superfamily Ligand Expression on APC Subsets over the Course of a Chronic Viral Infection in Mice. ImmunoHorizons, 2(11), 407-417. doi:10.4049/immunohorizons.1800047

3. Chang, Y. H., Wang, K. C., Chu, K. L., Clouthier, D. L., Tran, A. T., Torres Perez, M. S., . . . Watts, T. H. (2017). Dichotomous Expression of TNF Superfamily Ligands on Antigen- Presenting Cells Controls Post-priming Anti-viral CD4(+) T Cell Immunity. Immunity, 47(5), 943-958 e949. doi:10.1016/j.immuni.2017.10.014

4. Kirkham, C. L., Aguilar, O. A., Yu, T., Tanaka, M., Mesci, A., Chu, K. L., . . . Carlyle, J. R. (2017). Interferon-Dependent Induction of Clr-b during Mouse Cytomegalovirus Infection Protects Bystander Cells from Natural Killer Cells via NKR-P1B-Mediated Inhibition. J Innate Immun, 9(4), 343-358. doi:10.1159/000454926

Abbreviations

4-1BBL 4-1BB Ligand Ab Antibody Ad Adenovirus ADCC Antibody-Dependent Cell-Mediated Cytotoxicity AICD Activation Induced Cell Death Akt Ak Thymoma AlvMF Alveolar Macrophages APC Antigen Presenting Cells Batf Basic Leucine Zipper Transcription Factor, ATF-like Bcl B Cell Lymphoma BCR B Cell Receptor Blimp B Lymphocyte-Induced Maturation Protein BMDC Bone Marrow-Derived Dendritic Cells CCL C-C Chemokine Ligand CCR C-C Chemokine Receptor CD Cluster of Differentiation cDC Conventional Dendritic Cells CFSE Carboxyfluorescein Succinimidyl Ester CMV Cytomegalovirus COX Cyclooxygenase Cre Cre Recombinase CSF2 Colony-Stimulating Factor 2 CTL Cytotoxic T Lymphocyte CX3CR1 C-X3-C Chemokine Receptor 1 CXCL C-X-C Chemokine Ligand CXCR C-X-C Chemokine Receptor DC Dendritic Cells DR Death Receptor ERK Extracellular Signal-Regulated Kinase FACS Fluorescence Activated Cell Sorting

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Fc Fragment Crystallizable FcεRI Fragment Crystallization Region Epsilon Receptor 1 Fl Flox FMO Fluorescence Minus One FoxP3 Forkhead Box P3 FV Friend Virus GITR Glucocorticoid-Induced TNFR-Related Protein GITRL GITR Ligand HA Hemagglutinin HIV Human Immunodeficiency Virus HSV Herpes Simplex Virus InfAPC Inflammatory Antigen Presenting Cells ICAM-1 Intercellular Adhesion Molecule 1 ICOS Inducible T Cell Costimulator IFITM Interferon-Inducible Transmembrane Protein IFN Interferon IFNAR Type I Interferon Receptor Ig Immunoglobulin IκBα Inhibitor of κBα IKK IκB Kinase IL Interleukin ILC Innate Lymphoid Cells i.n. Intranasal InfDC Inflammatory Dendritic Cells InfMF Inflammatory Macrophages iNOS Inducible Nitric Oxide Synthase IntMF Interstitial Macrophages i.p. Intraperitoneal IRF Interferon Regulatory Factor JNK Jun N-terminal Kinase KLF2 Kruppel-Like Factor 2 KLRG1 Killer Cell Lectin‑Like Receptor G1 xvii

LCMV Lymphocytic Choriomeningitis Virus LFA-1 Lymphocyte Function-Associated Antigen 1 LT Lung Tissue LTβR Lymphotoxin-β Receptor LV Lung Vascular Ly6C Lymphocyte Antigen 6C LysM Lysozyme-M MAVS Mitochondrial Antiviral-Signaling Protein M1 Matrix Protein 1 M2 Matrix Protein 2 MAPK Mitogen-Activated Protein Kinase Medul MF Medullary Macrophages MFI Median Fluorescence Intensity MHC Major Histocompatibility Complex MLC Memory Lymphocyte Clusters mLN Mediastinal Lymph Node MMP Matrix Metalloproteinases MPEC Memory Precursor Effector Cells mRNA Messenger Ribonucleic Acid mTOR Mammalian Target of Rapamycin MyD88 Myeloid Differentiation 88 NA Neuraminidase NCR Natural Cytotoxicity Receptor NF-1 Nuclear Factor-1 NF-κB Nuclear Factor-κB NIK NF-κB-Inducing Kinase NK Natural Killer Cells NKT Natural Killer T Cells NP Nucleoprotein NS1 Non-Structural Protein 1 NS2 Non-Structural Protein 2 OVA Ovalbumin xviii

OX40L OX40 Ligand PA Polymerase Acidic Protein PB1 Polymerase Basic Protein 1 PB2 Polymerase Basic Protein 2 PBS Phosphate Buffered Saline PD-1 Programmed Cell Death Protein 1 pDC Plasmacytoid Dendritic Cells PDCA-1 Plasmacytoid Dendritic Cell Antigen 1 p.i. Post-Infection pS6 Phosphorylated-Ribosomal Protein S6 PRR Pattern Recognition Receptor PSGL-1 P-Selectin Glycoprotein Ligand 1 RAMD Repair-Associated Memory Depots RBC Red Blood Cells RIG-I Retinoic Acid-Inducible Gene 1 RLR RIG-I-Like Receptor RNA Ribonucleic Acid Runx3 Runt-Related Transcription Factor 3 S1P Sphingosine 1-Phosphate S1PR1 Sphingosine-1-Phosphate Receptor 1 siRNA Short Interference RNA SLEC Short-Lived Effector Cells Subcap MF Subcapsular Sinus Macrophages TAK-1 Transforming Growth Factor β-Activated Kinase 1 TCID50 Tissue Culture Infectious Dose 50 Tcm Central Memory T Cells TCR T Cell Receptor Tem Effector Memory T Cells Tg Transgenic TGFβ Transforming Growth Factor β Tpm Peripheral Memory T Cells Tfh Follicular Helper T Cells xix

Th1, 2, 17 T Helper Cell Type 1, 2, 17 Tim3 T Cell Immunoglobulin and Mucin Domain-Containing Protein 3 TLR Toll-Like Receptor TNF Tumour Necrosis Factor TNFR Tumour Necrosis Factor Receptor TNFRSF Tumour Necrosis Factor Receptor Superfamily TRAF TNFR-Associated Factor TRAIL TNF-Related Apoptosis-Inducing Ligand Treg Regulatory T Cells TRIF TIR Domain-Containing Adaptor-Inducing Interferon β Trm Tissue-Resident Memory T Cells TWEAK TNF-Related Weak Inducer of Apoptosis VacV Vaccinia Virus vRNP Ribonucleoprotein WT Wildtype

Chapter 1

Introduction

1 Introduction 1.1 Influenza virus

1.1.1 Influenza

Influenza is an acute viral infection of the respiratory system caused by an enveloped RNA virus known as the “influenza virus” (1, 2). Following infection with influenza, symptoms include the sudden onset of fever, muscle aches, headache, fatigue, and cough (3). Most people will recover from the infection in about a week. However, younger children, older people, and those with chronic conditions have a higher risk of developing severe complications such as pneumonia or even death after influenza infection. Influenza results in between 250,000 and 500,000 deaths around the world annually and is associated with a considerable economic burden due to the medication and hospitalization required (4). Four human influenza pandemics have occurred in the last 100 years and the worst pandemic happened in 1918 (2). This is known as the “Spanish Flu” which killed about 50 million people worldwide (5). Humans employ different mechanisms to protect themselves against influenza virus. These mechanisms include the rapid- acting innate immune system as well as the subsequent slower-acting but antigen-specific adaptive immune system which includes both antibody-mediated (humoral) immunity and cell- mediated immunity (6). Humoral immunity is controlled by B cells while cell-mediated immunity is carried out by T cells (6). These mechanisms will be covered in detail in subsequent sections.

1.1.2 Influenza structure and function

Influenza virus belongs to the Orthomyxoviridae family and contains eight negative sense single-stranded RNA segments (2, 7, 8), which encode 10 viral proteins: hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), matrix protein 2 (M2), nucleoprotein (NP), polymerase acidic protein (PA), polymerase basic protein 1 (PB1), polymerase basic protein 2 (PB2), non-structural protein 1 (NS1), and non-structural protein 2 (NS2). The influenza viral envelope consists of a lipid bilayer derived from the plasma membrane of the host and contains HA, NA, and the ion channel M2 (Figure 1.1) (7). M1 is the matrix protein beneath the viral envelope and plays an important role in maintaining the shape of the virus (9). M1 also plays a 2

critical role in holding the 8 viral ribonucleoproteins (vRNPs) in the virion (7). In the core of the virus, each vRNP contains one single-stranded RNA wrapped around many copies of NP. The heterotrimeric RNA-dependent RNA polymerase containing PA, PB1, and PB2 is bound at one end of the vRNPs (Figure 1.1) (7). Due to the error prone influenza RNA polymerase, point mutations are introduced into the viral genome during viral replication and this is known as the antigenic drift (2). In contrast, when a host cell is co-infected with two different influenza strains, reassortment of genes between the two strains can occur and this is known as antigenic shift (2). Antigenic shift can result in a major antigenic change and may cause an influenza pandemic.

Figure 1.1. Structure of influenza virion. The envelope of the influenza virus is derived from the plasma membrane of the host and contains three viral proteins HA, NA, and M2. Underneath the envelope, the M1protein serves as a matrix to hold the eight vRNPs in the core. Each vRNP consists of a single-stranded RNA wrapped around NP. Each vRNP is bound by the heterotrimeric RNA polymerase containing PA, PB1, and PB2. This figure was adapted from (2).

1.1.3 Influenza life cycle

After reaching a potential target cell, usually airway epithelial cells (10), the influenza HA binds to sialic acid residues of glycoproteins found on the surface of the target cell (7, 8). This binding leads to the receptor-mediated endocytosis of the virion (Figure 1.2). During the endosomal journey, acidification of the endosome leads to unpacking of the viral envelope and the release of vRNPs into the cytoplasm (7, 8). After vRNPs are transported to the nucleus, the viral RNA-dependent RNA polymerase performs the transcription and replication of viral RNAs. The viral mRNAs are exported to the cytoplasm for translation. Some of these newly synthesized viral proteins are transported to the plasma membrane for viral assembly while other viral proteins are transported back into the nucleus for generation of new vRNPs. After numerous rounds of viral protein synthesis and vRNP replication, the influenza NS2 mediates the export of newly synthesized vRNPs to the plasma membrane for viral assembly (11). After budding of the newly assembled virion, the influenza NA cleaves sialic acid residues from host glycoproteins, allowing the release of progeny viruses (7, 8).

Figure 1.2. Life cycle of influenza. Upon finding a potential target cell, the influenza HA binds to the sialic acid molecules of the host receptor, leading to the internalization of the virus. Acidification of the endosome causes the release of vRNPs into the cytoplasm of the host cell. vRNPs are exported into the nucleus where replication and transcription of the viral genome take place. After many rounds of viral protein synthesis and viral genome replication, new viral proteins and vRNPs are exported to the plasma membrane for viral assembly. After budding of the newly assembled virion, influenza NA cleaves off host sialic acid molecules to release the progeny viruses. This figure was modified from (2).

1.2 Immune responses against influenza virus

1.2.1 Innate immune responses

The innate immune responses against influenza virus are mediated by a wide range of different cell types as well as different cytokines and chemokines. Upon infection with influenza virus, lung epithelial cells secrete type I IFN (IFN-I). In mice, IFN-I collectively refers to a family of 14 IFN-α subtypes, a single IFN-β subtype, and other subtypes (12, 13). All of the above IFN-I subtypes bind to the type I interferon receptor (IFNAR) and collectively play an important role in inducing an “antiviral state” in cells as well as blocking viral replication (12, 13). Infected lung epithelial cells also secrete a wide array of different cytokines and chemokines such as TNF-α, IL-6, IL-8, CXCL10, CCL2, and CCL5 (14-18). These soluble mediators play a critical role in recruiting different innate immune cells to the site of infection to facilitate the control of the viral infection. Lung resident alveolar macrophages (AlvMF) are among the first line of defense against influenza. AlvMF produce IFN-I and have been shown to play a critical role in the maintenance of lung function during influenza infection (19, 20). Neutrophils are among the first innate immune cells recruited to the lung upon infection and play an important role in controlling viral spread, as depletion of neutrophils leads to an increase in viral titre (21- 23). Upon activation, natural killer cells (NK) lyse virally infected cells through perforin and granzyme B (24, 25). Upon influenza infection, innate lymphoid cells (ILC) are recruited to the lung and are involved in restoring the integrity and homeostasis of the lung tissue (26). Other types of innate immune cells with crucial functions to play during influenza are the different subsets of dendritic cells (DC). Plasmacytoid dendritic cells (pDC) are potent producers of IFN-I in response to viral infections (27). Conventional dendritic cells (cDC) play a critical role in the activation of the adaptive immune system which plays an important role in clearing influenza virus at a later stage of infection (discussed later in this chapter).

The innate immune system has a wide variety of pattern recognition receptors (PRR) for detection of influenza virus. RIG-I, a member of the RIG-I-like receptors (RLR), recognizes viral RNA containing 5’-triphosphate (28) and signals through MAVS leading to the production of pro-inflammatory cytokines downstream of NF-κB and production of IFN-I downstream of IRF3/7 (29). Many cell types such as lung epithelial cells, cDC, and AlvMF use RIG-I for viral detection (30). However, the influenza virus NS1 protein is a virulence factor that blocks RIG-I

signaling and inhibits IFN-I production (31, 32). The influenza virus is recognized by different toll-like receptors (TLR). TLR3 senses the secondary structure present in viral RNA while TLR7 binds to single-stranded viral RNA in the endosome. TLR3 signals through TRIF while TLR7 signals through MyD88 to induce IFN-I and pro-inflammatory cytokine production via IRF3/7 and NF-κB activation, respectively (33, 34).

1.2.2 APC subsets and priming of T cells

Antigen presenting cell (APC) subsets in the lung and mediastinal lymph node (mLN) are important players in immunity against influenza infection. Macrophages are a type of phagocytic cells capable of presenting antigens to T cells. Some macrophages originate during embryonic development while others arise from bone marrow derived circulating monocytes (35). There are two major macrophage subsets in the lung. Alveolar macrophages (AlvMF) originate from fetal monocytes and they reside in the air space of the alveoli and provide the first line of defense against invading pathogens (36, 37). During pulmonary infections, AlvMF have been shown to be one of the major producers of IFN-I (19, 38). One study showed that AlvMF were completely absent in the lung of Csf2-/- mice and these mice had an increase in morbidity to influenza infection when compared to wildtype (WT) mice. Csf2-/- mice also had an increased accumulation of dead cells as well as cellular debris in the lung after influenza infection, suggesting that AlvMF play an important role in maintaining lung function during influenza infection by removing cellular debris and dead cells (20). In contrast to AlvMF, interstitial macrophages (IntMF) have a mixed origin, containing both an embryonic origin as well as a postnatal bone marrow derived origin (39). IntMF are located in the lung parenchyma (37) and they are thought to promote immunity by presenting antigens to T cells due to their phagocytic ability and expression of MHC-II (40-42). In the lymph nodes, subcapsular sinus macrophages (Subcap MF) line the bottom of the subcapsular sinus and they have dendritic protrusions that extend into the B cell follicles beneath (43). Subcap MF are responsible for shuttling antigens into the follicle area (43). In contrast to Subcap MF, medullary macrophages (Medul MF) reside in the medullary region of the lymph nodes and they capture antigens draining into the lymph node medulla (44).

During inflammation or infection, monocytes are recruited from blood to the site of inflammation where they differentiate into inflammatory dendritic cells (InfDC) and inflammatory macrophages (InfMF) (45-47). This migration process is dependent on the chemokine receptor CCR2 and there is a significant reduction in the number of inflammatory monocytes in inflamed tissues and draining lymph nodes in CCR2-/- mice (45, 48, 49). Monocyte-derived inflammatory cells are mainly involved in innate immune defense and T cell activation. Several studies have shown that InfDC can present antigens and activate both CD4 and CD8 T cells (45, 50-52). In this thesis, InfDC and InfMF are collectively referred to as inflammatory antigen presenting cells (InfAPC).

Dendritic cells are known for their potent activity for priming and presenting antigens to activate naïve CD4 and CD8 T cells (53). Different subsets of dendritic cells have been identified and characterized (54). Lung migratory CD103+ DC and migratory CD11b+ DC reside in the lung tissue and upon acquiring influenza-derived antigens, they migrate to the local mLN in a CCR7 dependent manner and present antigens to activate T cells (55-57). In contrast to the migratory DC, lymphoid tissue resident CD8α+ DC spend their lifetime residing in the secondary lymphoid organs such as lymph nodes and spleen where they survey antigens draining into the organs (58). Studies showed that Batf3-/- mice lacked both CD103+ DC and CD8α+ DC and were defective in inducing CD8 T cell responses against influenza (19, 59). Thus, CD103+ DC and CD8α+ DC are believed to be the major DC involved in presenting antigens to CD8 T cells (59, 60). In contrast, CD11b+ DC have a higher expression of genes associated with MHC II antigen-presentation pathway compared to CD8α+ DC (61). Consistent with this gene expression profile, migratory CD11b+ DC process and present influenza antigens to CD4 T cells predominantly during influenza infection (56).

Early after activation, T cells upregulate CD69 which is a major regulator of T cell migration (62). CD69 mediates the transient retention of activated T cells in the lymph nodes by downregulating sphingosine-1-phosphate receptor 1 (S1PR1), a receptor that promotes lymphocyte egress (63-65). This transient retention of lymphocytes in the lymph nodes allows them to become fully activated before egress. As the immune response proceeds, effector T cells egress from the lymph nodes via efferent lymphatic vessels and circulate towards the thoracic duct for drainage into the blood (66). Once in the blood, effector T cells traffic to the site of inflammation and enter the infected lung by passing through the lung endothelium (66). T cell 8

migration into the lung is dependent on adhesion molecules LFA-1 and PSGL-1 (67, 68) as well as the chemokine receptor CXCR3 (69). A recent report showed that T cells require CXCR6 to enter lung airways following influenza infection (70). In the lung, effector T cells carry out effector functions leading to the clearance of influenza virus.

1.2.3 CD8 T cell responses

Upon activation by influenza antigens in the draining mLN, naïve CD8 T cells proliferate, gain cytotoxic activity, and migrate to the infected lung tissue to kill virally-infected cells with infected respiratory epithelial cells being the main target (71). Activated effector CD8 T cells have two mechanisms of killing infected cells. One of the mechanisms is known as granule exocytosis and refers to the release of granules containing perforin and granzymes by effector CD8 T cells after MHC-restricted interaction with infected target cells (72). Perforin is responsible for forming pores in the cell membranes of target cells, allowing granzymes to enter the cells and trigger apoptosis (73). There is also evidence that granzymes can enter target cells after binding to mannose-6-phosphate receptor (74). The other mechanism is Fas-mediated cytotoxicity. It is known that Fas/FasL interaction results in the apoptosis of the target cells and is one of the primary mechanisms used by CD8 T cells to kill infected cells during influenza infection (72, 75, 76). Besides Fas/FasL, death of infected cells can be induced when the TNF superfamily member TRAIL on CD8 T cells interact with its receptors death receptor 4 (DR4) and/or death receptor 5 (DR5) on target cells (77, 78). In addition, CD8 T cells produce pro- inflammatory cytokines such as interferon-gamma (IFNγ) and TNF which can promote cell death of infected cells directly or indirectly (79).

Unlike antibodies which target the more variable surface proteins of influenza, CD8 T cells are known to have a broader protection against different influenza strains since they target more conserved internal proteins of the virus (80, 81). In murine models of influenza, CD8 T cells target multiple epitopes derived from conserved internal proteins of the virus. In C57BL/6 (B6) mice, the primary CD8 T cell responses against influenza are dominated by two epitopes, b b one derived from influenza NP (D /NP366-374) and the other derived from influenza PA (D /PA224- b 233) (82, 83). Other minor CD8 T cell epitopes derived from PB1 (K /PB1703-711) , from M1 b d (K /M1128-135), and from NS2 (K /NS2114-121) have been described (84). 9

1.2.4 CD4 T cell responses

In contrast to CD8 T cells, CD4 T cells play more diverse roles during viral infections. Upon activation with virus-derived peptides, naïve CD4 T cells undergo extensive proliferation and differentiation into different types of effector T helper (Th) subsets under the influence of specific cytokines produced locally (85, 86). During viral infections, the presence of IFN-I and IL-12 usually favours the generation of T helper type 1 (Th1) cells (87). As is the case with influenza infection, activated CD4 T cells differentiate into T-bet expressing Th1 cells and secrete the signature Th1 cytokine IFNγ which contributes directly and indirectly to protection (88). IFNγ plays an important role in setting an antiviral state in infected tissue as well as activating innate immune cells such as macrophages (89).

Besides Th1 cells, some activated CD4 T cells differentiate into follicular helper T cells (Tfh) which play an important role in supporting antibody affinity maturation and isotype switching by participating in the germinal centre reaction in the B cell follicles of the mLN (90). Tfh support germinal centre reactions by providing key costimulatory signals through ICOS as well as by producing IL-21 (90, 91). Optimal antibody generation against influenza virus requires the interaction between CD40L on activated CD4 T cells and CD40 on B cells (92-94).

Under certain conditions such as during chronic LCMV Clone 13 infection, CD4 T cells provide vital help for CD8 T cells. In contrast, CD4 T cell help is not required for effective primary CD8 T cell responses against influenza virus (95), possibly due to effective/direct activation of APC by the influenza virus (96). However, CD4 T cell help is required for optimal generation of memory CD8 T cells against influenza since memory CD8 T cells primed without CD4 T cells not only are reduced in number, but also have a reduced ability to respond upon secondary infection (95). One possible mechanism by which CD4 T cells promote the generation of memory CD8 T cells is through downregulation of TRAIL on CD8 T cells which renders these cells less susceptible to TRAIL-mediated apoptosis (97).

Besides providing help for other immune cells, some effector CD4 T cells acquire cytolytic capabilities and are able to directly lyse infected cells through perforin and Fas (98). Lastly, regulatory T cells (Treg) are a subset of CD4 T cells known for their ability to suppress pathological immune responses. In a murine influenza infection model, Treg have been shown to play a critical role in limiting pathology caused by the infection (99). 10

1.2.5 Central memory and effector memory T cells

In the ensuing days after clearance of influenza virus from the lung, effector T cells undergo a contraction phase where around 90%-95% of effector cells die by apoptosis, leaving a small proportion of long-lived memory T cells (100-102). Memory T cells are able to control reinfection faster as they have a higher proliferative potential and a more rapid effector response (103). The formation of memory CD8 and CD4 T cells requires both IL-7 and IL-15, which are cytokines required for memory T cell survival and homeostatic proliferation (104-107). Memory T cells are a heterogeneous population and can be classified into two groups based on proliferative capacity and differential expression of lymphoid tissue homing molecules CD62L and CCR7 (108, 109). Central memory T cells (Tcm) have a high level of CD62L and CCR7 expression. Both CD4 and CD8 Tcm are mainly found in the secondary lymphoid organs. Whereas, effector memory T cells (Tem) are CD62L- and CCR7- and they mainly circulate between blood and non-lymphoid tissues (108, 110). Generally, Tcm have a higher proliferative potential and a greater resistance to apoptosis than Tem. In contrast, Tem have a lower proliferative capacity but with a greater ability to produce effector cytokines such as IFNγ (111). Recently, it became apparent that some memory T cells have the ability to permanently reside in the peripheral tissue after an infection is cleared, including influenza infection. This population of T cells is discussed in the next section.

1.2.6 Tissue resident memory T cells

After influenza is cleared, some effector T cells differentiate into memory T cells that permanently reside in the lung tissue and these are known as tissue-resident memory T cells (Trm). Trm have been shown to be highly protective against influenza virus due to some of their unique features discussed below (112-115). Circulating memory T cells need to undergo adhesion and extravasation in order to enter the infected tissue to carry out effector functions (116). In contrast, Trm reside within peripheral tissues where viral infection first occurs. The location of Trm provides them with the advantage of responding more rapidly to invading pathogens when compared to circulating memory T cells. In addition, lung Trm cells have the ability to rapidly produce IFNγ upon re-infection, which facilitates the recruitment of circulating memory T cells from the blood (117-121). Lung Trm are more resistant to becoming targets of 11

influenza virus infection due to enhanced expression of the IFN-induced transmembrane protein IFITM3, an antiviral molecule that confers resistance to different viral infections (122). Tissue transplant and parabiosis experiments are considered gold standard experiments to confirm tissue residency (119, 123, 124). In parabiosis experiments, circulating T cells equilibrate between the two surgically joined animals whereas non-circulating tissue-resident T cells remain exclusively in the tissues of one partner (119, 123, 124). Staining for Trm markers in combination with the labeling of vascular cells by intravenous infusion of anti-T cell antibodies is commonly used to unambiguously identify cells that are resident within a particular tissue at a particular point in time (119, 125). This intravascular labeling technique is also known as the in vivo antibody labeling technique.

CD8 Trm in various non-lymphoid tissues, including lung tissue, express CD69 (CD69+CD103-) or CD69 and CD103 (CD69+CD103+). Some CD8 Trm are localized to the epithelial layers of lung airways (126-128). Other CD8 Trm reside within the lung interstitium, either maintained within the repair-associated memory depots (RAMD), which are niches created at the site of tissue regeneration after injury, or found sparsely across uninjured lung areas (129). It is thought that lung CD8 Trm maintained in RAMD are committed to protect the damaged site by exerting their function as CTLs (130). Both CD69 and CD103 play critical roles in tissue retention of lung CD8 Trm since CD69 or CD103 deficiency results in a reduced number of lung CD8 Trm after viral infection (131, 132). CD69 prevents tissue egress by blocking responsiveness to S1P gradients through downregulation of S1PR1 (133). CD103 on lung CD8 Trm binds to E-cadherin expressed by epithelial cells, allowing the retention of Trm within the epithelium (131, 134).

The development of lung CD8 Trm depends on multiple factors. One of the factors is local antigen which is required for lung CD8 Trm differentiation (114, 129). This is supported by the evidence that CD8 Trm precursors exhibit high levels of Nur77 expression which is an indication of TCR signaling (135-137). In addition to antigen recognition, the local cytokine environment is critical for the formation of lung CD8 Trm. TGF-β facilitates the development of lung CD69+CD103+ CD8 Trm by downregulating T-box transcription factors Eomes and T-bet (138, 139). Downregulation of T-bet leads to increased CD103 expression and increased tissue retention. In the lung, TGF-β is produced by many different cell types such as AlvMF, neutrophils, as well as activated alveolar epithelial cells (140). CD103+ DC may play a role in 12

promoting lung CD69+CD103+ CD8 Trm formation by converting TGF-β into its active form locally through secretion of MMP (114). Interestingly, TGF-β is dispensable for the development of lung CD69+CD103- CD8 Trm (114, 138). IL-15 is not required for the establishment of lung CD8 Trm (141).

Trm have a distinct transcriptional landscape compared to circulating memory T cells. In addition to downregulation of Eomes and T-bet, Trm precursors downregulate the transcription factor Kruppel-like factor 2 (KLF2) which results in reduced expression of S1PR1 and promotes tissue retention of Trm (142). In mice, the transcription factors Blimp1 and Hobit work cooperatively to promote Trm development in tissues such as skin, gut, and liver by repressing tissue exit pathways (143). Interestingly, Blimp1 but not Hobit is required for the differentiation of lung CD8 Trm after influenza infection (144). Notch contributes to the maintenance of lung Trm after influenza infection by regulating the expression of CD103 (145). The transcription factor Runx3 was recently reported to be a crucial regulator of the development of CD8 Trm in multiple tissues, including the lung tissue (146). Runx3 supports expression of genes involved in tissue retention and represses genes involved in tissue egress (146).

In addition to local cytokines and antigen, CD4 T cell help is required for optimal formation of lung CD69+CD103+ CD8 Trm (147). During influenza infection, CD4 T cells traffic to the lung before CD8 T cells and secrete IFNγ which influences the tissue microenvironment and helps direct CD8 T cells into lung airway epithelium (147). In the lung epithelium, CD8 T cells are then exposed to TGF-β and upregulate the tissue retention molecule CD103. In the same study, it was shown that unhelped CD8 T cells expressed more T-bet and had a reduced expression of CD103 and CD69 (147). In this thesis, out of the two lung CD8 Trm populations, I mainly focus on CD69+CD103+ lung CD8 Trm.

Similarly to CD8 Trm, lung CD4 Trm are highly protective against influenza virus. In mice, lung CD4 Trm have been shown to provide superior protection against influenza when compared to memory CD4 T cells from the spleen. CD4 Trm are typically found within clusters of cells known as memory lymphocyte clusters (MLC) below the epithelium (116, 148). MLC have been identified in the skin, intestine, and lung and are different from tertiary lymphoid organs since they lack lymphatics and B cells (112, 116, 148, 149). Compared to CD8 Trm, lung CD4 Trm express CD11a instead of CD103 and can be distinguished from circulating CD4 Tcm

and Tem since they have elevated expression of CD69 and CD11a (119, 150, 151). It is thought that CD69 expression by Trm is due to continual stimulation through the TCR by residual influenza antigen in the tissue (152, 153). Local and sustained expression of CD69 by Trm plays an important role in tissue retention since CD69 prevents tissue egress by downregulating S1PR1 (64). CD11a is an integrin that binds to ligands such as intercellular adhesion molecule 1 (ICAM-1) (154). CD11a is involved in cell adhesion and may also contribute to tissue retention (150, 154). The developmental program of lung CD4 Trm is similar to lung CD8 Trm as they share similar transcriptional programs. For instance, lung CD4 Trm express low levels of T-bet and Eomes (155). Cognate antigen plays a critical role in the formation of lung CD4 Trm and enhances the survival of CD4 Trm through upregulation of CD127 (IL-7Rα) (135, 156). Besides antigen, IL-15 is required for early differentiation, but dispensable for long-term maintenance of lung CD4 Trm in mice (151).

Most of the current knowledge about Trm differentiation, function, and maintenance stems from studies using mouse models. However, in humans, several studies showed that T cells expressing Trm associated molecules CD69 and CD103 could be found in multiple tissues such as the lung, skin, liver, and intestines (145, 157-163). A further study by Kumar and colleagues showed that human CD69+ memory T cells in the tissue have a transcriptional landscape that is different from the CD69- memory counterpart in the tissue (164). Human CD69+ CD4 and CD8 tissue memory T cells share a core gene signature with mouse Trm (164). This core gene profile includes the downregulation of genes involved in tissue egress such as S1PR1 and KLF2 as well as the upregulation of adhesion molecules CD103 and CD49a (164).

1.2.7 B cell responses

B cells help clear influenza virus by secreting both neutralizing and non-neutralizing antibodies. Most of the neutralizing antibodies target the influenza surface protein HA and they have the ability to interfere with viral infection of target cells by binding to the sialic acid binding site of HA (165). Non-neutralizing antibodies do not prevent viral infection of target cells, but they mediate viral clearance through antibody-dependent cell-mediated cytotoxicity (ADCC) or through activation of the complement system (166, 167). Non-neutralizing antibodies can target the HA stem as well as other influenza proteins NP, M1, and M2 (165). Some of these 14

antibodies have been shown to be broadly neutralizing against multiple influenza strains, and are in development as new vaccine candiates (168).

After activation by influenza antigen in the mLN, B cells migrate to the T cell-B cell border to receive help from T cells. Subsequently, B cells differentiate into rapidly proliferating short-lived extrafollicular plasma cells that secrete IgM, IgG, and IgA to facilitate viral clearance (169). Other B cells can participate in the germinal centre (GC) response in the B cell follicles and develop into long-lived antibody-secreting plasma cells or non-secreting memory B cells (170). These long-lived B cells provide a long lasting immunological memory against influenza (170).

1.3 The TNFR superfamily

1.3.1 Classifications and functions of the TNFR superfamily

Members of the tumour necrosis factor (TNF) and TNF receptor (TNFR) superfamily play important roles in many biological processes, from developmental processes such as regulation of osteoclasts in bone remodeling (171) to regulation of cellular differentiation and programmed cell death (172). They also play critical roles in many immunological processes such as controlling the differentiation and proliferation of different immune cells (173). TNFR family members also influence innate immunity, T cell responses, as well as the amount of inflammation during an immune response. Several members of TNF/TNFR superfamily, such as LTβR, are important in the development of secondary lymphoid organs (172). The superfamily consists of 19 ligands and 29 receptors (Figure 1.3). Some receptors can interact with multiple ligands and some ligands can interact with more than one receptor (172). TNFRs are generally type I transmembrane proteins with cysteine-rich repeats in their extracellular domains which are involved in the interaction with their cognate ligands (172, 174). TNF ligands are type II transmembrane proteins with a common domain known as the TNF homology domain which is involved in binding to the cysteine-rich motifs in the TNFRs (172).

Members of the TNFR superfamily can be classified into three different groups: death domain-containing receptors, decoy receptors, and TNFR-associated factor (TRAF) binding receptors (173). Out of all the known TNFR family members, 8 of them contain a death domain: TNFR1, death receptor (DR) 3, DR4, DR5, DR6, Fas, nerve growth factor receptor, and ectodysplasin A receptor. Upon ligand binding, the death domain of these receptors interact with death domain containing adaptor molecules which results in the formation of a membrane proximal complex that initiates a downstream signaling cascade leading to the induction of apoptosis (172). In the case of TNFR1, however, the death inducing signaling complex only forms as complex II, if the cell is unable to maintain survival signaling (175). Decoy receptors are unable to transmit downstream signals since they lack a cytoplasmic tail. Their main function is the sequestration of ligands. TRAF binding receptors such as TNFR2, do not contain death domain, but possess short motifs that can recruit with TRAF proteins (176). TRAF binding receptors are able to initiate multiple downstream signaling cascades implicated in cytokine

production, cell survival as well as cellular proliferation. This group of TNFRs includes molecules involved in T cell costimulation (Figure 1.3).

Figure 1.3. The TNF/TNFR superfamily. The tumour necrosis factor (TNF) and TNF receptor (TNFR) superfamily consists of 19 ligands and 29 receptors. Some receptors such as TNFR1 can interact with more than one ligand. Some ligands such as TRAIL can bind to multiple receptors. Black lines represent ligand and receptor interactions. Yellow stars represent known TNFR family members involved in T cell costimulation. This figure was modified from (172).

1.3.2 TNFR signaling

After initial T cell activation, several T cell costimulatory members of the TNFR superfamily promote and sustain T cell responses (177). They can enhance effector T cell activity and promote cell division and survival in T cells through NF-κB, MAPK, and Akt pathways (176, 178). Since these TNFR superfamily members do not possess any known catalytic activity, they recruit signaling adaptors such as TRAF proteins to their cytoplasmic tail to mediate downstream signaling cascades. In mammalian cells, there are six members of TRAF proteins (TRAF1-6) and they share a C-terminal domain known as the TRAF domain, which plays an important role in the oligomerization of TRAF proteins and association with the respective TNFRs (179, 180). Downstream of TNFRs, TRAFs are known to regulate major downstream signaling cascades such as canonical NF-κB, non-canonical NF-κB, and MAPK pathways (181). For NF-κB pathways, five structurally related mammalian NF-κB proteins (RelA/p65, RelB, c-Rel, NF-κB1 p50, NF-κB2 p52) assemble into homo and heterodimers to regulate the expression of the target genes which include a number of inflammatory cytokines as well as survival genes that can inhibit apoptosis (182, 183).

In the canonical NF-κB pathway (Figure 1.4), activated TGFβ-activated kinase 1 (TAK1) phosphorylates and activates the IκB kinase (IKK) complex which is composed of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ/NEMO) (181). Then, activated IKK complex phosphorylates the inhibitory protein known as the inhibitor of κBα (IκBα), which sequesters members of the NF-κB family in the cytoplasm under normal conditions (181). Upon phosphorylation, IκBα is targeted to the proteasome for degradation, leading to the translocation of members of the NF-κB family such as the p50-RelA heterodimer into the nucleus to regulate the expression of target genes (181).

Several TNFR family members such as CD40, CD27, LTβR, 4-1BB, TWEAK, BAFFR and TNFR2 are able to signal through the non-canonical NF-κB pathway (184-186). In the non- canonical NF-κB pathway (Figure 1.4), activated NF-κB-inducing kinase (NIK) phosphorylates and activates IKKα (187, 188). Subsequently, activated IKKα phosphorylates the C-terminal serine residues of NF-κB2 precursor protein p100, resulting in the degradation of IκB-like structure of p100 and the generation of NF-κB2 p52. RelB and p52 translocate to the nucleus to regulate target gene expression (187, 188).

Figure 1.4. The NF-κB pathways downstream of TNFRs. Members of the TNFR superfamily can signal through the canonical as well as the non-canonical NF-κB pathway. In the canonical NF-κB pathway, activation of the IKK complex leads to phosphorylation and degradation of IκBα, resulting in the translocation of NF-κB family members into the nucleus to regulate target genes. The non-canonical NF-κB pathway involves the processing of NF-κB2 precursor protein p100 into NF-κB2 p52 which translocates into the nucleus with RelB to regulate expression of target genes. This figure was modified from (188).

1.3.3 TNFRs and viral infections

Even though T cells play an important role in controlling viral infections, they must be tightly regulated in order to clear the infection successfully while at the same time avoiding excessive immunopathology. This fine balance is accomplished through production of pro- and anti-inflammatory mediators. In addition to that, co-stimulatory and co-inhibitory receptors are important in maintaining this balance (189). After initial T cell activation, several T cell costimulatory members of the TNFR superfamily play critical roles in controlling viral infections, in some contexts providing protection, in other situations leading to immune pathology (177, 190). These members include CD27, CD30, 4-1BB, OX40 and GITR and their ligands, CD70, CD30L, 4-1BBL, OX40L and GITRL, respectively (GITR will be discussed in detail in a subsequent section).

CD27 is constitutively expressed by naïve CD8 T cells and is transiently upregulated upon activation (191, 192). Early studies have shown that CD27 is required for optimal primary CD8 T cell responses against influenza as CD27-/- mice have decreased accumulation of b D /NP366-374-specific CD8 T cells in the lung compared to WT mice (193-195). CD27 is also required for optimal secondary T cell responses against influenza (195). Recent studies by Ballesteros-Tato et al. showed that migratory CD103-CD11b+ DC express CD70 and cross- present NP antigens to T cells during the late phase of a primary influenza infection (196, 197). T cells recognizing antigens presented by these DC receive signals through CD27, allowing them to differentiate into memory T cells with robust proliferative and cytokine-producing potential b b (196, 197). In contrast to D /NP366-374-specific CD8 T cells, D /PA224-233-specific CD8 T cells are not dependent on CD27 costimulation (197). During influenza infection, CD27 is required for b sustaining low affinity D /NP366-374-specific CD8 T cells since more NP tetramers bound to b -/- D /NP366-374-specific CD8 T cells in CD27 mice than WT mice, indicating expansion of higher affinity NP specific CD8 T cells in CD27-/- mice (198). In the same study, it was shown that these low affinity CD8 T cells are capable of providing protection against heterologous influenza viruses (198).

CD30 is not detected in resting T cells but can be induced on both CD4 and CD8 T cells after stimulation with anti-CD3 and anti-CD28 antibodies in vitro (199, 200). After influenza PR8 infection, CD30 was detected on Treg, minimally expressed by Th1 cells, and it was

b undetectable on D /NP366-374-specific CD8 T cells (199). The role of CD30 during influenza infection was further assessed. In contrast to CD27, CD30 is not required for primary and secondary CD8 T cell responses against influenza (199). In addition, CD30 is dispensable for CD44hiT-bet+ Th1 accumulation in the lung after influenza infection (199).

4-1BB is not expressed by resting naïve or memory CD8 T cells but is transiently induced upon T cell activation. The effect of 4-1BB on CD8 T cell responses is dependent on the severity of the influenza infection (201). During mild influenza X31 infection, there is a similar number b -/- of D /NP366-374-specific CD8 T cells in the lung and spleen between 4-1BBL and WT mice. In contrast, during more severe influenza PR8 infection, there is a significant reduction in the b -/- number of lung D /NP366-374-specific CD8 T cells in 4-1BBL mice (201). This can be explained by the differential expression pattern of 4-1BB on CD8 T cells between mild and severe influenza infections. In mild influenza infection, 4-1BB is detected on lung CD8 T cells at day 6 post-infection (p.i.), but is not detectable on these cells at day 8 p.i. However, during severe influenza infection, 4-1BB is expressed on lung CD8 T cells at day 6 p.i. and the expression persists at least through day 8 p.i. (201). The prolonged 4-1BB expression suggests a greater dependency of CD8 T cell responses on 4-1BB during severe influenza infection. Interestingly,

CD8 T cell responses to the immuno-dominant NP366-374 epitope and subdominant epitopes

PB1703-711, NS2114-121, and M1128-135 are dependent on 4-1BB during severe influenza infection, indicating that the sensitivity of CD8 T cell responses to endogenous 4-1BB is independent of the epitopes studied but dependent on the severity of the infection (201).

OX40 is also absent from naïve and memory T cells in the resting state, but transiently induced upon T cell activation with a higher expression on CD4 T cells than CD8 T cells (202, 203). In contrast to 4-1BB which has a predominant effect on CD8 T cells, OX40 has a predominant effect on CD4 T cells during influenza infection since OX40-/- mice exhibit a reduced number of lung infiltrating CD4 T cells but not CD8 T cells (202). Similar results were observed in OX40L-/- mice in which these mice did not have a defect in the accumulation of b D /NP366-374-specific CD8 T cells in the lung as well as the draining mLN (195). However, OX40L is required for secondary CD8 T cell responses against influenza, suggesting that OX40 may play a role in enhancing T cell survival during the memory phase of T cell responses (195).

1.3.4 TNFRs and Trm formation

It is widely thought that the size of the T cell memory pool is dependent on the size of the effector pool (204, 205). Building on this concept, it is plausible that T cell costimulatory members of the TNF/TNFR superfamily contribute to optimal Trm formation by affecting the size of effector T cells. Several studies have provided evidence that TNF/TNFR family members contribute to Trm formation in non-lymphoid tissue during viral infections.

Debenedette and colleagues first showed that 4-1BBL is critical for CD8 recall responses to influenza virus (206). Later, Bertram and colleagues showed that 4-1BB signals were temporally delayed when compared to CD28 signals in CD8 T cell responses to influenza (207). In the same study, 4-1BBL was again shown to be important for CD8 recall responses against influenza (207). Similarly, OX40 was shown to be important for CD4 recall responses to influenza virus (203). With the recent discovery of non-circulating memory T cells, it became important to revisit the role of TNFR family members in T cell memory.

A recent study by Zhou and colleagues demonstrated a CD8 T cell intrinsic role for 4- 1BB in accumulation of lung effector CD8 T cells and Trm after influenza infection (208). In this study, WT mice were intra-nasally primed with influenza A/HK-X31. At day 30, they were boosted intra-nasally with a replication defective adenovirus containing either the influenza NP gene alone (Ad-NP) or with NP and 4-1BBL (Ad-NP-4-1BBL) (208). In vivo antibody labeling technique was used in this study to distinguish lung parenchymal cells from lung vascular- associated cells. At 7 months post-boost, mice receiving Ad-NP-4-1BBL had more total b D /NP366-374-specific CD8 T cells in the lung parenchyma when compared to the Ad-NP treated b group (208). Within the lung parenchymal D /NP366-374-specific CD8 T cell compartment, there were more CD69+CD103+, CD69+CD103-, and CD69-CD103- cells in the Ad-NP-4-1BBL treated group when compared to Ad-NP treated mice (208). Using 4-1BB+/+/4-1BB-/- mixed bone marrow chimeras, the authors showed that the effects of 4-1BBL boost on lung NP-specific CD8 Trm required 4-1BB expression on CD8 T cells (208). Using the same mixed bone marrow chimera models, it was shown that endogenous 4-1BB on CD8 T cells is crucial for the accumulation of lung CD8 Trm during influenza infection since 4-1BB-/- CD8 T cells failed to contribute significantly to the lung CD8 Trm pool (208). Thus, either supraphysiological delivery of 4-1BBL or endogenous 4-1BB provides an important signal for the accumulation of lung CD8

Trm during influenza infection. A follow-up study showed that the effect of 4-1BB on the accumulation of lung CD8 Trm is largely due to its effect on the effector T cell precursors of Trm since the defect in 4-1BB-/- effector T cells is apparent and similar in magntitude at day 9 when compared to the defect in Trm accumulation at day 45 in the 4-1BB+/+/4-1BB-/- mixed bone marrow chimeras (209). This 4-1BB dependent accumulation of effector CD8 T cells in the lung parenchyma requires the 4-1BB signaling adaptor TRAF1, local antigen, as well as the mTOR pathway (209).

Besides 4-1BB, additional members of the TNF/TNFR superfamily have been implicated in the optimal accumulation of Trm in non-lymphoid tissues. One such member is LIGHT, which is expressed by activated effector CD4 and CD8 T cells (210, 211). Using the adoptive transfer model of transgenic OT-I T cells, Desai et al. showed that WT OT-I and LIGHT-/- OT-I expand equally well in the lung early in the effector T cell response against vaccinia virus (VacV) (212). However, most of the LIGHT-/- OT-I were lost by day 40 p.i., suggesting that LIGHT is dispensable for initial T cell expansion but is important for the generation of memory CD8 T cells in the lung (212). Using the same approach but with the intravenous antibody labeling technique for Trm detection, the authors discovered that adoptive transfer of LIGHT-/- OT-I cells gave rise to fewer lung CD69+CD103+ CD8 Trm when compared to transfer of WT OT-I cells (212). Recipient mice transferred with WT or LIGHT-/- OT-I had similar proportion of CD69+CD103+ to CD69+CD103- CD8 Trm, suggesting that LIGHT does not play a role in the differentiation of lung CD8 Trm (212). It was suggested that LIGHT provides survival signals to effector T cells as they transition into long-lived memory cells by downregulating pro-apoptotic factors such as caspase-2, Bid, and Bad, and by upregulating anti-apoptotic molecules such as Akt1 and Atf5 (212). Recently, it was reported that effector CD8 T cells can be subdivided into different subsets based on differential expression of the fractalkine receptor CX3CR1, with CX3CR1hi cells being the most terminally differentiated, whereas CX3CR1neg cells are the least differentiated and give rise to Trm (213, 214). Desai and colleagues showed that in the absence of LIGHT, the reduction in the number of effector CD8 T cells in the lung was largely due to a decrease in the number of CXCR3+CX3CR1neg CD8 T cell population and this is consistent with a role for LIGHT in optimal lung CD8 Trm accumulation during VacV infection (212).

One study by Salek-Ardakani and colleagues examined the effect of triggering OX40 on lung resident memory CD8 T cells during VacV infection (215). WT mice treated intra- 24

peritoneally with anti-OX40 antibody had increased numbers of IFNγ producing CD8 T cells in the lung day 8 post VacV infection compared to control treated mice (215). Anti-OX40 treatment also increased the number of IFNγ producing CD8 T cells in the lung 360 days post VacV infection, suggesting that OX40 plays an important role in generating long-lived memory CD8 T cells in the lung (215). Using MHC-II deficient mice, the authors showed that this process is independent of CD4 T cells (215). However, the intravenous labeling technique was not used when identifying cells isolated from the lung. Thus, it is likely that the population examined in this study was a mixture of circulating memory and tissue-resident memory T cells.

1.4 GITR and GITRL

1.4.1 GITR structure and expression

Glucocorticoid-induced TNFR-related protein (GITR) was identified in 1997 by comparing gene expression profiles between untreated murine T cell hybridoma cells with those treated with the synthetic glucocorticoid compound dexamethasone (216). The murine GITR gene is located within the same genomic region as murine CD30, 4-1BB, and OX40 on mouse chromesome 4 (217). The murine GITR gene comprises 5 exons, 4 introns, and is approximately 2.5 kb long (217). The first three exons encode the extracellular domain of GITR which is crucial for binding to GITRL. A small part of the extracellular domain, the transmembrane domain, and some of the cytoplasmic domain are encoded by exon 4. The rest of the cytoplasmic tail is encoded by exon 5 (217). Upon analysis of different mRNA splice variants of GITR, three additional novel mRNA splice variants were discovered (218). Interestingly, one of the variants encodes a soluble form of GITR which may act as a decoy receptor (218). However, further investigation is required to determine the functional relevance of the different splice variants of GITR. GITR is a 228 amino acid type I transmembrane protein with cysteine rich motifs in the extracellular domain, while the intracellular domain is similar to other members of the TNFR family such as CD27, 4-1BB, and OX40 (216, 217). GITR expression in various cell types is summarized in Table 1.1. GITR is constitutively expressed by naive murine CD4 and CD8 T cells, with Treg exhibiting the highest level of expression (219, 220). GITR is upregulated upon T cell activation and this process is dependent on the p65 subunit of NF-κB (219-221). GITR is also expressed by other immune cell types such as B cells, DC, and macrophages (222).

GITR-/- mice are vital and fertile (223). GITR-/- mice have normal lymphoid organ development (223). Under steady state conditions, GITR-/- mice have normal numbers of CD4 T cells, CD8 T cells, and B cells in the secondary lymphoid organs when compared to WT mice (223).

Table 1.1 Murine GITR expression

Cell type GITR expression Note Reference

CD8 T cells ++ (Resting) (220, 224, 225)

+++ (Activated)

CD4 T cells ++ (Resting) (220, 224, 225)

+++ (Activated)

Treg +++ (Resting) (220, 224, 225)

++++ (Activated)

B cells + (Naïve) Among different B cell subsets, B220-CD138+ plasma cells have the ++ (Plasma) (220, 226) highest GITR expression, whereas GC (B220+IgD-PNA+) and memory -/+ (GC) (B220+IgG+) B cells have lower -/+ (Memory) GITR expression than naïve B cells.

NK Cells ++ (Resting) (224)

+++ (Activated)

ILC1 ++ (Resting) During influenza infection, GITR- (227) expressing ILC1 can be subdivided +++ (Activated) hi lo into GITR ILC1 and GITR ILC1 with a similar finding in vitro.

ILC2 ++ (Resting) Resting and activated ILC2 express (228, 229) similar amount of GITR. Similar ++ (Activated) findings after papain induced lung inflammation.

DC -/+ (Resting) CD11chi DC upregulate GITR after (224) LCMV Clone 13 infection. ++ (Activated)

pDC -/+ (Resting) (224)

++ (Activated)

Monocytes/macrophages + (Resting) (220)

++ (Activated)

NKT cells ++ (Resting) (230)

+++ (Activated)

Mast cells ++ (Resting) (231)

++ (Activated)

Endothelial cells - (232)

Keratinocytes ++ (233)

Osteoclasts + (234) - = no expression + = low ++ = intermediate +++ = high ++++ = very high

1.4.2 GITR signaling

Similar to other T cell costimulatory TNFR family members, GITR does not possess any known intrinsic catalytic activity. Thus, signaling through GITR requires the recruitment of TRAF adaptor proteins. Studies using a yeast 2 hybrid system or co-immunoprecipitation analyses suggest that GITR can associate with TRAF1, TRAF2, TRAF3, TRAF4, and TRAF5 (235-238). GITR can signal through the ERK, JNK, and p38 MAPK pathways which play important roles in regulating cell survival (Figure 1.5) (239, 240). In addition to the MAPK pathways, GITR can activate both canonical as well as non-canonical NF-κB pathways (Figure 1.5) (240-242). Using short interference RNA (siRNA) targeting of TRAF2 or TRAF5, Snell and colleagues showed that TRAF2 and TRAF5 are both required for GITR mediated classical NF-

κB activation in CD8 T cells which leads to enhanced T cell survival through Bcl-xL upregulation (243). In contrast, TRAF1 is not required for GITR-dependent NF-κB activation, since TRAF1-/- CD8 T cells show unimpaired NF-κB activation in response to ligation with anti- GITR antibody (243).

In vitro treatment of splenocytes with the agonistic anti-GITR antibody DTA-1 leads to increased Akt activation as indicated by a higher level of phosphorylation at Thr308 of Akt 15 minutes and 30 minutes after treatment (244). Thus, in addition to NF-κB and MAPK pathways, GITR can signal through the Akt/mTOR/pS6 pathway, an important determinant of cell size and protein translation (Figure 1.5) (245).

Although most studies suggest a costimulatory role of GITR on T cells, some studies imply a negative role of GITR on T cells. When stimulated with plate bound anti-CD3 antibodies, GITR-/- T cells hyper-proliferate when compared to GITR+/+ counterparts (223). Even though GITR does not contain a death domain, GITR has been shown to induce apoptosis by interacting with a pro-apoptotic protein known as Siva under certain circumstances (246). This interaction occurs near the membrane distal TRAF2 binding site of GITR and requires the 30 amino acid residues in the C-terminal domain of GITR (246). Subsequently, Siva promotes apoptosis by binding to Bcl-xL and prevents its anti-apoptotic effects (247). It remains unclear whether or not this interaction occurs in murine cells and what conditions determine whether GITR interacts with TRAF2 or with Siva.

Figure 1.5. GITR signaling pathways. Upon interaction with its ligand, GITR recruits signaling adaptors TRAF2 and TRAF5 leading to downstream signaling. Signaling through the canonical NF-κB pathway leads to the degradation of IκB and the translocation of active NF-κB dimers into the nucleus to drive the expression of prosurvival molecules Bcl-xL and Bcl-2. Signaling through the Akt/mTOR/pS6 pathway regulates cell metabolism and leads to enhanced protein translation. GITR can also signal through the MAPK pathways which can regulate cell survival as well as apoptosis. GITR also weakly activates the non-canonical NF-κB pathway which leads to the processing of NF-κB precursor protein p100. This figure was adapted from: Clouthier, D.L. (2015). The T cell co-stimulatory molecule GITR in the control and treatment of a persistent viral infection (Doctoral dissertation). Retrieved from https://tspace.library.utoronto.ca/bitstream/1807/70827/3/Clouthier_Derek_L_201511_PhD_thesis.pdf.

1.4.3 GITRL structure and expression

Glucocorticoid-induced TNFR-related protein ligand (GITRL) was first identified in humans in 1999 (235, 248). Subsequently, murine GITRL was identified in 2003 by searching for homology with the amino acid sequence of human GITRL (249-251). The gene for murine GITRL spans 9.3kb, contains three exons, and is located in chromosome 1 near OX40L (249- 251). Murine GITRL is a 173 amino acid type II transmembrane protein with a 129 amino acid C-terminal extracellular region, a 23 amino acid transmembrane domain, and a 21 amino acid N- terminal cytoplasmic tail (249-251). Murine GITRL exon 1 encodes for the cytoplasmic and transmembrane domains, exon 2 encodes for a small part of the membrane proximal segment of the extracellular domain, while exon 3 encodes for most of the extracellular domain. Human GITRL, like other TNF family members, assembles into a trimer. In contrast, the mouse GITRL crystal structure reveals a unique dimeric structure stabilized by a strand-exchange interaction (252) .

Murine GITRL transcript is detected in various immune cell populations, with high levels of expression by macrophages, DC, and B cells and low levels of expression detected in activated peritoneal macrophages (249, 250). GITRL transcript is not detected in resting T cells but whether or not it is expressed by activated T cells is conflicting (250, 253, 254). Cell surface expression of murine GITRL has been detected on B220+ B cells, F4/80+ macrophages, and DC (249, 250, 253, 255-257). In addition, GITRL is detected on the mouse endothelioma cell line EOMA (251). A recent study has shown that IFN-β is a potent inducer of GITRL whereas IFNγ minimally induces GITRL (258). Upon stimulation with LPS, murine GITRL is transiently upregulated on the macrophage cell line RAW264 as well as bone marrow derived DC and this effect is dependent on the transcription factor NF-1 (250). LPS induction of GITRL is largely dependent on IFN-I since the induction is mostly blocked upon anti-IFNAR1 treatment (259). Several studies have shown that GITRL is upregulated during viral infections. During HSV-1 infection, GITRL expression is upregulated on CD11b+, CD11c+ as well as B220+ cells from the draining lymph nodes 24 hours post infection, but the expression is downregulated by 96 hours post infection (255). A recent study by Chang et al. showed that during chronic LCMV Clone 13 infection, InfAPC, characterized by co-expression of CD64 and FcεRI, are the major expressors of GITRL with peak expression at day 2 post infection (258). In contrast, cDC, a major cell type involved in initial T cell priming, expressed a minimal level of GITRL (258). In the same study, 31

using GITR as proof of principle, it was shown that signals from TNFR family members occur post T cell priming and are mainly provided by InfAPC. These signals are termed “signal 4” and will be discussed in detail in a subsequent section.

1.4.4 GITRL reverse signaling

Since the short cytoplasmic domains of TNF family members have high conservation among different species, it is plausible that many of the family members are capable of reverse signaling (260). There are several studies suggesting that GITRL is capable of reverse signaling. Treatment of murine pDC with soluble GITR initiates reverse signaling through GITRL, leading to activation of noncanonical NF-κb pathway followed by IDO-dependent immune regulation (261). Splenic DC isolated from GITR-/- mice have a higher TLR4 expression when compared to WT mice. In addition, GITR-/- splenic DC downregulate TLR4 expression upon treatment with recombinant GITR, confirming that reverse signaling through GITRL modulates TLR4 expression on DC (262). Stimulation of murine macrophages with soluble GITR leads to enhanced production of inflammatory mediators such as MMP-9, cyclooxygenase (COX)-2, and inducible nitric oxide (NO) synthase (iNOS), which results in enhanced production of NO (263- 265). Reverse signaling through GITRL is also involved in other processes such as osteoclastogenesis as well as induction of inflammatory activation in microglia leading to upregulated expression of pro-inflammatory genes (257, 266). GITRL reverse signaling studies often used GITR-Fc fusion proteins with monomeric IgG or other control Fc proteins as negative controls. One major caveat to this method is that the GITR-Fc fusion proteins have a different oligomeric structure than control Fc proteins and might therefore show differential Fc receptor engagement. Thus, the phenomena observed in these studies could be Fc receptor mediated. GITRL-/- mice or cells can be used to rule out Fc receptor mediated effects and provide more convincing evidence for the importance of reverse signaling through GITRL.

GITR is not overtly required during embryonic development since embryos from GITR-/- mice develop normally (223). GITRL is required for embryonic development in zebrafish (267), but whether or not GITRL is required for murine embryonic development remains to be examined. In a recent study, mice were designed to delete GITRL through deleting exon 2 and shifting exon 3 out of frame (258). However, I discovered that these mice still expressed GITRL 32

(258). I found that exon 1 was expressed from a different reading frame such that exon 3 was brought back into its normal reading frame, producing a truncated GITRL protein that can still bind to the anti-GITRL antibody (258). There may be a high selective pressure to express GITRL to maintain mouse embryo viability, but this remains to be formally determined.

1.4.5 Effect of GITR on DC and macrophages

GITR is not required for the differentiation of DC since GITR+/+ and GITR-/- BMDC have comparable levels of CD11c, CD80, CD86, and MHC-II before and after stimulation with LPS (268). However, CD4+CD25- T cells co-cultured with GITR+/+ BMDC proliferated more when compared to co-culture with GITR-/- BMDC, suggesting that GITR-/- BMDC are defective at activating T cells in vitro (268). The ability of GITR-/- BMDC to activate T cells was restored when a GITR expression construct was transfected into GITR-/- BMDC (268). After contacting T cells, GITR-/- BMDC produced a higher amount of anti-inflammatory cytokine IL-10 but a lower amount of pro-inflammatory cytokine IL-6 when compared to WT counterparts (268). This increased IL-10 production by GITR-/- BMDC resulted in an increased generation of FoxP3+ Treg in the BMDC-T cell co-culture system (268). The authors showed that GITR-/- BMDC were also defective at activating T cells in vivo since LPS stimulated GITR+/+ BMDC loaded with ovalbumin (OVA) induced more activated CD25+ T cells in the lymph nodes compared with OVA loaded GITR-/- BMDC (268).

The effect of GITR on murine macrophages was investigated in the context of toxoplasma gondii infection. Mice treated with anti-GITR agonist antibody DTA-1 during toxoplasma gondii infection produced more of the inflammatory cytokines IL-6, TNF-α, and IFNγ and had reduced parasite burden during the chronic phase of infection (269). However, macrophages treated with DTA-1 exhibited similar production of IL-12 when compared to control treated macrophages, suggesting that the beneficial effect of DTA-1 treatment is independent of macrophages (269). Thus, GITR does not seem to play a major role in stimulating macrophages in the context of toxoplasma gondii infection, even though GITR is upregulated on CD11b+ F4/80+ macrophages upon stimulation with toxoplasma gondii (269). In contrast, Kim and colleagues found that the murine macrophage cell line RAW264.7

constitutively expressed low levels of GITR and produced MMP-9 and TNF-α upon stimulation with anti-GITR antibodies or with recombinant mouse GITRL (270).

1.4.6 Effect of GITR on ILC

Innate lymphoid cells (ILC) are lymphocytes that do not express antigen specific receptors found on T cells and B cells. ILC are important regulators of immune homeostasis and inflammation (271). ILC can be grouped into three different subsets based on their function, cytokine profiles, as well as specific transcription factor requirements (271). Group 1 ILC contain both NK cells and ILC1 and are characterized by their ability to produce the Th1 cytokine IFNγ (271). Group 2 ILC contain a single subset (ILC2) and produce the Th2 cytokines IL-5 and IL-13 (271). Lastly, group 3 ILC contain two subsets, the NCR- and NCR+ ILC3, and produce the Th17 cytokine IL-17 (271). The effects of GITR on ILC1 and ILC2 were recently reported (227, 229, 272), whereas the effects of GITR on ILC3 remain unknown.

After influenza infection, there is an increase in the frequency of murine GITR- expressing ILC1 as well as the total GITR expression level on lung ILC1 (Table 1.1) (227). Interestingly, GITR-expressing ILC1 are more functional and express more IFNγ and CD69 than ILC1 that do not express GITR (227). GITR-expressing ILC1 can be divided into GITRhi and GITRlo subpopulations with the GITRhi subpopulation showing signs of immune exhaustion (227). Triggering GITR using the DTA-1 antibody results in an increase in the frequency of IFNγ+ and TNFα+ ILC1 in vivo, suggesting that GITR plays a positive role in ILC1 in addition to being a marker of ILC1 activity (227).

Both resting and activated ILC2 isolated from the lung express GITR (Table 1.1) (228). Even though GITR is expressed on ILC progenitors, GITR is dispensable for ILC2 development since no difference was observed in the number of lung ILC2 at steady state between GITR-/- and WT mice (229). However, during papain-induced or IL-33 induced lung inflammation, GITR plays a critical role in the proliferation as well as survival of lung ILC2 since GITR-/- mice had reduced number of lung ILC2 (229). GITR signaling in ILC2 is also important for production of Th2 cytokines IL-5 and IL-13 which are cytokines known to be important in allergy (229). Both mouse and human ILC2 isolated from visceral adipose tissue express GITR (272). In a type 2

diabetes model, triggering GITR on activated ILC2 led to increased Th2 cytokine production and ameliorated established insulin resistance (272).

1.4.7 Effect of GITR on NK and NKT cells

Very little is known about the effect of GITR on murine NK cells since most of the studies focused on human NK cells. However, the existing data on the effect of GITR on human NK cells are controversial. One study showed that human NK cell cytotoxicity and IFNγ production was enhanced after in vitro culture with GITRL expressing pDC and this effect was abrogated by GITRL blocking antibodies (273). Moreover, this NK cell activating signal through GITR was synergistic with IFN-I (273). In contrast, other groups have found that triggering GITR on NK cells with soluble GITRL or immobilized GITRL-Ig fusion proteins inhibit NK cell cytotoxicity, proliferation and IFNγ production (274-276).

The effect of GITR on natural killer T (NKT) cells has not been extensively studied. Freshly isolated murine hepatic NKT cells constitutively express GITR on their cell surface with increased GITR expression upon TCR engagement (Table 1.1) (230). In the presence of TCR engagement, in vitro stimulation of NKT cells with DTA-1 results in enhanced proliferation, increased nuclear translocation of NF-κb as well as a higher expression level of the activation markers CD25 and CD69 (230). Stimulating GITR on NKT cells also led to enhanced production of IL-4 and IFNγ in vitro, with similar findings in vivo (230).

1.4.8 Effect of GITR on T cells

The effect of GITR on T cells has been well characterized. Initial studies suggest that GITR is costimulatory in T cells since T cells overexpressing GITR were more resistant to TCR/CD3 mediated activation induced cell death (AICD) whereas GITR-/- T cells were more prone to AICD (216, 223, 248, 277). Further in vitro studies provide evidence that GITR has costimulatory effects on effector CD4 and CD8 T cells as GITR engagement on T cells through anti-GITR antibodies, soluble GITRL, or murine GITRL transfected cells promoted cytokine production, CD25 upregulation and cell proliferation as well as cell survival (241, 250, 253, 254,

278, 279). Some studies have suggested that GITR plays a differential role in CD8 versus CD4 T cells. In vitro studies showed that murine GITR dependent costimulation plays a critical role in the activation of CD8 T cells (253). GITR activity in CD8 T cells was independent of CD28 activation since T cells from CD28-/- mice could be costimulated normally by GITR (254). In contrast to CD8 T cells, the effect of GITR on CD4 T cells is dependent on CD28 co-triggering and the results can be pro-survival or pro-apoptotic, depending on the experimental conditions as well as the strength of TCR stimulus (241, 246, 250, 253, 280, 281).

Tregs show constitutively high expression of GITR, which is partly controlled by FoxP3, a master regulator of Tregs (282, 283). Due to its high expression on Tregs, GITR was originally thought to be a marker of Tregs. However, it was discovered that activated effector T cells can upregulate GITR to levels almost as high as on Tregs (284). Multiple studies have shown that GITR engagement by agonistic anti-GITR antibodies, by soluble GITRL, or by GITRL transfected cells abrogates the suppressive effect of murine Tregs (219, 220, 225, 249, 250, 253, 285). By using a combination of rat effector T cells and mouse Tregs, Shimizu and colleagues demonstrated that the agonistic anti-GITR antibody DTA-1 acted directly on Tregs to abrogate their ability to suppress effector T cells (220). Similarly, Ronchetti and colleagues showed that anti-GITR antibody treatment directly inhibited the suppressor activity of Tregs since abrogation of suppression was observed when anti-GITR antibody was added to GITR+/+ CD4+CD25+ Treg and GITR-/- CD4+CD25- effector T cell cocultures (225). Similar results were observed in a murine arthritis model and in an in vivo colitis model (286, 287). Granzyme B, a molecule involved in Treg mediated suppression, has been shown to be downregulated in Tregs upon anti- GITR treatment (288). Even though short term GITR triggering inhibits Treg suppression activity, long term GITR triggering has been shown to enhance Treg activity as well as the number of Tregs. For instance, GITR-/- mice have a lower number of CD4+CD25+ Tregs whereas GITRL transgenic mice have up to three times more Tregs than WT mice (223, 289). These observations support the notion that GITR regulates the suppressor activity of Treg. However, the above observations might have been confounded by lack of use of littermate controls. Indeed, Clouthier and colleagues showed using GITR+/+ and GITR-/- littermate controls as well as GITR+/+ and GITR-/- mixed bone marrow chimeras, that the absence of GITR did not affect the number of Tregs in the steady state in B6 mice (244). However, after LCMV Clone 13 infection, the absence of GITR resulted in a 2-fold defect in Tregs compared to their WT counterparts,

suggesting that under inflammatory conditions, GITR can maintain Treg numbers (244). Moreover, other studies have shown that GITR is not involved in Treg suppressor activity since GITR-/- Tregs do not exhibit a defect in mediating suppression when compared to WT Tregs in vitro (225, 253). By using combinations of WT and GITR-/- Treg, Stephens and colleagues discovered that GITR signaling on effector T cells, but not on Tregs, rendered them refractory to Treg-mediated suppression (253). Evidence to date suggests that GITR can regulate both effector T cells and Tregs, but whether GITR affects effector T cells or Tregs may depend on the level of inflammation and other contextual considerations.

1.4.9 Effect of GITR on B cells

GITR expression on B cells is detected during B cell development and is constitutively expressed by naïve mature B cells (226). Among different B cell subsets, murine B220-CD138+ plasma cells have the highest GITR expression whereas B220+IgD-PNA+ germinal center and B220+IgG+ memory B cells express less GITR than naïve B cells (Table 1.1) (226). Both LPS stimulation and BCR signaling trigger GITR upregulation on B cells (220, 226). Even though GITR expression is detected on B cells and upregulated upon B cell activation, GITR does not seem to play a major role in B cell development as well as B cell responses (226). GITR-/- B cells do not have a defect in survival nor proliferation when compared to WT counterparts (226). GITR-/- mice have normal early B cell development with a very small reduction in the number of mature B cells (226). However, GITR-/- mice have a normal level of serum immunoglobulins and mount normal antibody responses to both T cell-dependent and T cell-independent antigens when compared to WT mice (226). During chronic infection, the absence of GITR impairs the antibody response to LCMV Clone 13, likely through its intrinsic effect on the Tfh response (244). It remains to be determined whether or not GITR is required for Tfh or antibody responses against acute viral infections such as influenza virus infection.

1.4.10 GITR and viral infections

The role of GITR in the context of different viral infections has been studied, with emphasis on T cells. In the murine model of herpes simplex virus type I (HSV-1), mice treated 37

with DTA-1 had increased numbers of activated CD8 T cells as well as IFNγ-producing CD8 T cells in the draining lymph nodes with a similar results for CD4 T cells when compared to control Ig-treated mice (290). Both CD4 and CD8 T cells isolated from DTA-1 treated mice had enhanced proliferation as well as the ability to produce IFNγ when compared to control Ig- treated mice (290). Similar findings were observed in a separate study done by Suvas and colleagues using a mouse model of ocular HSV infection (255). In this study, GITR stimulation leads to enhanced production of IL-2 and IFNγ by CD4 T cells and granzyme B by virus specific CD8 T cells, even though no difference in viral load was observed between DTA-1 treated and control treated mice (255). During the early phase of Friend Virus (FV) infection, mice treated with DTA-1 had greater expansion of virus specific CD8 T cells and more activated CD4 T cells compared to control treated mice (291). DTA-1 treatment led to reduced infection levels and prevented FV-induced splenomegaly (291).

Mice lacking GITR succumbed to severe influenza infection, suggesting a role for GITR in protection against influenza virus infection (243). An adoptive transfer model of transgenic T cells (OT-I CD8 T cells) demonstrated the intrinsic requirement of GITR on CD8 T cells for maximal accumulation of CD8 T cells in multiple organs during the primary as well as during the recall response against mild influenza A/HK-X31-OVA infection (243). Specifically, transfer of GITR+/+ but not GITR-/- OT-I cells could protect against an otherwise lethal infection with influenza carrying the epitope of the transgenic T cells (243). In this model, GITR did not affect effector T cell function since the proportion of IFNγ-producing cells in GITR-/- OT-I cells was comparable to WT OT-I cells (243). GITR did not affect OT-I cell proliferation as measured by CFSE dilution, but enhanced the survival of OT-I T cells by upregulating the pro-survival factor

Bcl-xL (243). In the same study, the effect of delivering agonistic antibody DTA-1 was also examined using a non-infectious i.p. route of influenza infection. Upon treatment with DTA-1, adoptively transferred WT OT-I expanded whereas the GITR-/- counterpart did not, providing further evidence that GITR acts directly on CD8 T cells to allow enhanced accumulation (243). There was also a small but statistically significant increase in the number of endogenous activated CD44hi CD4 T cells upon DTA-1 treatment when compared to control rat IgG treated mice (243).

The role of GITR during chronic LCMV Clone 13 has been well characterized. Upon infection with LCMV Clone 13, GITR-/- mice had increased viral load in multiple organs when 38

compared to GITR+/+ mice (244). Further examination of GITR-/- mice during chronic LCMV infection revealed a defective accumulation of antigen-specific CD8 T cells and Th1 cells in the spleen as well as elevated PD-1 and Tim3 expression on CD8 T cells, indicating increased T cell exhaustion (244). GITR-/- mice also had a defective Tfh response and were impaired in the production of LCMV-specific IgG antibodies in the chronic phase of LCMV infection (244). Mixed bone marrow chimeras and adoptive transfer models with LCMV specific transgenic T cells revealed that the effect of GITR on CD4 T cells is largely CD4 T cell intrinsic whereas the effect of GITR on CD8 T cells is almost completely dependent on CD4 T cells and IL-2 (244). In the same study, it was shown that GITR does not affect the initial CD4 T cell proliferation, but plays a crucial role in supporting the accumulation of Th1 cells through activation of NF-κB p65 and phosphorylated-ribosomal protein S6 (pS6), leading to enhanced CD8 T cell accumulation and improved viral control (244). A follow up study by Chang and colleagues revealed that GITRL on LysM+ cells contributed significantly to the accumulation of Th1 and Tfh cells during LCMV Clone 13 infection (258). A complementary study by Pascutti and colleagues showed that overexpressing GITRL on B cells (GITRL transgenic mice) leads to early clearance of LCMV Clone 13 accompanied by enhanced helper function of virus specific CD4 T cells as well as increased number of virus specific CD8 T cells (292). Using CD4 T cell depletion experiments, Pascutti and colleagues showed that the enhanced expansion of virus specific CD8 T cells in GITRL transgenic mice is entirely dependent on CD4 T cells (292). Even though endogenous GITR is intrinsically required for CD4 T cell accumulation during chronic LCMV infection, the delivery of agonistic anti-GITR antibodies at the chronic stage of LCMV Clone 13 infection leads to expansion of CD8 T cells without having an effect of CD4 T cells, as at this stage of treatment, the CD4 T cells are likely too exhausted to respond (224). Mixed bone marrow chimeras with GITR+/+ and GITR-/- T cells showed that CD8 T cells can respond directly to anti- GITR at the chronic stage of LCMV Clone 13 infection (224), thus the absence of direct effects of endogenous GITRL on CD8 T cells likely reflect lack of access to the ligand by the CD8 T cells, perhaps because the GITRL expressing APC are poor at cross-presentation.

The involvement of GITR in enhancing T cell responses in different viral infection models suggests its potential for use in vaccine design. Indeed, when plasmids encoding HIV-1 antigens were co-administered with a plasmid encoding GITRL, CD4 T cell responses were strongly induced whereas CD8 T cells were weakly induced (293). In this case, the weak CD8

response likely reflects the nature of the antigen. Similar observations were obtained with a vaccine model against FV infection (294). In mice, co-immunization of FV antigens and GITRL expressing plasmids leads to enhanced protection against a FV challenge (294).

1.4.11 GITR and signal 4

The initial T cell activation requires the recognition of peptide-MHC complex (signal 1), costimulation through CD28 (signal 2) (295), as well as signals from cytokines (signal 3) (296). Early work suggests that a brief initial interaction between naïve T cells and APCs is able to trigger the differentiation of naïve T cells into effector and memory T cells (297, 298). Recently, it has become more appreciated that multiple encounters between T cells and APC are crucial for optimal primary T cell responses as well as optimal memory T cell formation (152, 299). Many members of the TNFR superfamily are upregulated upon T cell activation and play an important role in determining the magnitude as well as the duration of the T cell responses (176). As TNFRs are induced by TCR signaling, it makes sense that TNFR signals are post-priming events. As I will discuss below, this was formally demonstrated recently for GITR (258).

During chronic LCMV infection, InfDC and InfMF were the major expressors of TNFR family ligands GITRL, 4-1BBL, OX40L, and CD70 in the spleen, lymph node, liver, and lung (258, 259). In contrast, cDC expressed minimal levels of the 4 TNFR family ligands throughout LCMV Clone 13 infection (258, 259). Compared to the superior antigen presenting capacity of cDC, InfDC and InfMF were inferior at antigen presentation (258). Nevertheless, InfDC and InfMF could take up antigen in vivo and present it to naïve CD4 T cells ex vivo (258). For this reason, InfDC and InfMF are referred to as InfAPC. Since activated T cells are more responsive to low antigen load and InfAPC have highest expression TNFR ligands, it seemed plausible that TNFR signals were post-priming events and were provided by InfAPC. To test this hypothesis, Chang and colleagues adoptively transferred GITR+/+ and GITR-/- LCMV-specific SMARTA transgenic CD4 T cells into B6 mice followed by assessing their upregulation of T cell activation markers and intracellular signaling intermediates downstream of GITR after LCMV infection (258). By 12 hours p.i., the majority of the transferred SMARTA cells in the spleen had upregulated activation markers CD69 and 4-1BB, indicating that most of the transferred cells were activated by this time point (258). At this time point, there was no difference in the 40

expression of early T cell activation markers between GITR+/+ and GITR-/- transferred cells, suggesting that GITR is dispensable for initial T cell activation (258). Also at this time point, there was no difference in the levels of GITR signaling intermediates NF-κB p65 and pS6 between transferred GITR+/+ and GITR-/- cells (258). However, between 24 hours to 72 hours p.i., the transferred GITR+/+ SMARTA cells had higher levels of NF-κB p65 and pS6 when compared to their GITR-/- counterparts within the same mice, suggesting that GITR signaling occurred post-priming (258). In the same study, by using mice in which exon 2 of GITRL was deleted in LysM+ myeloid derived cells, the authors provided evidence that the GITR dependent signals require GITRL on InfAPC (258). This post-priming signal from TNFR family members was referred to as “signal 4” (Figure 1.6) (258).

Figure 1.6. Role of InfAPC in providing signal 4 for T cells. During initial T cell activation, TCR and CD28-dependent costimulatory signals provide signal 1 and 2, respectively. Initial T cell activation also requires signal 3 from cytokines. InfAPC upregulates TNFR family ligands GITRL, 4- 1BBL, OX40L, and CD70 in response to IFN-I. After T cells are activated, they receive post-priming signals from TNFR family members (signal 4) provided by InfAPC. This figure was modified from the manuscript “Monocyte- derived cells in tissue resident memory T cell formation” which was accepted for publication at the Journal of Immunology.

1.5 Thesis synopsis

From the studies examining the effect of GITR/GITRL costimulation in the context of viral infections, several questions remain to be elucidated. By using the OT-I CD8 transgenic model, Snell and colleagues showed that GITR is intrinsically required on CD8 T cells for their maximal accumulation during acute influenza infection (243). In the same study, a small increase in the number of activated CD4 T cells was observed after DTA-1 treatment (243), but due to the model used, it is possible that this could be an indirect effect since GITR is also expressed by many other immune cell types. Thus, at the time I began my thesis work the effect of GITR on the overall endogenous CD4 and CD8 T cell responses against influenza remained largely unknown. Trm are a newly described subset of memory T cells that reside for long periods of time in the peripheral tissue such as the lung tissue and they have been shown to be highly protective against influenza infection (112-115). At the time I began my thesis work, we knew from the transgenic CD8 T cell model, that GITR is required on CD8 T cells for secondary expansion against influenza infection. However, it was not known if GITR regulates the number/formation/maintenance of lung CD4 and CD8 Trm after influenza infection.

In the LCMV Clone 13 model, GITRL was originally described to as being highly expressed by splenic CD11bhighF4/80+ myeloid cells (224). However, these cells are heterogenous. In parallel to the work discussed in my thesis, as discussed above, Chang et al. showed that InfAPC exhibited the highest GITRL expression during LCMV Clone 13 infection (258). I contributed to that study by extending this work to show that GITRL is also expressed on InfAPC in the mLN during influenza infection. In my thesis, in chapter 2, I further explore the expression of GITRL in the lung during influenza virus infection. Using GITR as a prototype, Chang and colleagues had shown that GITR signaling can be detected in LCMV-specific CD4 T cells, temporally segregated from initial T cell activation (258). However, it remains unknown whether T cells can also receive signals through TNFR family members in the peripheral tissue during viral infections or alternatively perhaps these signals are confined to events that occur in secondary lymphoid tissues. This was a question I set out to ask in my thesis and I describe my findings in chapter 2.

In chapter 2, I provide evidence that GITR/GITRL costimulation not only takes place in secondary lymphoid organs, but also takes place in non-lymphoid tissues during influenza

infection. I show that this signal is required for rescuing low affinity influenza nucleoprotein specific CD8 T cells as well as the accumulation of both effector CD4 and CD8 T cells and optimal formation of CD4 and CD8 lung Trm after influenza infection. I further provide evidence that this signal is likely provided by InfAPC during influenza infection.

In chapter 3, I show that during influenza infection, lung effector CD4 T cells can be divided into two subsets whereas effector CD8 T cells can be divided into three subsets. I provide evidence that GITR differentially affects these lung effector T cell subpopulations. Although GITR equally affects the accumulation of the two effector CD4 subpopulations, it selectively regulates CD127 expression in CD4 T cells endowed with enhanced memory potential. In CD8 T cells, GITR differentially affects the accumulation of the three CD8 T cell subsets with the least differentiated CD8 subset being most dependent on GITR.

Together, this thesis expands our current knowledge of T cell costimulation by GITR/GITRL during influenza infection. I provide evidence that GITR/GITRL costimulation can occur in the lung for optimal effector T cell accumulation and optimal lung Trm formation. I also provide insight on how GITR affects different effector T cell subpopulations.

Chapter 2

GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T cell accumulation and tissue- resident memory T cell formation

This work was published in Mucosal Immunology:

Chu, K. L., Batista, N. V., Wang, K. C., Zhou, A. C. & Watts, T. H. GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T-cell accumulation and tissue-resident memory T-cell formation. Mucosal Immunol 12, 363-377, doi:10.1038/s41385-

018-0105-5 (2019).

Figure 2.11 is unpublished

Author contributions:

NV Batista, KC Wang, and AC Zhou contributed to this study by providing technical assistance.

NV Batista assisted with the generation of GITR mixed bone marrow chimeras.

2 GITRL on inflammatory antigen presenting cells in the lung parenchyma provides signal 4 for T cell accumulation and tissue-resident memory T cell formation

2.1 Summary

T cell responses in the lung are crucial for protection against respiratory pathogens such as influenza virus. TNFR superfamily members play important roles in providing survival signals to T cells during respiratory infections. However, whether these signals take place mainly during T cell priming in the secondary lymphoid organs and/or in the peripheral tissues remains unknown. Here we show that under competitive conditions, GITR provides a T cell intrinsic advantage to both CD4 and CD8 effector T cells in the lung tissue, as well as for the formation of CD4 and CD8 Trm during respiratory influenza infection in mice. In contrast, under non- competitive conditions, GITR has a preferential effect on CD8 over CD4 T cells. The nucleoprotein-specific CD8 T cell response partially compensated for GITR deficiency by expansion of higher affinity T cells, whereas the polymerase-specific response was less flexible and more dependent on GITR. Following influenza infection, GITR is expressed on T cells in the mLN and in the lung. GITRL is preferentially expressed on monocyte-derived inflammatory antigen presenting cells in the mLN as well as in the lung. Accordingly, we show that GITR+/+ T cells in the mLN express more pS6 than their GITR-/- counterparts, with larger effects in the lung parenchyma later in the infection. Thus, we show that this signal can take place in the mLN and then again in the lung tissue and this signal critically regulates effector and tissue-resident memory T cell accumulation during influenza infection.

2.2 Introduction

Influenza remains an important human pathogen. CD4 and CD8 T cells play important roles in control of influenza virus (72, 98). However, their activation must be tightly regulated in order to clear the infection and avoid pathology, while at the same time allowing for appropriate formation of memory T cells (300, 301). The initial activation of T cells requires a peptide antigen bound to MHC (signal 1), a costimulatory signal through co-receptor CD28 (signal 2) (295) as well as cytokines (signal 3) (296). While these signals are important in initiating T cell responses, additional post-priming signals from TNFR superfamily members play crucial roles in controlling the duration and magnitude of the T cell responses (193, 201, 258, 302-304), referred to as signal 4 (258).

GITR is a costimulatory member of the TNFR superfamily and has been shown to play an important role in the control of viral infections, including influenza virus and LCMV Clone 13 (243, 244, 258, 284). GITR is constitutively expressed at low levels on resting T cells and at high levels on CD4+ CD25+ regulatory T cells (219, 220), and is rapidly upregulated on effector CD4 and CD8 T cells upon activation (219, 220). GITR is also expressed by other immune cell types such as NK cells, B cells, and macrophages (222). GITRL is mainly expressed on antigen presenting cells (APC) and endothelial cells (277), with monocyte-derived inflammatory APC showing the highest level of expression during chronic LCMV Clone 13 infection (258).

The role of GITR during viral infection appears to be context dependent. In the chronic LCMV Clone 13 infection model, GITR is mainly required on CD4 T cells to provide a post- priming checkpoint for CD4 accumulation and shows only indirect effects on CD8 T cells (244, 258). In contrast, in a TCR transgenic T cell adoptive transfer model of influenza infection, GITR was shown to mainly affect CD8 T cell responses, with critical effects on viral control (243). However, the effect of GITR in the endogenous T cell responses to respiratory influenza virus, and when and where these signals take place are incompletely defined. Here, we provide evidence that GITRL on monocyte-derived inflammatory APC provides crucial signals through GITR on T cells in the mLN and then again in the lung tissue to allow effector T cell accumulation during respiratory influenza infection. This signal is critical for rescuing low b affinity D /NP366-374-specific CD8 T cells as well as for the optimal formation of lung Trm.

2.3 Materials and Methods

2.3.1 Mice

WT C57BL/6 mice were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). Female mice were used in all experiments except where indicated and were age- and sex-matched within each experiment. GITR-/- mice were previously described (244) and backcrossed one additional time to C57BL/6 mice or to CD45.1 OT-II mice (kindly provided by David Brooks, Princess Margaret Cancer Center, Toronto, Ontario) or to CD45.2 OT-I (C57BL/6-Tg(TcraTcrb)1100Mjb/J) mice obtained from Jackson Laboratories (Bar Harbor, USA) to generate littermate controls. GITRLexon2fl/fl mice were previously described (258). Briefly, after neomycin resistance gene removal, GITRLexon2fl/fl mice were bred to homozygosity before crossing to LysM-Cre (B6.129P2-Lyz2tm1(cre)Ifo/J) mice to generate LysM-Cre+/+/LysM- Cre-/- GITRLexon2fl/fl littermate controls or to CMV-Cre (B6.C-Tg (CMV-Cre)1Cgn/J) mice to delete GITRL ubiquitously. After confirming germline deletion, the CMV-Cre GITRLexon2Δ/Δ mice were maintained without the Cre transgene (referred to as GITRLexon2Δ/Δ mice). For bone marrow chimera experiments, Thy1.1 (B6.PL-Thy1a/CyJ) and CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mice were purchased from Jackson Laboratories (Bar Harbor, USA). All mice were housed under specific pathogen-free conditions in sterile microisolator cages in the Division of Comparative Medicine at the Terrence Donnelly Centre for Cellular and Biomolecular Research (University of Toronto). Animal studies were approved by the animal care committee at the University of Toronto in accordance with the Canadian Council on Animal Care.

2.3.2 Reagents and antibodies

b b Biotinylated H-2D /NP366-374:ASNENMETM and H-2D /PA224-233:SSLENFRAYV monomers were obtained from the National Institute for Allergy and Infectious Disease tetramer facility (Emory University, Atlanta, GA), and tetramerized with streptavidin APC (ProZyme, San Leandro, CA). All antibodies used for flow cytometry are listed in Table 2.1. Fixable viability dye was purchased from eBioscience (San Diego, CA). Anti-mouse IFNAR1 antibody (Clone: MAR1-5A3) and Mouse IgG1 isotype control were purchased from Bio X Cell (West Lebanon, NH).

Table 2.1 Anti-mouse antibodies for flow cytometry

Antibody Fluorochrome Clone Source

B220 AF488, BV605 RA3-6B2 eBioscience, Biolegend

CCR2 PE 475301 R&D

CD103 PerCP-eF710 2E7 eBioscience

CD11a BV786 M17/4 BD Biosciences

CD11b APC-eF780 M1/70 eBioscience

CD11c AF700 N418 eBioscience

CD16/32 (Fc Block) Unlabeled 93 eBioscience

CD169 AF647 3D6.112 Biolegend

CD19 BV605 6D5 Biolegend

CD25 AF700 PC61.5 eBioscience

CD3 BV605 17A2 Biolegend

CD3ε PE-Cy7, eF450 145-2C11 eBioscience

CD4 BV605 RM4-5 Biolegend

CD44 FITC, PerCP-Cy5.5 IM7 eBioscience

CD45.1 APC-eF780 A20 eBioscience

CD45.2 BUV395, PE, FITC, 104 BD Biosciences, eBioscience AF700

CD62L PE-Cy7, APC-eF780 MEL-14 eBioscience

CD64 BV711 X54-5/7.1 Biolegend

CD69 PE-Cy7, APC-eF780 H1.2F3 eBioscience

CD8α BV605, AF700, PerCP- 53-6.7 Biolegend, eBioscience eF710

CXCR5 PerCP-eF710 SPRCL5 eBioscience

F4/80 BV785 BM8 Biolegend

FcεR1 PE-Cy7 MAR-1 eBioscience

Foxp3 eF450 FJK-16s eBioscience

GITR PE, APC, BV421 DTA-1 eBioscience, BD Biosciences PerCP-eF710

GITRL PE MIH44 BD Biosciences

IFNγ PE XMG1.2 eBioscience

IL-2 eF450 JES6-5H4 eBioscience

Ly6C APC-eF780 HK1.4 eBioscience

MHC-II eF450 M5/114.15.2 eBioscience

PD-1 PE J43 eBioscience

PDCA-1 APC eBio927 eBioscience phospho-S6 Ser 235/236 PE Cupk34k eBioscience

Siglec-F AF647 E50-2440 BD Biosciences

T-bet eF660 eBio4B10 eBioscience

TCR Vα2 APC B20.1 Biolegend

TCR Vβ5.1/5.2 FITC MR9-4 BD Biosciences

Thy1.2 FITC, APC, PE, PerCP- 53-2.1 eBioscience eF710

Thy1.2 BV785 30-H12 Biolegend

2.3.3 Influenza virus infection

Influenza A/PR8-OVA and A/PR8-OVAII were kindly provided by Paul Thomas and Peter Doherty (St. Jude Children's Research Hospital, Memphis, Tennessee). The tissue culture infectious dose 50 (TCID50) was determined for influenza A/HK-X31, A/PR8, A/PR8-OVA and A/PR8-OVAII viruses by MDCK assays (305). Six- to ten-week old mice were anaesthetized with isofluorane and infected intranasally (i.n.) with 30 μL of diluted virus at a dose of 5 HAU 4 5 (equivalent to 3.86 x 10 TCID50) of X31, or 5 x 10 TCID50 of PR8, PR8-OVA or PR8-OVAII. For PR8, PR8-OVA, or PR8-OVAII infection, mice were monitored daily and sacrificed when moribund.

2.3.4 Tissue harvest and processing

At the indicated time points after infection, lung, mLN and spleen were harvested. Lung was perfused with 10 mL of PBS, and then minced and digested with 2 mg mL-1 of collagenase IV (Invitrogen, Carlsbad, CA) for 45 minutes at 37oC while shaking. Lung tissue was mechanically disrupted through a 70 μm cell strainer and leukocytes were enriched by isolation over an 80/40% Percoll gradient (GE healthcare, Chicago, IL) after RBC lysis. Spleen (after RBC lysis) and mLN were mechanically disrupted through a 70 μm cell strainer to generate single cell suspensions. For APC isolation, mLN was digested with 1 mg mL-1 of collagenase IV for 45 minutes at 37oC while shaking. For Trm detection, mice were injected intravenously with 3 μg of anti-mouse Thy1.2 antibody conjugated to FITC and sacrificed 10 minutes later with tissues processed as described above. Blood was collected from the saphenous vein and treated with RBC lysis buffer.

2.3.5 Flow cytometry

Freshly isolated single cell suspensions from the lung, mLN, and spleen were treated with Fc Receptor Block for 15 minutes at 4oC followed by surface staining for 30 minutes at 4oC. Staining with H-2Db-restricted tetramers were performed simultaneous with surface staining for 30 minutes at 4oC. Samples were fixed with 4% paraformaldehyde following surface staining.

Intracellular staining, where applicable, was performed for 30 min at 4oC following surface staining described above and permeabilization with FoxP3 Transcription Factor Staining Buffer Set (eBioscience). For intracellular cytokine staining, lung and spleen samples were restimulated d b ex vivo with 1 μM of the I-A -restricted OVA323-339 peptide or the H-2D -restricted peptides: o NP366-374 or PA224-233 for 6 h with GolgiStop (BD Biosciences, San Jose, CA) at 37 C. For endogenous CD4 T cell cytokine detection, lung and spleen samples were restimulated ex vivo with 500 HAU/mL of live influenza virus for 18 h at 37oC with GolgiStop added for the final 6 h of incubation. After restimulation, cells were surfaced stained, fixed and permeabilized (BD Biosciences), and stained for intracellular cytokine production. Unstimulated samples (no peptide) were used as negative controls. Samples were acquired with LSR Fortessa or LSR Fortessa X20 (BD Biosciences) with FACSDiva software and analyzed with Flowjo VX (Tree Star, Inc., Ashland, OR).

2.3.6 T cell isolation and adoptive transfers

Naive CD4 T cells were purified from spleens of GITR+/+ and GITR-/- CD45.1 OT-II TCR-Tg mice with EasySep Mouse CD4 T Cell Isolation Kit (StemCell Technologies, Vancouver, BC). Naive CD8 T cells were purified from spleens of GITR+/+ and GITR-/- CD45.2 OT-I TCR-Tg mice with EasySep Mouse CD8 T Cell Isolation Kit (StemCell Technologies, Vancouver, BC). Samples of either genotype were counted by trypan blue exclusion at least 3 times and mixed in a 1:1 ratio. OT-I TCR-Tg CD8 T cell purity, OT-II TCR-Tg CD4 T cell purity, and GITR+/+:GITR-/- ratios were confirmed by flow cytometry. Cells from the mixed sample were adoptively transferred intravenously in 200 μL volume into recipient mice 1 day prior to infection. For OT-II transfers, 2 x 106 cells were transferred for day 3 and day 5 p.i. analyses, 5 x 104 cells were transferred for day 10 p.i. analyses. For OT-I transfers, 2 x 106 cells were transferred for day 5 p.i. analyses.

2.3.7 Phosphoflow assays

Lung tissue was processed as described above, except that the digestion with 2 mg mL-1 of collagenase IV was reduced to 30 minutes at 37oC in a shaker. Phosphoflow staining was 52

performed according to Protocol I described in BD Phosflow Protocols for Mouse Splenocytes. Briefly, following viability dye staining, samples were fixed with BD Phosflow Lyse/Fix Buffer for 11 minutes at 37oC, permeabilized with BD Phosflow Perm/Wash Buffer I for 30 minutes at RT, then stained with antibody mixture for 1 h at RT.

2.3.8 Mixed bone marrow chimeras

Thy1.1 or CD45.1 recipient mice were lethally irradiated with two doses of 550 rad and reconstituted with a 1:1 mixture of GITR-/- Thy1.2 CD45.2:GITR+/+ Thy1.2 CD45.1 bone marrow cells delivered intravenously for a total of 5 x 106 cells. Reconstituted chimeric mice were given water supplemented with 2 mg mL-1 neomycin sulfate (Bio-Shop, Burlington, ON, Canada) for two weeks consecutively, and were rested for a total of 90 days before chimerism in the blood was checked. Chimeric mice were infected according to the indicated schedules.

2.3.9 In vivo IFNAR-1 blockade

For in vivo IFNAR-1 blockade, mice were injected i.p. with 500 μg of α-IFNAR-1 blocking antibody or IgG1 isotype control at day -1 and day 0 (total of 1mg/mouse) prior to infection with influenza virus.

2.3.10 Data analysis and statistics

All statistical analyses were performed using GraphPad Prism 6 (San Diego, CA), with the specific test performed indicated elsewhere. n.s. not significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 were applied.

2.4 Results

2.4.1 GITR is required for optimal effector CD4 and CD8 T cell accumulation in a competitive influenza model

To determine the intrinsic effect of GITR on endogenous CD4 and CD8 T cell responses during respiratory influenza infection, we generated mixed bone marrow chimeras in which Thy1.1 CD45.2 recipients were lethally irradiated and reconstituted with bone marrow from Thy1.2 CD45.1 GITR+/+ and Thy1.2 CD45.2 GITR-/- mice mixed in a 1:1 ratio. This model allows us to assess the competition between GITR+/+ and GITR-/- cells within the same recipient/mouse (Figure 2.1A, B). After reconstitution, mice were allowed to rest for 90 days and then pre-infection ratios of donor CD45.1 and CD45.2 were determined in the blood. In another set of bone marrow chimeras, we analyzed the reconstitution ratio of GITR+/+ to GITR-/- CD4 or CD8 T cells in the spleen and mLN and found that these ratios were indistinguishable from the ratio measured in the blood (Figure 2.2A, B). Therefore, for each mouse, normalization was done using the pre-infection ratio of GITR+/+ to GITR-/- T cells measured in the blood. Following intranasal infection with influenza A/HK-X31, GITR+/+ Th1 cells in the reconstituted mice showed a competitive advantage over GITR-/- Th1 cells in the lung, mLN and spleen, with a greater effect in the lung (Figure 2.1C, D). These effects were more dramatic at the peak of the response, day 10 post-infection (p.i.), compared to day 7 p.i. GITR+/+ Tregs had a 1.5- to 2-fold advantage over GITR-/- Tregs in the three organs analyzed at day 7 p.i., with a similar result at day 10 p.i. (Figure 2.1E). In contrast, there was a minimal or no effect of GITR on CXCR5+ PD- 1+ FoxP3- T follicular helper (Tfh) cells (Figure 2.1F).

The effects of GITR on expansion of T cells specific for the two immunodominant CD8 b b epitopes of influenza virus in B6 mice, D /Nucleoprotein (NP)366-374 and D /Polymerase acidic protein (PA)224-233, were also more apparent at day 10 than at day 7 with the PA epitope more +/+ b dependent on GITR than the NP epitope. For example, the GITR D /PA224-233-specific CD8 T -/- cells exhibited a 4.2-fold advantage over their GITR counterparts, compared to a 2.7-fold for +/+ b GITR D /NP366-374-specific CD8 T cells at day 10 p.i. in the lung (Figure 2.1G-I, p = 0.021, Wilcoxon Test).

GITR+/+ IFNγ-producing CD4 T cells detected after whole influenza virus restimulation ex vivo also showed increased proportions compared to their GITR-/- counterparts (Figure 2.3A,

B) and similar results were obtained with IFNγ-producing CD8 T cells after restimulation with

NP366-374 or PA224-233 peptides (Figure 2.3C, D). GITR had a significantly greater effect on accumulation of PA- as compared to NP-specific IFN-γ-producing cells (comparison of fold effect of GITR on NP vs PA Lung day 10 p.i., p=0.029 Mann-Whitney Test).

Thus, under a competitive setting, GITR is intrinsically required for the accumulation of Th1 cells and antigen-specific CD8 T cells but minimally affects Tfh cells during influenza b infection and D /PA224-233-specific CD8 T cells exhibited a greater dependence on GITR than b D /NP366-374-specific CD8 T cells.

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)

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(

Ratio r

. 5 +/+ -/- N

4 GITR :GITR 0.0 +/+ +/+

D mLN Spleen CD45.1 (GITR ) CD45.1 (GITR ) C CD45.1 (GITR+/+)

H NP366-374 CD8 T Cell NP CD8 T Cell I PA CD8 T Cell PA CD8 T Cell 366-374 224-233 224-233 Day 7 p.i. Day 10 p.i. Day 7 p.i. Day 10 p.i.

12 n.s. 10 10 10 ****

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2 2 a 2 2

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r r 1 1 r 1

1 o

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N N 0 0 N 0 0 Lung mLN Spleen Lung mLN Spleen Lung mLN Spleen Lung mLN Spleen

Figure 2.1. GITR is required for effector CD4 and CD8 T cell accumulation in a competitive influenza model. (A) Schematic showing that lethally irradiated Thy1.1 CD45.2 recipients were reconstituted with a 1:1 mixture of GITR-/- Thy1.2 CD45.2: GITR+/+ Thy1.2 CD45.1 bone marrow cells. Chimeric mice were rested for 90 days and chimerism assessed in the blood of each mouse, before intranasal (i.n.) infection with influenza A/HK-X31 followed by analysis at day 7 or day 10 post-infection (p.i.) (B) Representative flow cytometry gating strategy for donor CD44hi T-bet+ Th1, CD25hi FoxP3+ Treg, and tetramer+ CD8 T cells. (C) Representative flow cytometry plots showing proportions of GITR+/+: GITR-/- of blood CD4 T cells before infection and of lung Th1 day 10 p.i. The normalized GITR+/+: GITR-/- ratio in Th1 (D) and Treg (E) compartments was evaluated in the lung, mediastinal lymph node (mLN), and spleen day 7 or day 10 p.i. Normalization was done by dividing the post-infection ratio in each mouse in each tissue by the pre-infection ratio of CD4 T cells in blood from the same mouse. (F) The normalized GITR+/+: GITR-/- ratio in CXCR5+ PD-1+ FoxP3- Tfh compartment was evaluated in the mLN and spleen day 7 p.i. and normalization was done as described above. Representative gating strategy for donor Tfh is shown on the left. (G) Representative flow cytometry plots showing proportions of GITR+/+: GITR-/- of blood CD8 T cells b +/+ -/- before infection and of lung D /PA224-233-specific CD8 T cells day 10 p.i. The normalized GITR : GITR ratio in b b D /NP366-374-specific CD8 T cell (H) and D /PA224-233-specific CD8 T cell (I) compartments was evaluated in the lung, mLN, and spleen day 7 or day 10 p.i. Normalization was done using pre-infection ratio of blood CD8 T cells as described for CD4 T cells above. Each symbol represents an individual mouse, with bars indicating median with interquartile range (IQR). Statistical analyses were performed using the Wilcoxon test comparing pre- and post- infection ratios. Data are pooled from 11 to 16 individual chimeric mice from at least two independent experiments.

A CD4 T cells

5 Blood mLN Spleen -

/ 4

I G

: 3

R T I n.s. n.s.

G 2

t a

R 1

0 Blood mLN Spleen Total CD4 Total CD4 Total CD4 B CD8 T cells

3 Blood mLN Spleen

R T

I 2

G :

+ n.s. /

+ n.s.

i 1

a R

0 Blood mLN Spleen Total CD8 Total CD8 Total CD8

Figure 2.2. Reconstitution ratio of GITR+/+ to GITR-/- CD4 and CD8 T cells in the blood, mLN, and spleen. Lethally irradiated Thy1.1 CD45.2 recipients were reconstituted with a 1:1 mixture of GITR-/- Thy1.2 CD45.2: GITR+/+ Thy1.2 CD45.1 bone marrow cells. After resting for 90 days, chimeric mice were euthanized to compare the reconstitution ratio of GITR+/+ to GITR-/- cells in the total CD4 T cell compartment (A) and total CD8 T cell compartment (B) in the blood, mLN, and spleen within each mouse. Each symbol represents an individual mouse. Statistical analyses were performed using the Wilcoxon test comparing the ratio in the mLN or in the spleen to the ratio in the blood. Data are pooled from 4 individual chimeric mice from one independent experiment.

A B Lung, Day 7 p.i. CD4+ IFNγ+ T Cells CD4+ IFNγ+ T Cells Day 7 p.i. Day 10 p.i.

5 5 o

Of CD4 T cell o i

i ***

a a

*** -

0.75% 7.39% 4.25% - /

/ 4 -

- 4

: + + 3 3

*** /

R T

T **

I G

G 2 2

γ a

a 1 1

I N N 0 0 Unstimulated CD45.1 GITR+/+ CD45.2 GITR-/- Lung Spleen Lung Spleen

CD8+ IFNγ+ T Cells CD8+ IFNγ+ T Cells CD8+ IFNγ+ T Cells CD8+ IFNγ+ T Cells (NP restim) (NP restim) (PA restim) C 366-374 366-374 D 224-233 (PA224-233 restim) Day 7 p.i. Day 10 p.i. Day 7 p.i. Day 10 p.i.

8 8 ** 8 8 **

o o o

*** o

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t t

a a a

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- -

- ****

/ / /

- - -

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: :

5 5 5 : 5

+ +

R R R

4 n.s. 4 4 R 4

T T

I I

3 3 3 3

d d

n.s. d

e e

l l

2 2 2 l 2

m m

r r

1 1 1 r 1

o o

N N 0 0 0 N 0 Spleen Lung Spleen Spleen Lung Spleen Figure 2.3. GITR is required for accumulation of influenza-specific IFNγ-producing CD4 and CD8 T cells. Mixed bone marrow chimeras were generated and infected as described in Figure 2.1. Freshly isolated single cell suspension from the lung and splenocytes from chimeric mice were restimulated with live influenza A/HK-X31 b virus for 18 h (A and B) or with D -restricted influenza peptides: NP366-374 or PA224-233 for 6 hr (C and D), after which intracellular cytokines were measured by flow cytometry. (A) Representative flow cytometry plots depicting CD4+IFNγ+ T cells in the GITR+/+ and GITR-/- compartments day 7 p.i. in the lung. The normalized GITR+/+: GITR-/- ratio in CD4+IFNγ+ T cell (B), CD8+IFNγ+ T cell (NP restim) (C), and CD8+IFNγ+ T cell (PA restim) (D) compartments was evaluated at day 7 or day 10 p.i. in the lung and spleen. Normalization was done as described in Figure 2.1. Each symbol represents an individual mouse, with bars indicating median with IQR. Statistical analyses were performed using the Wilcoxon test comparing pre- and post-infection ratios. Data are pooled from 10 to 16 individual chimeric mice from at least two independent experiments.

b 2.4.2 GITR signaling rescues low affinity D /NP366-374-specific CD8 T cells

During an immune response, multiple factors determine which T cells among a polyclonal repertoire expand and dominate the effector response. One such factor is access to costimulatory signals (197, 198). For example, the TNFR family member CD27 plays an important role in rescuing low affinity CD8 T cells during influenza infection (198). Previous studies have established that TCR affinity strongly correlates with the amount of tetramer binding (198, 306, 307). A higher affinity T cell will bind more tetramer than a low affinity T cell, due to the lower valency requirement for the tetramers to bind to the TCR. Accordingly, we analyzed the level of tetramer binding per cell to ask whether GITR affects the affinity of b b D /NP366-374-specific and D /PA224-233-specific CD8 T cells. At day 10 p.i. of the mixed bone marrow chimeras, we observed a significant increase in the median fluorescence intensity (MFI) -/- b of NP tetramer bound to GITR D /NP366-374-specific CD8 T cells in the lung, spleen and mLN, +/+ b when compared to GITR D /NP366-374-specific CD8 T cells (Figure 2.4A). This was not due to -/- b a higher level of TCR per cell, as we observed even lower CD3 levels on GITR D /NP366-374- specific CD8 T cells compared to their GITR+/+ counterparts in the same mouse (Figure 2.4A). b exon2Δ/Δ Similarly, D /NP366-374-specific CD8 T cells from the lung and mLN of GITRL mice bound more tetramer than T cells from GITRL+/+ mice, whereas there was no change in the CD3 level (Figure 2.4B). A similar result was seen in the lung with mice lacking exon 2 of GITRL only in myeloid cells (LysM+ cells) compared to their Cre-negative littermates (Figure 2.4C). GITRLexon2Δ/Δ mice lack an 11 amino acid sequence encoded by exon 2, resulting in a thermally unstable protein and a partial defect in activation of GITR signaling (258).

b In contrast to the results with the D /NP366-374-specific T cells, there was only a minimal, -/- b albeit statistically significant, increase in the amount of PA tetramer bound to GITR D /PA224- +/+ 233-specific CD8 T cells when compared to their GITR counterparts (Figure 2.4D). Moreover, b there was no difference in the amount of PA tetramer bound to D /PA224-233-specific CD8 T cells between GITRLexon2Δ/Δ and GITRL+/+ mice (Figure 2.4E) or LysM-Cre+/+ and LysM-Cre-/- GITRLexon2fl/fl littermate mice (Figure 2.4F).

Together, these results suggest that GITR allows the accumulation of low-affinity b D /NP366-374-specific effector CD8 T cells during influenza infection and this is largely due to + b GITRL on LysM myeloid cells. In contrast, the D /PA224-233-specific T cells, despite more

costimulation-dependence, show only a minimal difference in tetramer binding on GITR+/+ and GITR-/- T cells, suggesting similar affinity. This difference between the NP and PA epitope- specific T cells was most evident in the lung (Figure 2.4A, D).

A B C

NP366-374 CD8, Day 10 p.i. NP366-374 CD8, Day 9 p.i. NP366-374 CD8, Day 9 p.i.

) )

8000 ) I

I 4000 8000

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p= 0.0909

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GITR+/+ GITRL+/+

x x a

a -/- exon2Δ/Δ

x M

M GITR GITRL -/-

a LysM-Cre %

% exon2fl/fl M

GITRL %

LysM-Cre+/+ exon2fl/fl NP Tetramer NP Tetramer GITRL NP Tetramer D E F

PA224-233 CD8, Day 10 p.i. PA224-233 CD8, Day 9 p.i. PA224-233 CD8, Day 9 p.i.

)

) )

I 4000 5000 I 8000

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n.s. F n.s.

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) )

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C C 0 0 0 Lung mLN Spleen Lung mLN Spleen Lung mLN Spleen

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GITR+/+ GITRL+/+

x x

a a

-/- exon2Δ/Δ x -/-

a M

M GITRL LysM-Cre

GITR

M exon2fl/fl

% GITRL %

LysM-Cre+/+ GITRLexon2fl/fl PA Tetramer PA Tetramer PA Tetramer

b Figure 2.4. GITR rescues low affinity D /NP366-374-specific CD8 T cells. (A and D) GITR+/+:GITR-/- mixed bone marrow chimeras were generated and infected as described in Figure 2.1. (B and E) WT C57BL/6 (GITRL+/+) mice were co-housed with GITRL exon2Δ/Δ mice from weaning and infected intranasally with influenza A/HK-X31 and analyses were done at day 9 p.i. (C and F) LysM-Cre+/+ and LysM-Cre-/- GITRLexon2fl/fl littermates were infected intranasally with influenza A/HK-X31 and analyzed at day 9 p.i. The MFI of b + +/+ D /NP366-374 tetramer binding or CD3 binding to tetramer CD8 T cells in the lung, mLN, and spleen of GITR and GITR-/- donor origin was determined on day 10 (A), of GITRL+/+ and GITRL exon2Δ/Δ mice on day 9 (B), and of +/+ -/- exon2fl/fl b LysM-Cre and LysM-Cre GITRL mice on day 9 (C). MFI of D /PA224-233 tetramer binding or CD3 binding of tetramer+ CD8 T cells in the lung, mLN, and spleen of GITR+/+ and GITR-/- donor cells was determined on day 10 (D), of GITRL+/+ and GITRL exon2Δ/Δ mice on day 9 (E), and of LysM-Cre+/+ and LysM-Cre-/- GITRLexon2fl/fl mice on day 9 (F). Representative histogram plots are shown below the summary plots for each set of data. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Statistical analyses were performed using the Wilcoxon test (A and D) or the Mann-Whitney test (B, C, E, and F). Data from (A and D) are pooled from 12 individual chimeric mice with two independent experiments. Data from (B and E) are pooled from 3 independent experiments with a total of 6 to 7 mice per group. Data from (C and F) are pooled from 2 independent experiments with a total of 8 mice per group.

2.4.3 GITR is required for optimal lung CD4 and CD8 Trm formation following influenza infection

CD4 and CD8 Trm, subsets of highly protective non-circulating memory T cells that reside in the peripheral tissue such as the lung tissue, are crucial for protective immunity to influenza infection (113, 150, 308, 309). A recent study showed that the TNFR superfamily member 4-1BB plays a critical role in the establishment of CD8 Trm in the lung after influenza infection (208). To investigate, whether GITR is also required for the optimal establishment of lung CD4 and CD8 Trm following influenza infection, we again used competitive mixed bone marrow chimeras where GITR+/+ and GITR-/- cells compete within the same mouse (Figure 2.5A). Prior to harvest, we used intravascular infusion of fluorescently labeled anti-Thy1.2 to distinguish lung vascular (LV) exposed cells from cells located within the lung tissue (LT) (115, 310). CD4 Trm were defined based on CD69 and CD11a co-expression and CD8 Trm based on CD69 and CD103 co-expression (Figure 2.5B). At day 30 p.i., GITR+/+ LT CD4 Trm had a 5- fold advantage over GITR-/- LT CD4 Trm, while GITR+/+ and GITR-/- LV CD4 T cells competed equally well (Figure 2.5C, D). Similarly, in the lung tissue, the total GITR+/+ CD8 Trm, as well as the tetramer positive subpopulations had a 3- to 3.5-fold advantage over their GITR-/- counterparts, whereas the CD8 T cells in the vasculature showed no such difference (Figure 2.5C, E). GITR did not influence the number of splenic CD4 or CD8 Trm (Figure 2.6A-C). In fact, there were very few NP-specific CD8 Trm detectable in the spleen (Figure 2.6D). Moreover, GITR had a less dramatic effect on splenic CD4 and CD8 central memory T cells (Tcm) and effector memory T cells (Tem) than on the lung Trm (Figure 2.5F, G). In sum, GITR is required for the optimal formation of CD4 and CD8 Trm in the lung tissue after influenza infection.

A Transfer 1:1 B Donor CD4/CD8 T cell GITR+/+:GITR-/- 2 × 550 cGy Infection

v Lung Vasculature (LV)

90 days i

2 (reconstitution) .

CD45.1 1

+/+ Day 30 p.i. y GITR h Lung Tissue (LT)

Analysis T Irradiated CD45.1 Hosts In Vivo Ab Labeling FSC-A

CD45.2 T L g u -/- n e is n GITR u u s g L s u is e T C Pre-infection Day 30 p.i. + hi + + Of Blood CD4 T cell Of LV CD4 T cell Of LT CD4 Trm CD69 CD11a CD4 Trm CD69 CD103 CD8 Trm

) 12.1%

- 40.1% /

- 41.9%

a 3

1 0

1 1

D D

(

C C

. 5

4 58.0% 56.6% 86.7% D

C CD69 CD69

CD45.1 (GITR+/+)

)

- / Pre-infection Day 30 p.i. - +

R Tetramer Trm T

Of Blood CD8 T cell Of LV CD8 T cell Of LT PA CD8 Trm I

(

)

- 38.3% 14.1%

2 -

39.1% .

( T

+/+ 2

. CD45.1 (GITR ) 5 4 57.8% 84.1% D 60.8% FSC-A C Ratio GITR+/+:GITR-/- CD45.1 (GITR+/+)

D Day 30 p.i. E Day 30 p.i.

8 *** 30 *

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2.5 18 n.s.

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o N N 0.0 0 Spleen Spleen Spleen Spleen Spleen Spleen CD4 Tcm CD4 Tem NP CD8 PA CD8 CD8 Tcm CD8 Tem

Figure 2.5. GITR is required for optimal lung CD4 and CD8 Trm formation after influenza infection. (A) Schematic showing that lethally irradiated CD45.1 recipients were reconstituted with a 1:1 mixture of GITR-/- CD45.2: GITR+/+ CD45.1 bone marrow cells. Chimeric mice were rested for 90 days and chimerism checked in the blood before intranasal infection with influenza A/HK-X31 and analyses were done at day 30 p.i. (B) Representative flow cytometry gating strategy for donor CD69+ CD11ahi CD4 Trm, polyclonal CD69+ CD103+ CD8 Trm, and tetramer+ CD8 Trm from the lung parenchyma. (C) Top: Representative flow cytometry plots showing proportions of GITR+/+:GITR-/- of CD4 T cells in blood before infection, of lung vascular (LV) CD4 T cell at day 30 p.i., and of lung tissue (LT) CD4 Trm at day 30 p.i. Bottom: Representative flow cytometry plots showing proportions of +/+ -/- b GITR :GITR of CD8 T cells before infection, of LV CD8 T cell day 30 p.i., and of LT D /PA224-233-specific CD8 Trm day 30 p.i. (D) The normalized GITR+/+:GITR-/- ratio in the LV CD4 T cell and LT CD4 Trm compartments shown for individual mice. (E) The normalized GITR+/+: GITR-/- ratio in the LV CD8 T cell, LT polyclonal CD8 b b Trm, LT D /NP366-374-specific CD8 Trm, and LT D /PA224-233-specific CD8 Trm compartments is shown for each mouse. (F) The normalized GITR+/+: GITR-/- ratio in the CD44hi CD62L+ CD4 Tcm and CD44hi CD62L- CD4 Tem +/+ -/- b compartments was evaluated in the spleen. (G) The normalized GITR : GITR ratio in the D /NP366-374-specific b hi + hi - CD8 T cell, D /PA224-233-specific CD8 T cell, CD44 CD62L CD8 Tcm and CD44 CD62L CD8 Tem compartments was evaluated in the spleen. Normalization was done as described in Figure 2.1. Each symbol represents an individual mouse, with bars indicating median with IQR. Statistical analyses were performed using the Wilcoxon test comparing pre- and post-infection ratios. Data are pooled from 2 independent experiments with 11 individual chimeric mice.

A B Day 30 p.i. 3

Transfer 1:1 o i +/+ -/- 2 × 550 cGy t

GITR :GITR Infection a

- /

90 days -

R T Thy1.2 CD45.1 (reconstitution) I 2

Day 30 p.i. G

+/+ : +

GITR /

Analysis +

Irradiated R T

I n.s.

CD45.1 Hosts In Vivo Ab Labeling

d 1 e

Thy1.2 CD45.2 z i -/- l

GITR a

r o

N 0 Spleen CD4 Trm C D Spleen, day 30 p.i. Day 30 p.i. hi - +

3 Gated: CD44 CD62L CD69 CD8 Trm

- /

- n.s.

R T

I 2

6 R

d 1

r o

N 0 Spleen CD8 Trm FSC-A

Figure 2.6. GITR is not required for the accumulation of splenic CD4 and CD8 Trm. (A) Lethally irradiated Thy1.1 CD45.2 recipients were reconstituted with a 1:1 mixture of GITR-/- Thy1.2 CD45.2: GITR+/+ Thy1.2 CD45.1 bone marrow cells. Chimeric mice were rested for 90 days and chimerism checked in the blood before intranasal infection with influenza A/HK-X31 and analyses were done at day 30 p.i. with intravascular staining with anti-Thy1.2 FITC. (B) After gating on the donor CD4 T cells negative for intravascular Thy1.2 staining, the normalized GITR+/+: GITR-/- ratio in the CD44hi CD62L- CD69+ CD4 Trm compartment was evaluated in the spleen. (C) After gating on the donor CD8 T cells negative for intravascular Thy1.2 staining, the normalized GITR+/+: GITR-/- ratio in the CD44hi CD62L- CD69+ CD8 Trm compartment was evaluated in the spleen. (D) b hi - Representative flow cytometry plot showing D /NP366-374 tetramer staining after gating on donor CD44 CD62L CD69+ CD8 Trm in the spleen. Normalization was done as described in Figure 2.1. Each symbol represents an individual mouse, with bars indicating median with IQR. Statistical analyses were performed using the Wilcoxon test comparing pre- and post-infection ratios. Data are pooled from 1 experiment with 4 individual chimeric mice.

2.4.4 Preferential effect of GITR on CD8 over CD4 T cell responses in a non-competitive influenza model

To investigate whether GITR was also required for T cell responses in a non-competitive setting, we infected GITR+/+ and GITR-/- littermate mice with influenza A/HK-X31 and assessed CD4 and CD8 T cell responses. At day 9 p.i., there was a significant decrease in the total number b b of CD8 T cells in the lung, as well as D /NP366-374-specific and D /PA224-233-specific CD8 T cells in GITR-/- mice when compared to their GITR+/+ littermates (Figure 2.7A), whereas there was no deficit in GITR-/- T cell responses in the mLN and spleen (Figure 2.8A). At day 30 p.i. with influenza, there was a significant reduction in the total number of polyclonal CD69+ CD103+ b -/- CD8 Trm in the lung tissue, as well as D /PA224-233-specific CD8 Trm in GITR mice when compared to GITR+/+ littermates (Figure 2.7B). GITR-/- mice exhibited a trend towards fewer b +/+ D /NP366-374-specific CD8 Trm in the lung parenchyma in comparison to GITR littermates (Figure 2.7B). In contrast to the results from the mixed bone marrow chimeras, there was no difference in the total number of CD4 T cells, Th1, and Treg in the lung, mLN, and spleen between GITR+/+ and GITR-/- mice (Figure 2.7C and 2.8B). Similarly, at day 30 p.i., there was no significant difference in the number of lung parenchymal CD69+ CD11ahi CD4 Trm between GITR+/+ and GITR-/- mice (Figure 2.7D). Again, the Tfh response in the mLN and spleen of GITR-/- mice showed no detectable differences when compared to GITR+/+ littermate controls (Figure 2.7E). These data show that while GITR is required for optimal lung CD8 effector and resident memory T cell responses against influenza under both competitive and non-competitive conditions, the effects of GITR on CD4 T cell responses are evident only under conditions of competition.

A Lung CD8 T cell, day 9 p.i.

6 5×10 1×106 * 7×105 *

*** 5 s

s 6×10

l l

l 5 e e 5×10

4×106 8×105

l 5

l 8 8 4×10

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2×10 6 5 2

6 4×10

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2×10

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/ /

b 5 6 b

1×10 2×10 5 D

D 1×10

# # 0 0 0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

B Lung CD8 Trm, day 30 p.i. n.s. ***

1.2×105 *** 5×103 1.5×104

m m

T 3

r r

4×10 T

4 T

9.0×10

8 8

D 4

D D C 1.0×10

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4 /

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# 0.0 0 0.0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

C Lung CD4 T cell, day 9 p.i. n.s. 2.5×106 8×105 2.0×105 n.s.

n.s.

6 g 1

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h 6×105 1.5×105

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6 e

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# D

C 5 4

2×10 5.0×10 C

5.0×10 # #

0.0 0 0.0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

D Lung CD4 Trm, day 30 p.i. E mLN Tfh, day 9 p.i. Spleen Tfh, day 9 p.i.

5 n.s. 6 6

3.5×10 1×10 n.s. 3×10 h

5 h n.s.

f f

m 3.0×10

- - T 1.5×10

5 3

3 8×10

D x

x 6

o o

2×10

F F

h 6×10 + 5 +

a 1.0×10

D P

P 5

4×10

+ +

5 5 + 1×10

9 4 R

5.0×10 R

6 C

C 5 D

2×10 X

C C

# # 0.0 0 0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

Figure 2.7. Preferential effect of GITR on CD8 over CD4 T cells in a non-competitive influenza model. GITR+/+ and GITR-/- littermates were infected i.n. with influenza A/HK-X31 and sacrificed at day 9 p.i. (A, C, and b b E) or at day 30 p.i. (B and D). (A) Total number of CD8 T cells, D /NP366-374-specific, and D /PA224-233-specific CD8 + + b b T cell in the lung. (B) Total number of polyclonal CD69 CD103 CD8 Trm, D /NP366-374-specific, and D /PA224-233- specific CD69+ CD103+ CD8 Trm in the lung parenchyma. (C) Total number of CD4 T cells, CD44hi T-bet+ Th1, and CD25hi FoxP3+ Treg in the lung. (D) Total number of CD69+ CD11ahi CD4 Trm in the lung parenchyma. (E) Total number of CXCR5+ PD-1+ FoxP3- Tfh in the mLN and spleen. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Statistical analyses were performed using the Mann-Whitney test. Data from (A) are pooled from 4 independent experiments with a total of 13 to 14 mice per group. Data from (C and E) are pooled from 3 independent experiments with a total of 6 to 7 mice per group. Data from (B and D) are pooled from 3 independent experiments with a total of 10 to 11 mice per group. 69

A mLN CD8 T cell, day 9 p.i.

6 6×10 n.s. 1×105 6×104 n.s.

n.s.

s s

l l l

6 l 4 e 5×10 e 5×10

8×104

C C

T T

l 6

l 4 8

4×10 8 4×10

e D D 4

C 6×10

C C

T 6

4 3

3×10 4 3

7 3×10

3 2

- -

D 4

6 4 2

6 4×10

C 2 6 3

2×10 2×104

N P

/ / b b 4

6 2×10 4 D

1×10 D 1×10

# # 0 0 0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

Spleen CD8 T cell, day 9 p.i.

4×107 6 n.s. 6 n.s.

n.s. 3×10 1.5×10

s s

e C

7 C

3×10

l 6 6 8

2×10 8 1.0×10

T 7

4 3

2×10

C 3 6 2 5

1×10 5.0×10

P #

7 A

N P /

1×10 /

# # 0 0 0.0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/- B mLN CD4 T cell, day 9 p.i.

8×106 1×106 8×105 n.s. n.s.

n.s.

5 g 1 8×10 e

6 r

6×10 h 6×105

3 e

e 5

6×10 P

- T

6 o 5

4×10 4×10

h i

5 h D

4 4×10

# D

6 C 5

2×10 2×10

5 C

# 2×10 #

0 0 0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

Spleen CD4 T cell, day 9 p.i.

3.0×107 5×106 5×106

n.s. n.s. n.s.

6 g 6 1

4×10 e 4×10 r

7 h

s 2.5×10

3 e

e 6 6

3×10 P 3×10

F 4

7 h

2.0×10 i D 6 h 6

4 2×10 2×10

6 C 6

# 1×10 1×10 1.5×107 # 1×107 0 0 0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

Figure 2.8. Influenza-specific T cell responses in secondary lymphoid organs in GITR-deficient mice. GITR+/+ and GITR-/- littermate mice were infected i.n. with influenza A/HK-X31 and sacrificed at day 9 p.i. (A) b b Total number of CD8 T cells, D /NP366-374-specific, and D /PA224-233-specific CD8 T cell in the mLN and spleen (B) Total number of CD4 T cells, CD44hi T-bet+ Th1, and CD25hi FoxP3+ Treg in the mLN and spleen. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Statistical analyses were performed using the Mann-Whitney test. Data from (A) are pooled from 4 independent experiments with a total of 13 to 14 mice per group. Data from (B) are pooled from 3 independent experiments with a total of 6 to 7 mice per group.

2.4.5 GITR is transiently upregulated on Th1, Treg, and influenza-specific CD8 T cells in the lung and mLN

To gain insight into where GITR costimulation takes place, we examined the expression of GITR on T cells isolated from the mLN and the lung during influenza infection. At day 3 p.i. in the mLN, activated effector CD4 T cells (CD44hi CD69+ FoxP3-) and effector CD8 T cells (CD44hi CD69+) upregulated GITR relative to naïve controls (Figure 2.9A, B). Consistent with the published literature (219, 220), Treg had the highest GITR expression in the mLN and the lung (Figure 2.9A, C). We observed GITR expression on the total lung CD4 T cell population as well as the Th1 population at day 5 p.i., with peak expression at day 7 p.i., declining again by b b day 9 p.i. (Figure 2.9C). D /NP366-374-specific and D /PA224-233-specific CD8 T cells showed a high of GITR expression at day 5 p.i. in the lung, declining by day 7, and returning to baseline by day 9 (Figure 2.9D). Thus, GITR is transiently upregulated after T cell activation and is detected in both the mLN and the lung, with peak expression at day 5-7 p.i. in the lung.

A mLN CD4 T cell, day 3 p.i. B mLN CD8 T cell, day 3 p.i.

GITR dMFI GITR dMFI

FMO FMO GITR-/- 13 GITR-/- -3 Naive CD4 1843 Naive CD8 1337 Total CD4 T cell 2224

Total CD8 T cell

CD44hi CD69+ FoxP3- 1470 x

x 4474

a a

hi +

M M

CD44 CD69

CD25hi FoxP3+ Treg 12204 1763

% %

GITR GITR C D Lung day 7 p.i. Lung day 5 p.i.

Lung CD4 T Cell Lung CD8 T Cell

45000 Total CD4 T Cell 6000 Total CD8 T Cell CD44hi T-bet+ Th1 NP CD8 40000 366-374 hi + CD25 FoxP3 Treg FMO 5000 PA224-233 CD8 FMO

35000

)

I I F

F -/- -/- M

M 30000 GITR 4000 GITR

( (

n n

o o i

i 25000 s

s Naive CD4 Naive CD8 s

s 3000

e r

r 20000

p p

x x

Total CD4 T cell Total CD8 T cell R

15000 R 2000

I G

10000 hi + G x

x CD44 T-bet Th1 NP366-374 CD8 a

a 1000 M

5000 M

hi +

CD25 FoxP3 Treg PA224-233 CD8

% % -1 1 3 5 7 9 -1 1 3 5 7 9 Days p.i. Days p.i. GITR GITR Figure 2.9. GITR upregulation on activated CD4 and CD8 T cells in the mLN and lung following influenza infection. WT C57BL/6 mice were infected i.n. with influenza A/HK-X31 and expression level of GITR was determined in the mLN (A and B) and the lung (C and D). (A) GITR expression on total CD4 T cell (green), CD44hi CD69+ FoxP3- CD4 T cell (blue), and CD25hi FoxP3+ Treg (red) in the mLN at day 3 p.i. (B) GITR expression on total CD8 T cell (green), CD44hi CD69+ CD8 T cell (blue) in the mLN at day 3 p.i. (C) Kinetics of GITR expression on total CD4 T cell (green), CD44hi T-bet+ Th1 (blue), and CD25hi FoxP3+ Treg (red) in the lung with representative staining at day b 7 p.i. shown on the right. (D) Kinetics of GITR expression on total CD8 T cell (green), D /NP366-374-specific (blue), b and D /PA224-233-specific CD8 T cell (red) with representative staining at day 5 p.i. shown on the right. Dashed lines represent baseline/naïve expression level of GITR. dMFI refers to the MFI for the specific antibody stain minus the fluorescence minus one (FMO) control. Data points indicate mean ± SEM. Data from (A and B) are representative of two independent experiments, each with 3 mice. Data from (C and D) are pooled from two independent experiments with a total of six mice per time point.

2.4.6 GITRL expression is highest on inflammatory APC subsets after influenza infection

We next examined GITRL expression on different immune cell subsets in the lung and mLN from pre-infection (day -1) through the first 8 days of influenza A/HK-X31 infection. A 12-parameter flow cytometry panel was used to delineate different immune cell subsets in the lung and mLN, with emphasis on different APC subsets (Figure 2.10A, B). Inflammatory dendritic cells (InfDC) and inflammatory macrophages (InfMF) were distinguished from other immune cell subsets using the high affinity IgE receptor, FcεRI (Figure 2.10A, B) (47, 52). Consistent with the published literature (47, 311), InfDC and InfMF expressed high levels of the chemokine receptor CCR2 among different immune cell subsets in the lung and mLN (Figure 2.11A, B). We observed highest GITRL expression on InfDC and InfMF with peak expression day 4 to day 5 p.i. (Figure 2.12A). Alveolar macrophages (AlvMF, CD11c+Siglec-F+) also appeared to stain with GITRL. However, examination of the flow histograms showed that the AlvMF have a high background fluorescence and did not show a major shift in stain over fluorescence minus one (FMO) control and the apparent change in MFI is not reflective of significant GITRL expression (Figure 2.12A, bottom row). In the mLN, we observed a similar pattern of GITRL expression with InfDC and InfMF exhibiting highest GITRL expression among different immune cell subsets, albeit with slightly earlier kinetics when compared to the lung (Figure 2.12B). Since InfDC and InfMF exhibited the highest GITRL expression among different APC subsets in the lung, we tracked the number of InfDC and InfMF in the lung over the course of influenza infection. We observed the highest number of InfDC and InfMF in the lung at day 5 p.i. and the number declined by day 8 p.i. (Figure 2.12C). Next, we asked whether a stronger influenza infection model (influenza A/PR8) would induce higher expression of GITRL. However, we observed a similar pattern of GITRL expression with marginally lower GITRL expression during PR8 infection when compared to X31 infection. Of note, the number of InfDC and InfMF was lower during the more severe PR8 infection, but the inflammatory APC persisted through to day 8 p.i. (Figure 2.13A-C). B220+ PDCA-1+ plasmacytoid dendritic cells (pDC) were also examined in a separate flow panel and found to express minimal levels of GITRL in the lung and mLN (Figure 2.13D, E). These results show that GITRL expression is highest on InfAPC subsets in both the mLN and lung during influenza infection.

A Lung, day 3 p.i. CD103+ DC Naive

InfDC

I I

R R

ε ε

c c

F F

C M CD11b+ DC Gated: live CD11c CD11b FSC-A FSC-A CD45.2+ CD3- CD19- B220- population Naive

InfMF

I I

R R

ε ε

c c

F F C IntMF CD11c FSC-A FSC-A FSC-A

B mLN, day 3 p.i. + CD8α DC InfDC

Naive

I I

R R

ε ε

c c

F F

C M

Gated: live CD11c CD103 + CD11b CD11b CD103 DC CD11b+ DC CD3- CD19- B220- population

InfMF Medul MF

0 0

8 8

/ /

4 4

F F

F C

CD11c FSC-A FSC-A CD169

Naive Subcap MF

c F

FSC-A Figure 2.10. Gating strategy for APC subsets in the lung and mLN. (A) Gating strategy to identify different APC subsets in the lung at day 3 p.i. with influenza A/PR8. A sequential gating strategy was first used to gate on singlet, live, CD45.2+CD3-CD19- population. Then, lung alveolar macrophages (CD11c+Siglec-F+) and lung eosinophils (CD11b+Siglec-F+) were gated out before different lung dendritic cell and macrophage subsets were sequentially identified from the B220- population. (B) Gating strategy to identify different cell subsets in the mLN at day 3 p.i. with influenza A/PR8. A sequential gating strategy was first used to gate on singlet, live, CD3-CD19- population. Then, different mLN dendritic cell and macrophage subsets were sequentially identified from the B220- population. Identical lung and mLN APC gating strategies were used in influenza A/HK-X31 infection.

A Lung

Eosinophil CD103+ DC CD11b+ DC IntMF AlvMF InfMF InfDC

FMO FMO FMO FMO FMO FMO FMO

% CCR2

B mLN

CD8α+ DC CD103+ DC CD11b+ DC Subcap MF Medul MF InfMF InfDC

FMO FMO FMO FMO FMO FMO FMO

% CCR2

Figure 2.11. CCR2 expression in the lung and mLN during influenza infection. WT C57BL/6 mice were infected i.n. with influenza A/PR8 and CCR2 expression determined in the lung and mLN at day 5 p.i. (A) CCR2 expression on different immunce cell subsets from the lung defined in Figure 2.10A at day 5 p.i. (B) CCR2 expression on different immune cell subsets from the mLN defined in Figure 2.10B at day 5 p.i. Grey filled histograms represent FMO and open histograms represent CCR2 staining. Data from (A and B) are representative of a single experiment with 3 mice.

A Lung, X31 Eosinophil CD103+ DC CD11b+ DC IntMF AlvMF InfMF InfDC

400 400 400 400 400 400 400

)

M d

( 300

300 300 300 300 300 300

i s

s 200 200 200

e 200 200 200 200

p x

E 100 100 100

L 100 100 100 100

R T I 0 G 0 0 0 0 0 0 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. Eosinophil CD103+ DC CD11b+ DC IntMF AlvMF InfMF InfDC

FMO FMO FMO FMO FMO FMO FMO

% GITRL

B mLN, X31 CD8α+ DC CD103+ DC CD11b+ DC Subcap MF Medul MF InfMF InfDC

300 300 300 300 300 300 300

)

I F

M 250 250 250 250 250 250 250

(

n 200 200 200

o 200 200 200 200

i s

s 150 150 150

e 150 150 150 150 r

p 100

x 100 100

100 100 100 100

L 50 50 50

R 50 50 50 50 T I 0

G 0 0 0 0 0 0 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. CD8α+ DC CD103+ DC CD11b+ DC Subcap MF Medul MF InfMF InfDC

FMO FMO FMO FMO FMO FMO FMO

% GITRL

C Lung, X31

InfMF InfDC

40 15

)

0 1

( 30

n 10

u o

C 20

l e

C 5

a 10

o T 0 0 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 Days p.i. Days p.i. Figure 2.12. GITRL expression is highest on inflammatory APCs after influenza X31 infection. WT C57BL/6 mice were infected i.n. with influenza A/HK-X31 and GITRL expression kinetics determined in the lung and mLN. (A) GITRL expression kinetics on different immune cell subsets from the lung defined in Figure 2.10A between day -1 and 8 p.i. with representative flow staining from day 5 p.i. shown below the kinetics plot. Grey filled histograms represent FMO and open histograms the GITRL staining. (B) GITRL expression kinetics on different cell subsets from the mLN defined in Figure 2.10B between day -1 and 8 p.i. with representative staining shown at day 2 p.i., with GITRL staining shown as open histograms and FMO as grey filled histograms. Horizontal dashed lines in the kinetics plots in A and B indicate the baseline/naïve expression level of GITRL. (C) Total number of InfMF and InfDC in the lung between day 2 and 8 p.i. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Data points indicate mean ± SEM. Data in (A-C) are pooled from two independent experiments with a total of 6 to 12 mice per time point. 76

A Lung, PR8 Eosinophil CD103+ DC CD11b+ DC IntMF AlvMF InfMF InfDC

400 400 400 400 400 400 400

)

M d

( 300

300 300 300 300 300 300

i s

s 200 200 200

e 200 200 200 200

p x

E 100 100 100

L 100 100 100 100

FMO FMO FMO FMO FMO FMO FMO

% GITRL

B mLN, PR8 CD8α+ DC CD103+ DC CD11b+ DC Subcap MF Medul MF InfMF InfDC

250 250 250 250 250 250 250

)

F M

d 200 200 200 200 200 200 200

(

n o i 150 150 150

s 150 150 150 150

e r

p 100 100 100 100 100 100 100

L 50 50 50 50 50

R 50 50

T I G 0 0 0 0 0 0 0 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. Days p.i. CD8α+ DC CD103+ DC CD11b+ DC Subcap MF Medul MF InfMF InfDC

FMO FMO FMO FMO FMO FMO FMO

% GITRL

C Lung, PR8 D Lung, day 3 p.i. E mLN, day 3 p.i.

InfMF InfDC + - - - -

15 5 Gated: live CD45.2 CD3 CD19 population Gated: live CD3 CD19 population ) 4 pDC pDC

0 4

(

t FMO FMO

n 10

u 3 pDC

o pDC

l l

e 2

C -

5 -

t C

1 C

P % 0 0 % -1 0 1 2 3 4 5 6 7 8 -1 0 1 2 3 4 5 6 7 8 B220 GITRL B220 GITRL Days p.i. Days p.i. Figure 2.13. GITRL expression is highest on inflammatory APCs after influenza PR8 infection. Male WT C57BL/6 infected i.n. with influenza A/PR8 and GITRL expression determined in the lung and mLN. (A) GITRL expression kinetics on lung cell subsets defined in Figure 2.10A between day -1 and 8 p.i. with representative staining from day 5 p.i. shown below the kinetics plot. (B) GITRL expression kinetics on mLN cell subsets defined in Figure 2.10B between day -1 and 8 p.i. with representative staining from day 2 p.i. shown below the kinetics plot. Horizontal dashed lines in the kinetics plots in A and B indicate the baseline/naïve GITRL expression level. (C) Total number of Lung InfMF and InfDC between day 2 and 8 p.i. (D) At day 3 p.i., GITRL expression was analyzed on lung B220+ PDCA-1+ pDC. (E) At day 3 p.i., GITRL expression was analyzed on mLN B220+ PDCA-1+ pDC. Grey filled histograms represent FMO and open histograms the GITRL staining. dMFI refers to the MFI for the specific antibody stain minus FMO. Data points indicate mean ± SEM. Data in (A-C) are pooled from two independent experiments with a total of 5 to 8 mice per time point. Data in (D and E) are representative of three mice in a single experiment. 77

2.4.7 GITRL expression in the lung is partially mediated by IFN-I during influenza infection

Recent work by Chang et al. showed that GITRL expression during chronic LCMV Clone 13 infection is induced on APC by type I interferon (IFN-I) (258). To determine whether this is also the case in the lung during respiratory influenza virus infection, we analyzed InfAPC and conventional DC (cDC) using the markers FcεRI, CD64, Ly6C, CD11c, and MHC II (Figure 2.14A). WT mice were treated with two doses of isotype control or blocking antibodies to the interferon receptor subunit 1 (IFNAR1), administered one day before and on the day of infection, and GITRL expression was assessed in the lung at day 3 p.i. The expression of GITRL during influenza infection was substantially blocked on InfAPC and cDC by anti-IFNAR1 treatment (Figure 2.14B). These results show that GITRL expression during influenza infection in the lung is mediated in part by IFN-I.

A B Lung, day 3 p.i. Lung, day 3 p.i. 800 ) ** I **

Gated: live CD3- CD19- B220- population F

d (

600

s s

e 400

r I

p **

L 200

T I G 0 cDC InfAPC CD64 IgG Control α-IFNAR InfAPC

0 FMO

2 2

B InfAPC

I IgG Control

9 1

C cDC α-IFNAR

M /

C M

CD11c Ly6C % GITRL

Figure 2.14. Lung GITRL expression is mediated in part by IFN-I during influenza infection. (A and B) WT C57BL/6 mice were injected i.p. with IFNAR blocking or isotype control antibodies at day -1 and 0 prior to influenza A/PR8 infection. Flow cytometry gating strategy is shown in (A) and GITRL expression on InfAPC and cDC in the lung at day 3 p.i. summarized in (B), with representative flow staining shown below. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Statistical analyses were performed using the Mann-Whitney test. Data from (B) are pooled from two independent experiments with a total of 6 mice per group.

2.4.8 GITR-dependent signals in T cells take place in the mLN and then in the lung tissue

The finding that GITR and GITRL were detected early in the mLN combined with the finding that GITR on T cells and GITRL on lung inflammatory APC are present as the T cells start to accumulate in the lung, suggests that GITR costimulation may first take place in the mLN and then again in the lung tissue. To more directly test this hypothesis, we analyzed GITR- dependent signaling in T cells from mLN and lung directly ex vivo. Previous studies have shown that GITR signaling in CD4 T cells specifically induces phosphorylated-ribosomal protein S6 (pS6) (244, 258), a downstream target of mTOR that is important in regulating cell size and glucose homeostasis (245). To analyze GITR-dependent induction of pS6 in the mLN and lung, we adoptively transferred a 1:1 mixture of GITR+/+ and GITR-/- CD45.1 OT-II cells from littermate mice into WT CD45.2 C57BL/6 mice followed by intranasal infection with influenza PR8 carrying the OT-II epitope (PR8-OVAII) (Figure 2.15A). GITR expression on donor CD45.1 OT-II cells was used to distinguish GITR+/+ and GITR-/- T cells pre- and post-infection (Figure 2.15B). By co-transferring GITR+/+ and GITR-/- OT-II into the same recipient, they are exposed to the same level of the virus, inflammation, and soluble mediators, therefore signals directly downstream of GITR can be determined by comparing GITR+/+ and GITR-/- OT-II T cells within each mouse. At day 3 p.i., GITR+/+ OT-II cells isolated from the mLN had a higher level of pS6 than GITR-/- OT-II T cells from the same mice (Figure 2.15C). Thus, GITR signaling can occur in the mLN by day 3 p.i.

We next examined OT-II responses in the lung after influenza infection. At day 5 p.i., there was an equal frequency and total number of GITR+/+ and GITR-/- OT-II in the lung (Figure 2.16A), suggesting that there is no defect in GITR-/- T cells reaching the lung post-priming. However, the GITR+/+ OT-II cells isolated from the lung had a higher level of pS6 than GITR-/- counterparts from the same mice (Figure 2.15D). Similar experiments done with Thy1.2 infusion showed that in the lung parenchyma GITR+/+ OT-II cells had a higher level of pS6 than GITR-/- OT-II cells (Figure 2.17A, B). At day 10 p.i., there was a lower frequency and total number of GITR-/- OT-II cells in the lung, mLN, and spleen than their GITR+/+ counterparts, with a greater effect in the lung, similar to what we observed in the bone marrow chimera model (Figure 2.15E, F). There was also a lower frequency and total number of IL-2 producing GITR-/- OT-II in the lung but not in the spleen (Figure 2.16B-D). At day 10 p.i., there was no difference

in the IL-2 production per cell in the lung and there is a marginal but statistically significant decrease in the IL-2 production per cell in GITR-/- OT-II in the spleen (Figure 2.16E). There was also no difference in the frequency and total number of IFNγ producing OT-II between GITR+/+ and GITR-/- donor cells (Figure 2.16B, F). Thus, the decreased pS6 signal in GITR-/- OT-II cells correlated with decreased accumulation of the GITR-/- OT-II cells as well as GITR-/- IL-2 producing OT-II cells compared to their GITR+/+ counterparts. These results suggest that GITR costimulation on T cells can occur in the mLN and then in the lung tissue to promote accumulation of the T cells during influenza infection.

Next, we used a similar approach to assess if GITR costimulation can occur in CD8 T cells in the lung during influenza infection. We adoptively transferred a 1:1 mixture of GITR+/+ and GITR-/- CD45.2 OT-I cells isolated from littermate mice into CD45.1 mice followed by infection with influenza PR8 carrying the OT-I epitope (PR8-OVA) (Figure 2.15G). At day 5 p.i., GITR+/+ OT-I T cells isolated from the lung exhibited a small but significantly higher level of pS6 than GITR-/- OT-I T cells from the same mice (Figure 2.15H). Thus, GITR signaling on CD8 T cells can also take place in the lung tissue.

A B Gated: live CD4+ CD45.1+ OT-II Adoptive Transfer: Day -1 p.i. Lung, day 10 p.i. Transfer 1:1 Initial input ratio FMO GITR+/+:GITR-/- GITR+/+ 47.6% GITR+/+ 66.4%

CD45.1 Infection: Day 0 +/+ Day 3, 5, 10 p.i. GITR OT-II Analysis

Naive R

R -/-

T T I I GITR 32.7%

C57BL/6 GITR-/- 52.1%

G G CD45.1 FSC-A FSC-A GITR-/- OT-II

C mLN, day 3 p.i. FMO D Lung, day 5 p.i. FMO +/+ +/+ Gated: live CD4+ CD45.1+ OT-II GITR Gated: live CD4+ CD45.1+ OT-II GITR GITR-/- GITR-/- 80000 ** 10000 **

8000

60000

I F

F 6000

40000

6 S

S 4000

p p

20000 x

2000 x

a a

M M

% 0 0 % GITR+/+ GITR-/- pS6 GITR+/+ GITR-/- pS6

E Day 10 p.i. F Day 10 p.i. Lung mLN Spleen 100 2.5×105 2.0×105 4×105 ** GITR+/+ -/- *** *

*** GITR s

+ +

s 2.0×10

l 5

** e 5 l 4 75 **

4 1.5×10 3×10

C T

+ 5

+ 1.5×10

- . 50 - 5 5

4 1.0×10 2×10

D C

4 5

C C

T 1.0×10

I T 5 - 4

% 25 O

# 5.0×10 1×10

T 5.0×104 O

0 0.0 0.0 0 Lung mLN Spleen GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

FMO G H Lung, day 5 p.i. GITR+/+ Adoptive Transfer: Day -1 p.i. Gated: live CD8+ CD45.2+ OT-I GITR-/- Transfer 1:1 GITR+/+:GITR-/- 9000 * 8000

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Figure 2.15. GITR signaling increases pS6 levels in antigen-specific OT-II and OT-I T cells. (A) WT CD45.2 C57BL/6 mice received a 1:1 mixture of 1 million each (C and D) or 25,000 each (E and F) of GITR+/+ and GITR-/- CD45.1 OT-II isolated from littermates and were infected i.n. the following day with influenza A/PR8-OVAII. (B) Representative flow cytometry plots depicting the initial and the post-infection GITR+/+: GITR-/- ratio. (C) At day 3 p.i., cells isolated from the mLN were stained directly ex vivo for the level of phosphorylated- ribosomal protein S6 (pS6) with representative flow staining on the right. (D) At day 5 p.i., cells isolated from the lung were stained directly ex vivo for the level of pS6 with representative flow staining on the right. At day 10 p.i., proportions (E) and total numbers (F) of GITR+/+ and GITR-/- OT-II were determined in the lung, mLN, and spleen. (G and H) Naive CD45.1 mice received a 1:1 mixture of 1 million each of GITR+/+ and GITR-/- CD45.2 OT-I isolated from littermates, and were infected i.n. the following day with influenza A/PR8-OVA. (H) At day 5 p.i., cells isolated from the lung were stained directly ex vivo for the level of pS6 with representative flow staining on the right. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Statistical analyses were performed using the Wilcoxon test. Data in (C and D) are pooled from two independent experiments with 8 mice. Data in (E and F) are pooled from two independent experiments with 12 mice. Data in (H) are pooled from two independent experiments with 7 mice.

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# F M 0 0 0 GITR+/+ GITR-/- GITR+/+ GITR-/- Lung Spleen Figure 2.16. GITR is required for optimal IL-2 producing OT-II accumulation. WT CD45.2 C57BL/6 mice received a 1:1 mixture of 1 million each (A) or 25,000 each (B-F) of GITR+/+ and GITR- /- CD45.1 OT-II isolated from littermates and were infected i.n. the following day with influenza A/PR8-OVAII. (A) At day 5 p.i., the proportions and the total number of GITR+/+ and GITR-/- OT-II cells were evaluated in the lung. (B) At day 10 p.i., the proportions of GITR+/+ and GITR-/- OT-II cells were evaluated in IFNγ+ or IL-2+ compartments in the lung and spleen following six hours of OVA323-339 peptide restimulation, with representative flow staining shown in (C). (D) The total number of IL-2+ GITR+/+ and GITR-/- OT-II cells was evaluated in the lung and spleen at day 10 p.i. (E) The MFI of IL-2 in GITR+/+ and GITR-/- IL-2+ OT-II cells was evaluated in the lung and spleen at day 10 p.i. (F) The total number of IFNγ+ GITR+/+ and GITR-/- OT-II cells was evaluated in the lung and spleen at day 10 p.i. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data from (A) pooled are from two independent experiments with 8 mice. Data from (B, D, E and F) are pooled from two independent experiments with 10 mice.

A Adoptive Transfer: Day -1 p.i. B Lung, day 5 p.i. FMO Transfer 1:1 +/+ Gated: live CD4+ CD45.1+ OT-II GITR GITR+/+:GITR-/- GITR-/- * CD45.1 Infection: Day 0 30000 +/+ Day 5 p.i. GITR OT-II Analysis 25000 Naive

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5000 a M 0 GITR+/+ GITR-/- % pS6 Figure 2.17. GITR signaling increases pS6 levels in antigen-specific OT-II T cells in the lung parenchyma. (A) WT CD45.2 C57BL/6 mice received a 1:1 mixture of 1 million each of GITR+/+ and GITR-/- CD45.1 OT-II isolated from littermates and were infected i.n. the following day with influenza A/PR8-OVAII. Analyses were done at day 5 p.i. with intravascular staining with anti-Thy1.2 FITC. (B) pS6 expression in GITR+/+ and GITR-/- OT-II cells in the lung parenchyma at day 5 p.i. with representative flow staining on the right. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Statistical analyses were performed using the Wilcoxon test. Data in (B) are pooled from two independent experiments with 7 mice.

2.5 Discussion

The activation of naïve T cells is widely thought to require 3 signals, the peptide-MHC complex, the B7 family costimulatory signal, as well as cytokines (295, 296). It has long been known that TNFR family members provide additional crucial signals to sustain the survival of activated T cells (193, 201, 258, 302-304), however, the timing of this signal relative to initial T cell priming, the APC type involved and the role of TNFR signaling in secondary lymphoid organs versus the tissues, have not been fully elucidated. Recently, in a chronic infection model with LCMV Clone 13, it was shown that GITR signaling on CD4 T cells in the spleen could be temporally and spatially segregated from initial priming, with monocyte-derived inflammatory APCs providing GITRL to primed T cells. Thus, it was proposed that TNF family ligands provide a post-priming signal 4 for T cell accumulation (258). However, the role of signal 4 during an acute infection and its importance in non-lymphoid tissues was unclear. Here, in this chapter, I provide evidence that GITRL on monocyte-derived inflammatory APC allows increased accumulation of GITR+/+ over GITR-/- T cells in the lung during respiratory influenza infection. I also provide evidence that this signal can take place in the mLN and then again in the b lung parenchyma. I further show that GITR costimulation can rescue low affinity D /NP366-374- specific CD8 T cells and is crucial for the optimal formation of Trm in the lung.

In the context of competition, GITR is required for both CD4 and CD8 T cell responses to influenza virus (Figure 2.1 and 2.3). In contrast, in the absence of competition, GITR is required for the CD8 T cell response, but does not significantly affect the accumulation of the CD4 T cells (Figure 2.7). A recent study showed that during viral infection in mice, monocyte- derived inflammatory APC are the main expressers of 4 TNFR superfamily ligands, GITRL, 4- 1BBL, OX40L, and CD70, dependent on IFN-I (258). In the same study, it was shown that GITR signaling in T cells can regulate molecules involved in cellular adhesion (258). Thus, when GITR+/+ and GITR-/- CD4 T cells compete within the same mouse, it is possible that GITR+/+ CD4 T cells are more efficient at engaging the inflammatory APC than their GITR-/- counterparts, thereby having access to survival signals from all 4 TNF family ligands. On the other hand, in the absence of competition, GITR-/- CD4 T cells do not have to compete with their GITR+/+ counterparts and are more likely to be able to access the signals from the other TNF family ligands. Thus, other costimulatory molecules such as OX40 and CD27 likely compensate for the lack of GITR on the CD4 T cells in the non-competitive setting (193, 202). In contrast to 85

the apparent redundancy of GITR with other signals for CD4 T cells, GITR impacts CD8 T cell accumulation even in a non-competitive setting. Similarly, non-redundant roles for accumulation of lung influenza specific CD8 T cells have also been shown for CD27 and 4-1BB (196, 197, 201, 208). This greater dependency of CD8 T cell responses on multiple TNFRs compared to CD4 T cells might reflect the higher demand for these cells to accumulate to resolve a viral infection.

Tfh cells are known to play a critical role in the formation of GC as well as in the generation of a protective antibody response (312). In the competitive mixed bone marrow chimera model, GITR played no apparent role in the accumulation of Tfh cells in the mLN and spleen during influenza infection (Figure 2.1). Similar to the results from the competitive model, in the non-competitive model (GITR+/+/GITR-/- littermate mice), GITR-/- mice had no defect in the number of Tfh cells in the mLN and spleen when compared to GITR+/+ littermates (Figure 2.7). These results are consistent with a previous report showing that GITR is dispensable for B cell development as well as for antibody responses to both T-dependent and T-independent model antigens (226). Interestingly, in the chronic LCMV Clone 13 model, GITR is required for sustaining Tfh cells as well as optimal LCMV-specific IgG production (244). It remains to be determined why GITR is required for the optimal Tfh responses in a chronic infection, but dispensable for Tfh responses in an acute respiratory infection. Perhaps the level of inflammation may play a role in this discrepancy.

Lung resident CD4 and CD8 Trm have been shown to play an important role in providing protection against influenza infection (113, 150), but the factors affecting their establishment in the lung parenchyma remain incompletely defined. We showed here that the absence of GITR severely impaired the formation of memory T cells in the lung tissue as read out at day 30 p.i. (Figure 2.5). As the magnitude of the effect of GITR is already substantial at the effector phase, it appears that the effect of GITR on Trm is in large part due to its impact on the effector precursors. However, further work with conditional deletion of GITR or GITRL will be required to determine if there is an additional effect of GITR/GITRL on the maintenance of Trm.

We discovered that GITR+/+ and GITR-/- OT-II cells enter the lung equivalently by day 5 but differences in their accumulation are noted by day 10 (Figure 2.15 and 2.16). Similarly, a previous study showed that OT-I T cells enter the lung in equal numbers by day 7 post-influenza

infection with defects in GITR-/- OT-I accumulation detected by day 10 p.i. (243). Thus, the defect in GITR-/- T cells does not appear to be due to a failure to traffic to the lung, but rather a failure in accumulation or persistence over time. As GITR affects the accumulation/survival rather than the initial rate of division of CD4 and CD8 T cells (243, 244), the present study supports the hypothesis that GITR provides important survival signals to the effector cells in the lung tissue as they differentiate into Trm.

During the early phase of vesicular stomatitis virus infection, CD8 Trm formation in the small intestine requires signaling through the mTOR pathway (313). The data presented here show that GITR+/+ CD4 and CD8 T cells in the lung tissue exhibit higher levels of pS6, a downstream target of mTOR, than their GITR-/- counterparts. Previous studies have shown a role for GITR in upregulating pS6 as well as NF-κB, leading to enhanced survival signals, such as

Bcl-XL, and enhanced expression of other TNFRs and cytokine receptors associated with T cell longevity (243, 244, 258).

Monocyte-derived inflammatory APC are the major cell subsets expressing GITRL during acute influenza virus infection, as shown here (Figure 2.12 and 2.13). During chronic LCMV infection there is coordinate induction of GITRL, 4-1BBL, OX40L, and CD70, with preferential expression on inflammatory APC, largely dependent on IFN-I (258). Thus, it is possible that this pattern of expression is a general feature of viral infections. A previous study by Ballesteros-Tato et al. suggested that CD70 was mainly expressed by CD103- CD11bhi DCs during influenza infection (196). In that study, FcεR1 and/or CD64, markers of monocyte- derived inflammatory APC, were not used to distinguish cDC from InfAPC. Thus, it is possible that the CD103- CD11bhi population reported by Ballesteros-Tato et al. also contained the monocyte-derived inflammatory APC population. Recently, using CCR2 knockout mice, Desai et al. showed that inflammatory monocytes were required for persistence of CX3CR1- tissue resident memory T cells in the lung after respiratory Vaccinia infection (314). These findings are consistent with our result that GITRL on inflammatory monocytes is important for Trm formation.

During primary influenza virus infection, a diverse array of TCRs are used to recognize antigens and there is more or less equivalent expansion of T cells specific for the PA224-233 and

NP366-374 epitopes of influenza virus. However, from primary to secondary influenza infection,

the NP response becomes dominant (83) and this is also accompanied by a narrowing of the TCR repertoire to the NP366-374 epitope with an increased overall TCR affinity (315, 316). In contrast, there is no narrowing of the TCR repertoire to PA224-233 epitope in the memory response (317).

These results suggest that the TCR repertoire to NP366-374 epitope is more flexible than the TCR repertoire to PA224-233 epitope. Here we found that PA epitope-specific T effector cells are more dependent on GITR than the NP-specific effector T cells (Figure 2.1 and 2.3). The level of -/- tetramer bound per T cell can be used as a surrogate for TCR affinity (198). GITR NP366-374- specific T cells had an average of 50% increase in MFI for tetramer binding compared to their GITR+/+ counterparts within the same mice (Figure 2.4) Thus, in the absence of GITR, the TCR repertoire to NP366-374 can compensate, by expanding T cells with higher affinity, whereas there was only a minor change in tetramer MFI for the TCR repertoire to PA224-233. These findings are consistent with previous work suggesting that the PA-specific TCR repertoire is less flexible, and likely explains the greater dependence of PA versus NP epitope specific T cells on GITR.

Interestingly, an increase in TCR affinity to the NP366-374 epitope was also observed in CD27- deficient mice, but whether or not the TCR affinity to PA224-233 epitope is affected by the lack of CD27 was not evaluated (198).

In this study, we used mice expressing a conditional deletion of GITRL exon 2 to test the role of GITRL in rescuing low affinity influenza-specific CD8 T cells (Figure 2.4). This mouse was previously shown to express normal levels of GITRL on the cell surface, but with less structural stability, resulting in reduced activity of GITRL, albeit a partial defect compared to mice completely lacking GITR (258). Here we found that mice with mutant GITRL in all cells, exon2Δ/Δ GITRL mice, had an approximately 25% increase in TCR binding to NP366-374-specific T cells and a similar effect (20% increase) was observed in mice in which exon 2 was deleted only in LysM+ cells. The relatively similar effect when exon 2 was deleted in all cells as compared to only in LysM+ cells, is consistent with the idea that the majority of the GITRL signal comes from monocyte derived APC rather than from cDC. The reduced effect in GITRLexon2Δ/Δ mice compared to GITR-/- mice is likely due to the residual function of GITRLΔexon2 (258).

There has been growing evidence that T cells, instead of a brief encounter, need multiple encounters with APCs for optimal response against viral infections, including signals from APC in the lungs (45, 299, 318). However, very little is known about the costimulatory requirements for APC interactions in the lung, or whether TNFR family signals are confined to the secondary 88

lymphoid organs. Here, we detected GITR and GITRL early in the mLN as well as in the lung at a time when T cells start to accumulate there. To provide further support that GITR/GITRL is signaling within the mLN and then in the lung, we compared the level of pS6 in GITR+/+ and GITR-/- T cells competing within the same mouse. As phospho-signaling is generally transient (319), the finding that GITR+/+ OT-II isolated from mLN and lung had a higher level of pS6 than their GITR-/- counterparts argues that GITR/GITRL signaling is occurring first in the mLN and then again in the lung tissue. Thus, we provide evidence that signal 4 provides important additional survival signals to T cells in the mLN as well as when T cells enter the influenza- infected lung, and that these signals sustain the effectors as well as allowing them to survive the transition to Trm.

Chapter 3

GITR differentially affects lung effector T cell subpopulations during influenza virus infection

This work was submitted to the Journal of Leukocyte Biology except for Figure 3.5 and

3.14:

Chu, K. L., Batista, N. V., Girard, M., Law, J. C. & Watts, T. H. GITR differentially affects lung effector T cell subpopulations during influenza virus infection.

Author contributions:

NV Batista, M Girard, and JC Law contributed to this study by providing technical assistance.

NV Batista assisted with the generation of GITR mixed bone marrow chimeras.

NV Batista, M Girard, and JC Law assisted with processing lung tissues for effector cell sorting.

3 GITR differentially affects lung effector T cell subpopulations during influenza virus infection

3.1 Summary

Tissue resident memory T cells are critical for local protection against reinfection. The accumulation of T cells in the tissues requires a post-priming signal from TNFR superfamily members, referred to as signal 4. GITR signaling is important for this post-priming signal and for Trm formation during respiratory infection with influenza virus. As GITR/GITRL interactions take place in the lung parenchyma at a time when effector T cells first accumulate in the lung, we asked if GITR differentially affects subsets of effector cells with different memory potential. Effector CD4 T cells can be subdivided into 2 populations based on expression of Ly6C, whereas effector CD8 T cells can be divided into 3 populations based on Ly6C and CX3CR1. The Ly6C and CX3CR1-positive T cell populations represent the most differentiated effector T cells. Here we show that GITR has a similar effect on the accumulation of both the Ly6Chi and Ly6Clo CD4 T cell subsets but selectively increases CD127 expression on the Ly6Clo subset, a population of T cells that we show has a greater propensity to enter the lung parenchyma upon adoptive transfer. GITR increased the accumulation of all three CD8 T cell subsets defined by CX3CR1 and Ly6C expression, with a more substantial effect on the least differentiated Ly6Clo CX3CR1lo subset. Moreover, GITR selectively upregulated CXCR6 on the less differentiated CX3CR1lo CD8 T cell subsets. These data show that GITR is important for both effector and memory T cell responses in the lung, with unique effects on memory precursors.

3.2 Introduction

Influenza remains a major cause of morbidity and mortality worldwide. T cells play a critical role in defense against influenza virus. CD8 T cells contribute to influenza clearance by killing virally infected cells through Fas or perforin dependent pathways (72). CD4 T cells can also acquire perforin dependent cytotoxicity, but their major functions are cytokine production as well as providing help for B cells (98, 320, 321). During an immune response, T cells require 4 signals to become fully activated and accumulate. Signal 1 comes from the antigen-specific interaction of the TCR with peptide-MHC, signal 2 comes from costimulation by B7 family molecules (295), while signal 3 is derived from cytokines (296). As will be discussed below, T cells also require a 4th signal from TNFR superfamily members for their sustained accumulation particularly in extra-lymphoid tissues (258, 322). TNFR family members such as CD27, 4-1BB, OX40, and GITR are upregulated upon T cell activation and play important roles in controlling the magnitude and duration of T cell responses during viral infections (193, 201, 202, 206, 207, 244, 302-304). Recent data show that during viral infection, the TNF superfamily ligands, CD70, 4-1BBL, OX40L and GITRL are coordinately induced by type I interferon on monocyte-derived cells with minimal expression on conventional dendritic cells (258). Using GITR (TNFRSF18) /GITRL as a proof of principle, we recently showed that inflammatory monocyte-derived cells provide signal 4 to T cells post-priming, allowing T cell accumulation (258, 322). GITR signaling not only takes place in secondary lymphoid organs but can also take place in non- lymphoid tissues (322). Moreover, signal 4 from GITR and 4-1BB is crucial for the optimal formation of lung CD4 and CD8 Trm after influenza infection (208, 209, 322).

During an immune response, the responding effector CD8 T cells are heterogeneous. Some T cells will become short-lived effector cells (SLEC) that confer immediate protection against pathogens but rapidly decline in number after pathogen clearance, while others will give rise to memory precursor effector cells (MPEC) that can give rise to a pool of long-lived memory cells (205, 323). Costaining for surface expression of CD127 (IL-7Rα) and KLRG1 has been used to distinguish SLEC (CD127lo KLRG1hi) and MPEC (CD127hi KLRG1lo) (323, 324). More recently, new markers have been used to identify and dissect different effector CD8 T cell populations. One such marker is the chemokine receptor CX3CR1, which identifies three different effector CD8 T cell subsets in LCMV or VSV infection (214). CX3CR1hi effector CD8 T cells are the most differentiated and give rise to effector memory T cells (Tem), while the 92

CX3CR1lo subset is the least differentiated and gives rise to central memory T cells (Tcm) as well as tissue-resident memory T cells (Trm) (214). Trm are a subset of highly protective non- circulating memory T cells that reside in non-lymphoid tissues and provide a first line of defense against reinfection (113, 150, 308, 309). At the memory stage, CD8 T cells expressing an intermediate level of CX3CR1 (CX3CR1int) are referred to as peripheral memory T cells (Tpm) and are mainly responsible for patrolling non-lymphoid tissues (214). Less is known about the heterogeneity of effector CD4 T cells. During infections with LCMV or γ-herpesvirus, Ly6Chi CD4 effector cells have a more terminally differentiated effector cell phenotype, whereas Ly6Clo cells have more potential to become long-lived memory cells (325, 326). However, the signals that regulate the population size and survival of different effector CD4 and CD8 T cell subsets remain incompletely defined.

As discussed above, GITR affects the accumulation of effector T cells to allow viral control and also provides key signals for the formation of Trm (243, 322). As GITRL expression is mainly limited to the first week of influenza virus infection, the signals that GITR delivers for both effector T cell survival and Trm formation most likely occur at the effector stage of the response. Therefore, we asked if GITR differentially affects different subpopulations of effector T cells. Here, we show that, similar to results in other infection models, following influenza infection the activated effector CD4 T cells can be subdivided into two subsets based on Ly6C expression (Ly6Chi and Ly6Clo), whereas the effector CD8 T cells can be divided into three subsets based on the markers Ly6C and CX3CR1 (Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo, and Ly6Clo CX3CR1lo). Further analysis of the subsets revealed that the Ly6Chi CD4 subset and the Ly6Chi CX3CR1hi CD8 subset have the most differentiated phenotype based on T-bet, CX3CR1 and/or KLRG1 expression. The less differentiated Ly6Clo CD4 subset preferentially enters the lung parenchyma and has a tendency to generate more lung CD4 Trm. We found that GITR increases CD127 surface expression on the Ly6Clo subset but not on the Ly6Chi subset of CD4 T cells. While GITR affected the accumulation of all three subsets of CD8 effector T cells in the lung, it had the largest effect on accumulation of the less differentiated Ly6Clo CX3CR1lo population and also uniquely increased CXCR6 on the less differentiated CX3CR1lo CD8 T cell subsets. These studies provide evidence that GITR contributes to accumulation of both highly differentiated effector cells as well as Trm precursors, but with differential effects on effector subpopulations.

3.3 Materials and Methods

3.3.1 Mice

C57BL/6 mice were purchased from Charles River Laboratories (St. Constant, Quebec, Canada). For generating mixed bone marrow chimeras, Thy1.1 (B6.PL-Thy1a/CyJ) and CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mice were purchased from Jackson Laboratories (Bar Harbor, USA). GITR-/- mice were previously described (244) and backcrossed to CD45.1 OT-II mice (kindly provided by David Brooks, Princess Margaret Cancer Center, Toronto, Ontario) or to CD45.2 OT-I (C57BL/6-Tg(TcraTcrb)1100Mjb/J) mice purchased from Jackson Laboratories (Bar Harbor, USA) to generate littermate controls. Age-matched female mice were used in all experiments. All experimental mice were housed under specific pathogen-free conditions in the Division of Comparative Medicine at the Terrence Donnelly Centre for Cellular and Biomolecular Research at the University of Toronto. All animal procedures were approved by the University of Toronto animal care committee in accordance with the Canadian Council on Animal Care.

3.3.2 Reagents and antibodies

Anti-CD11a (clone: M17/4), anti-OX40 (clone: OX86), anti-Runx3 (clone: R3-5G4) and anti-TCR Vβ5.1/5.2 (clone: MR9-4) were purchased from BD Biosciences. Anti-CD4 (clone: RM4-5), anti-CD8α (clone: 53-6.7), anti-CX3CR1 (clone: SA011F11), anti-CXCR6 (clone: SA051D1), and anti-TCR Vα2 (clone: B20.1) were purchased from BioLegend. Anti-CD127 (clone: A7R34), anti-CD16/32 (Fc Block, clone: 93), anti-CD3ε (clone: 145-2C11), anti-CD45.1 (clone: A20), anti-CD45.2 (clone: 104), anti-CD69 (clone: H1.2F3), anti-GITR (clone: DTA-1), anti-IFNγ (clone: XMG1.2), anti-IL-2 (clone: JES6-5H4), anti-KLRG1 (clone: 2F1), anti-Ly6C (clone: HK1.4), anti-T-bet (clone: eBio4B10), anti-Thy1.2 (clone: 53-2.1), streptavidin PE, and fixable viability dye eF506 were purchased from ThermoFisher Scientific.

3.3.3 Influenza virus infection

Influenza A/HKx31-OVA and A/HKx31-OVAII were kindly provided by Paul Thomas and Peter Doherty (St. Jude Children's Research Hospital, Memphis, Tennessee). MDCK assays were used to determine the tissue culture infectious dose 50 (TCID50) for influenza A/HKx31, A/HKx31-OVA, and A/HKx31-OVAII viruses (305). After anesthesia with isofluorane, 6 to 10 week old mice were infected intranasally (i.n.) with 30 μL of diluted virus at a dose of 5 HAU 4 (equivalent to 3.86 x 10 TCID50) of HKx31, HKx31-OVA, or HKx31-OVAII.

3.3.4 Tissue harvest and processing

Mice were euthanized at the indicated time points after infection and lung, mLN and spleen were harvested. After perfusion with 10 mL of PBS, lung was minced and subjected to enzymatic digestion with 2 mg mL-1 of collagenase IV (Invitrogen, Carlsbad, CA) for 45 minutes at 37oC while shaking. After digestion, lung tissue was mechanically disrupted through a 70 μm pore size cell strainer and lymphocytes were enriched by isolation over an 80/40% Percoll gradient (GE healthcare, Chicago, IL) after red blood cell lysis with ACK lysis buffer. mLN and spleen (after red blood cell lysis) were mechanically disrupted through a 70 μm pore size cell strainer to generate single cell suspensions. For detection of Trm, mice were injected intravenously with 3 μg of FITC-conjugated anti-mouse Thy1.2 antibody and sacrificed 10 minutes later with tissues prepared as described above. Blood was collected from the saphenous vein and treated with ACK lysis buffer.

3.3.5 Flow cytometry

Freshly prepared single cell suspensions from the lung, mLN, and spleen were treated with Fc Receptor Block (anti-CD16/CD32) for 15 minutes at 4oC followed by extracellular staining for 30 minutes at 4oC. Samples were fixed with 4% paraformaldehyde following extracellular staining. Where applicable, after extracellular staining and permeabilization with FoxP3 Transcription Factor Staining Buffer Set (ThermoFisher Scientific), intracellular staining was performed for 30 min at 4oC. For intracellular cytokine staining, lung samples were

d restimulated ex vivo with 1 μM of the I-A -restricted OVA323-339 for 6 h with GolgiStop (BD Biosciences) at 37oC. After peptide restimulation, cells were surfaced stained, fixed and permeabilized (BD Biosciences), and stained for intracellular cytokine production. Unstimulated samples (no peptide) were used as negative controls. Samples were acquired with LSR Fortessa or LSR Fortessa X20 (BD Biosciences) with FACSDiva software and then analyzed with Flowjo VX (Tree Star, Inc.).

3.3.6 T cell isolation and adoptive transfers

Naive CD8 T cells were purified from spleens of GITR+/+ and GITR-/- CD45.2 OT-I TCR-Tg littermates by negative selection with EasySep Mouse CD8 T Cell Isolation Kit (StemCell Technologies, Vancouver, BC). Naive CD4 T cells were purified from spleens of GITR+/+ and GITR-/- CD45.1 OT-II TCR-Tg littermates by negative selection with EasySep Mouse CD4 T Cell Isolation Kit (StemCell Technologies, Vancouver, BC). Samples of either genotype were counted by trypan blue exclusion 3 times before mixing in a 1:1 ratio. OT-I TCR- Tg CD8 T cell purity, OT-II TCR-Tg CD4 T cell purity, and GITR+/+:GITR-/- ratios were confirmed by flow cytometry. Cells from GITR+/+ and GITR-/- mixed sample were adoptively transferred intravenously in 200 μL of volume into congenically marked recipient mice 1 day prior to infection. The number of OT-I or OT-II cells transferred is indicated for each experiment in the figure legends.

3.3.7 Effector T cell transfers

Naïve C57BL/6 mice were infected i.n. with 5 HAU of influenza A/HKx31. Then, at day 7 p.i., mice were euthanized, lungs were processed as described and lung effector CD4 T cells (CD45.2+ CD44hi Ly6Chi and CD45.2+ CD44hi Ly6Clo) were sorted using FACS Aria (BD Biosciences). The purity of sorted samples was at least 94%. 2 x 105 Ly6Chi or Ly6Clo effector

CD4 T cells were adoptively transferred intravenously in 200 μL of volume into infection- matched CD45.1 recipient mice. 24 days post-transfer, recipient mice were sacrificed for Trm analysis.

3.3.8 Mixed bone marrow chimeras

Thy1.1 recipient mice were lethally irradiated with two doses of 550 rad before reconstitution with a 1:1 mixture of GITR+/+ Thy1.2 CD45.1:GITR-/- Thy1.2 CD45.2 bone marrow cells delivered intravenously for a total of 5 x 106 cells. Reconstituted chimeric mice received water supplemented with 2 mg mL-1 neomycin sulfate (Bio-Shop, Burlington, ON, Canada) for two weeks consecutively, and were rested for a total of 90 days before chimerism was checked in the blood. Chimeric mice were infected according to schedules indicated elsewhere.

3.3.9 Data analysis and statistics

GraphPad Prism 6 (San Diego, CA) was used to perform all statistical analyses with specific test performed indicated elsewhere. n.s. not significant, * p < 0.05, ** p < 0.01 were applied.

3.4 Results

3.4.1 During influenza infection, CD4 T subsets can be divided into Ly6Chi and Ly6Clo subpopulations

Ly6C and CX3CR1, two markers associated with monocytes, are also expressed on effector T cells and have been individually used to define different T cell subsets (214, 325-327). To determine whether influenza virus infection induces similar effector CD4 T cell heterogeneity, we stained CD4 T cells from C57BL/6 mice with antibodies to Ly6C and CX3CR1. CX3CR1 was only weakly expressed on CD4 T cells during influenza infection, whereas Ly6C delineated two clear populations of CD4 effector T cells at day 7 post-infection (p.i.). (Figure 3.1A). Thus, we subdivided lung CD44hi effector CD4 T cells into Ly6Chi and Ly6Clo subsets (Figure 3.1A). The Ly6Chi subset exhibited higher levels of T-bet and CX3CR1 proteins than the Ly6Clo subset (Figure 3.1B). Following their adoptive transfer and infection of mice, OT-II cells isolated from influenza A/HKx31-OVAII infected lungs could also be subdivided into Ly6Chi and Ly6Clo subpopulations (Figure 3.1C). Similar to the results from the endogenous response, lung Ly6Chi OT-II T cells expressed higher levels of T-bet and CX3CR1 than Ly6Clo T cells at day 7 p.i. (Figure 3.1D). In addition, lung Ly6Chi OT-II cells produced more IFNγ and IL-2 on a per cell basis than the Ly6Clo subset (Figure 3.1E). These data show that during influenza infection both OT-II transgenic and endogenous CD4 effector T cells can be subdivided into Ly6Chi and Ly6Clo subpopulations. The Ly6Chi subset, which expresses more T-bet, CX3CR1 and cytokines than the Ly6Clo subset, likely represents the most differentiated effector T cell subset.

Figure 3.1. Effector CD4 T cells in the lung can be divided into Ly6Chi and Ly6Clo subpopulations. (A) WT C57BL/6 mice were infected i.n. with influenza A/HKx31 and analyzed at day 7 p.i. Left: Representative flow plot showing Ly6C and CX3CR1 staining after gating on lung CD44hi CD4 T cells. Dashed horizontal and vertical lines indicate boundaries set by FMO controls. Right: Ly6Chi (red) and Ly6Clo (blue) subpopulations were identified from lung CD44hi CD4 T cells. (B) T-bet and CX3CR1 expression in Ly6Chi or Ly6Clo CD44hi CD4 T cells isolated from the lung of C57BL/6 mice 7 days p.i. with influenza A/HKx31. Representative histograms shown below each summary plot. (C) Flow cytometry plot depicting Ly6Chi and Ly6Clo subpopulations among the OT-II cells in the lung 7 days after influenza A/HKx31-OVAII infection. (D) At day 7 p.i. with influenza A/HKx31- OVAII, T-bet and CX3CR1 expression were evaluated in OT-II cells in the Ly6Chi or Ly6Clo compartments in the lung. (E) At day 7 p.i. with influenza A/HKx31-OVAII, IFNγ and IL-2 MFI were evaluated in OT-II cells in the hi lo Ly6C or Ly6C compartments in the lung following six hours of OVA323-339 peptide restimulation. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Grey filled histograms indicate FMO controls or unstimulated (no peptide) controls. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data from (B) are pooled from two independent experiments with 7 mice. Data from (D and E) are pooled from two independent experiments with 8 mice.

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3.4.2 Ly6Chi or Ly6Clo CD4 subsets are similarly dependent on GITR costimulation for their accumulation

To examine the effect of GITR on the Ly6Chi and Ly6Clo effector CD4 T cells, we adoptively transferred a 1:1 mix of CD45.1 GITR+/+:GITR-/- transgenic OT-II CD4 T cells into naïve C57BL/6 mice followed by infection with influenza A/HKx31-OVAII virus and analysis at day 7, 10, and 17 p.i. (Figure 3.2A). We used GITR expression on donor CD45.1 OT-II cells to distinguish GITR+/+ from GITR-/- T cells within the same hosts (Figure 3.2B). The initial input ratio of GITR+/+ to GITR-/- OT-II was about 1:1; however, by day 7 p.i. about 60% of both Ly6Clo and Ly6Chi OT-II cells were GITR+/+, with a similar result at day 10 p.i. (about 1.5 fold advantage over their GITR-/- counterparts) (Figure 3.2B-D). At day 17 p.i., too few Ly6Chi OT-II cells were recovered for analysis, but we observed a similar defect for GITR-/- Ly6Clo OT-II cells when compared to earlier time points (Figure 3.2D). Similarly, there was a reduction in the total number of GITR-/- Ly6Chi and GITR-/- Ly6Clo OT-II cells in the lung at all time points examined when compared to their GITR+/+ counterparts (Figure 3.2E).

It was possible that the results observed were affected by the use of TCR transgenic T cells. Therefore, to determine the effect of GITR on endogenous CD44hi Ly6Chi or Ly6Clo CD4 T cell subsets, we generated mixed bone marrow chimeras. We reconstituted lethally irradiated Thy1.1 CD45.2 hosts with bone marrow from Thy1.2 CD45.1 GITR+/+ and Thy1.2 CD45.2 GITR-/- mice in a 1:1 ratio (Figure 3.3A, B). Similar to the results from the OT-II transgenic system, the lung GITR+/+ Ly6Chi or Ly6Clo subset had a similar 1.5-fold accumulation advantage over their GITR-/- counterparts at day 10 p.i. (Figure 3.3C, D). Together, these data suggest that the accumulation of both the Ly6Chi and Ly6Clo effector CD4 T cell subpopulations are equally affected by GITR costimulation.

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Figure 3.2. Similar effect of GITR on accumulation of Ly6Chi or Ly6Clo OT-II CD4 T cells in the lung during influenza infection. (A) WT CD45.2 C57BL/6 mice received a 1:1 mixture of 1 x 105 each of GITR+/+ and GITR-/- CD45.1 OT-II isolated from littermates and were infected i.n. the following day with influenza A/HKx31-OVAII, followed by analysis at day 7, 10, and 17 p.i. (B) Representative flow cytometry plots showing the initial input ratio and the post- infection GITR+/+:GITR-/- ratio in Ly6Chi or Ly6Clo OT-II compartments in the lung day 7 p.i. (C) At day 7 and 10 p.i., the proportions (left) and ratio (right) of GITR+/+ and GITR-/- OT-II cells were evaluated in Ly6Chi compartments in the lung. (D) At day 7, 10, and 17 p.i., the proportions (left) and ratio (right) of GITR+/+ and GITR- /- OT-II cells were evaluated in Ly6Clo compartments in the lung. (E) The total number of Ly6Chi or Ly6Clo OT-II cells in the GITR+/+ or GITR-/- compartments was evaluated in the lung day 7, 10, and 17 p.i. Ratios were calculated by dividing the proportion of GITR+/+ cells by the proportion of GITR-/- cells in each mouse within each specific cell compartment. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two independent experiments totaling 8 mice.

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Figure 3.3. Similar effect of GITR on accumulation of endogenous Ly6Chi or Ly6Clo effector CD4 T cells in the lung during influenza infection. (A) Schematic indicating that lethally irradiated Thy1.1 CD45.2 recipients were reconstituted with a 1:1 mixture of GITR+/+ Thy1.2 CD45.1: GITR−/− Thy1.2 CD45.2 bone marrow cells. Chimeric mice were rested for 90 days and chimerism checked in the blood from each mouse, before i.n. infection with influenza A/HKx31 followed by CD4 T cell analysis at day 10 p.i. (B) Gating strategy to identify GITR+/+ and GITR-/- cells from Ly6Chi or Ly6Clo subpopulations within the donor CD44hi CD4 T cell compartments. (C) The normalized GITR+/+:GITR−/− ratio in CD44hi Ly6Chi or CD44hi Ly6Clo CD4 T cell compartments was evaluated in the lung at day 10 p.i. Normalization was done by dividing the post-infection ratio in each mouse in the lung by the pre-infection ratio of CD4 T cells in blood from the same mouse. (D) Representative flow cytometry plots showing proportions of GITR+/+: GITR−/− of blood CD4 T cells before infection and of lung CD44hi Ly6Chi or CD44hi Ly6Clo CD4 T cells day 10 p.i. Statistical analyses were performed using the Wilcoxon test comparing pre- and post-infection ratios. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from 8 individual chimeric mice from two independent experiments.

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3.4.3 Cytokine producing Ly6Chi CD4 T cells are more dependent on GITR costimulation than cytokine producing Ly6Clo CD4 T cells

Next, we investigated whether cytokine producing Ly6Chi or Ly6Clo effector CD4 T cells are equally dependent on GITR costimulation. We adoptively transferred a 1:1 mix of CD45.1 GITR+/+:GITR-/- transgenic OT-II CD4 T cells into naïve C57BL/6 mice, infected mice with influenza A/HKx31-OVAII virus, and then, at day 7 p.i., we re-stimulated lung T cells with hi lo OVA323-339 peptide to examine the effect of GITR on Ly6C and Ly6C subpopulations within the IFNγ producing or IL-2 producing OT-II subfraction (Figure 3.4A, B). The absence of GITR resulted in a larger decrease in the frequency and total number of IFNγ producing OT-II cells in the Ly6Chi subpopulation than in their Ly6Clo counterparts in the lung at day 7 p.i. (Figure 3.4C- E). We observed a similar result in the IL-2 producing OT-II T cell subset, where the absence of GITR resulted in a greater loss of the Ly6Chi CD4+ IL-2-producing T cells compared to the Ly6Clo T cells (Figure 3.4F-H). In summary, these data suggest that cytokine producing Ly6Chi CD4 T cells are more dependent on GITR costimulation than the cytokine producing Ly6Clo CD4 T cells.

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Figure 3.4. IFNγ producing or IL-2 producing Ly6Chi OT-II CD4 T cells are more dependent on GITR costimulation than their Ly6Clo counterparts. (A) WT CD45.2 C57BL/6 mice received a 1:1 mixture of 5 x 105 each of GITR+/+ and GITR-/- CD45.1 OT-II isolated from littermates and were infected i.n. the following day with influenza A/HKx31-OVAII, followed by cytokine analysis at day 7 p.i. (B) Gating strategy to identify GITR+/+ and GITR-/- cells from the Ly6Chi or Ly6Clo subpopulation within the IFNγ producing or IL-2 producing OT-II compartment. (C) At day 7 p.i., the proportions (left) and ratio (right) of GITR+/+ and GITR-/- OT-II cells were evaluated in IFNγ+ Ly6Chi or IFNγ+ Ly6Clo compartments in the lung following six hours of OVA323-339 peptide re-stimulation. (D) Representative flow cytometry plots showing the initial input ratio and the post-infection GITR+/+:GITR-/- ratio in IFNγ+ Ly6Chi or IFNγ+ Ly6Clo compartments in the lung day 7 p.i. (E) The total number of IFNγ+ Ly6Chi or IFNγ+ Ly6Clo OT-II cells in the GITR+/+ or GITR-/- compartments was evaluated in the lung day 7 p.i. (F) At day 7 p.i., the proportions (left) and ratio (right) of GITR+/+ and GITR-/- OT-II cells were evaluated in IL-2+ Ly6Chi or IL-2+ Ly6Clo compartments in the lung following six hours of OVA323-339 peptide restimulation. (G) Representative flow cytometry plots showing the initial input ratio and the post-infection GITR+/+:GITR-/- ratio in IL-2+ Ly6Chi or IL-2+ Ly6Clo compartments in the lung day 7 p.i. (H) The total number of IL-2+ Ly6Chi or IL-2+ Ly6Clo OT-II cells in the GITR+/+ or GITR-/- compartments was evaluated in the lung day 7 p.i. Ratios were calculated by dividing the proportion of GITR+/+ cells by the proportion of GITR-/- cells in each mouse within each specific cell compartment. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two independent experiments with 8 mice.

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3.4.4 GITR differentially regulates CD127 (IL-7Rα) in CD4 effector T cell subsets

To gain further insight into how GITR affects CD4 T cell accumulation, we investigated several molecules associated with T cell survival. The choice of molecules was based on a previous RNA-sequencing study of transcripts regulated by GITR on CD4 T cells during chronic LCMV infection (258). GITR signaling in LCMV-specific CD4 T cells was previously shown to increase OX40 expression and also to limit the downregulation of CD127 (IL-7Rα) in the effector T cells (258), receptors that provide important survival signals to memory T cells (328- 330). OX40 is normally absent on naïve CD4 T cells (Figure 3.5), but upregulated upon T cell activation (331). CD127 is normally present on naïve CD4 T cells (Figure 3.5), downregulated on effector T cells and then re-expressed on memory T cells (332, 333). Moreover, memory precursors are enriched in the CD127+ subset (323, 334). To determine if GITR is involved in the regulation of OX40 and CD127 expression on the two CD4 subsets during influenza infection, we adoptively transferred GITR+/+ and GITR-/- OT-II cells into naïve C57BL/6 mice, followed by infection with influenza A/HKx31-OVAII (Figure 3.6A). At day 5 p.i., the GITR+/+ cells had increased OX40 expression when compared to their GITR-/- counterparts for both the Ly6Chi and Ly6Clo subpopulations (Figure 3.6B, C). We did not observe a difference in the expression of CD127 between GITR+/+ and GITR-/- naïve OT-II cells (Figure 3.5). However, at day 5 p.i., GITR+/+ Ly6Chi OT-II cells had a trend toward higher expression of CD127 than their GITR-/- Ly6Chi OT-II cells, but this was not statistically significant (Figure 3.6D, E). In contrast, the GITR+/+ Ly6Clo OT-II cells exhibited a small but significant increase in the level of CD127 compared to their GITR-/- counterparts at day 5 p.i. (Figure 3.6D, E).

Again, it was possible that the results observed were affected by the use of OT-II transgenic T cells. Thus, we investigated the level of OX40 and CD127 expression on endogenous GITR+/+ and GITR-/- Ly6Chi or Ly6Clo effector CD4 T cells during influenza infection using mixed bone marrow chimeras (Figure 3.7A). Similar to the results in the OT-II transfer model, the endogenous GITR+/+ Ly6Chi or Ly6Clo CD4 subsets had higher surface expression of OX40 than their GITR-/- counterparts in the lung, mLN, and spleen day 6 p.i. (Figure 3.7B, C). At both day 6 and day 10 p.i., there was a small but significant increase in the level of CD127 on the lung GITR+/+ Ly6Clo subset when compared to their GITR-/- counterparts, whereas no significant difference was observed in the Ly6Chi subset (Figure 3.7D-F). In the

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lung at day 10 p.i., there was also a higher frequency of CD127+ cells in the GITR+/+ Ly6Clo subset when compared to their GITR-/- counterparts, whereas GITR+/+ and GITR-/- Ly6Chi cells had a similar frequency of CD127+ cells (Figure 3.7G, H). Together, these data show that during influenza infection GITR induces a small but significant increase in CD127 expression on the less differentiated Ly6Clo but not on the more highly differentiated Ly6Chi effector CD4 T cell sub-population in the lung. Additionally, GITR signaling increased OX40 expression on both the Ly6Chi and Ly6Clo CD4 subsets.

3.4.5 Effect of GITR signaling on CXCR6 expression on CD4 T cells during influenza infection

Chemokine receptors can be important in the localization of T cells, ensuring that they enter the appropriate environment to receive the signals needed for differentiation into memory T cells. Although previous work had shown that CX3CR1 is upregulated by GITR on CD4 T cells during LCMV Clone 13 infection (258), we did not find an increase in CX3CR1 on GITR+/+ compared to GITR-/- CD4 T cells during influenza infection (Figure 3.8). Moreover, in the influenza model, there is only minimal expression of CX3CR1 on the CD4 T cells (Figure 3.1A). CXCR6 is a chemokine receptor that has been implicated in the formation of skin CD8 Trm (335). CXCR6 is expressed at a low level on resting CD4 T cells (Figure 3.5) but is upregulated upon activation (336, 337). CXCR6 expression was not different between GITR+/+ and GITR-/- naïve CD4 T cells (Figure 3.5). Next, we asked if GITR regulates CXCR6 in the CD4 T cells after influenza infection. In the mixed bone marrow chimeras, GITR+/+ cells exhibited a higher level of CXCR6 than GITR-/- cells, for both the Ly6Chi and Ly6Clo CD4 subsets in all three organs examined day 6 p.i. (Figure 3.7I, J). Thus, GITR may contribute to Trm formation through inducing higher levels of CXCR6, although the effects of this small increase in CXCR6 remains to be investigated.

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Naive mLN Gated: live CD45.1+ CD4+ OT-II

Total OT-II, OX40 Expression Total OT-II, CD127 Expression Total OT-II, CXCR6 Expression 400 400 n.s. 400 GITR+/+ GITR-/-

300 300 300

n.s. n.s. 6

200 7 200 200

C C 100 100 100

0 0 0 GITR+/+ GITR-/- GITR+/+ GITR-/- GITR+/+ GITR-/-

Naive mLN Naive mLN Naive mLN FMO FMO FMO GITR+/+ GITR+/+ GITR+/+

GITR-/- GITR-/- GITR-/-

x x x

a a a

M M M

% % %

OX40 CD127 CXCR6 Figure 3.5. OX40, CD127, and CXCR6 expression on naïve OT-II CD4 T cells. (A) WT CD45.2 C57BL/6 mice received a 1:1 mixture of 5 x 105 each of GITR+/+ and GITR-/- CD45.1 OT-II isolated from littermates. One day post transfer, expression of OX40, CD127, and CXCR6 was evaluated in GITR+/+ or GITR-/- OT-II cells isolated from the mLN. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from a single experiment with 4 mice.

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Figure 3.6. Regulation of OX40 and CD127 expression by GITR in antigen specific OT-II CD4 T cells during influenza virus infection. (A) WT CD45.2 C57BL/6 mice received a 1:1 mixture of 5 x 105 each of GITR+/+ and GITR-/- CD45.1 OT-II isolated from littermates and were infected i.n. the following day with influenza A/HKx31-OVAII, followed by analysis at day 5 p.i. (B) At day 5 p.i., OX40 expression was evaluated in GITR+/+ or GITR-/- OT-II cells in the Ly6Chi or Ly6Clo compartments with representative histograms shown in (C). (D) At day 5 p.i., CD127 expression was evaluated in GITR+/+ or GITR-/- OT-II cells in the Ly6Chi or Ly6Clo compartments with representative histograms shown in (E). dMFI refers to the MFI for the specific antibody stain minus the FMO control. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two independent experiments with 8 mice.

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Figure 3.7. Regulation of OX40, CD127, and CXCR6 expression by GITR in endogenous effector CD4 T cells during influenza infection. (A) Schematic indicating that lethally irradiated Thy1.1 CD45.2 recipients were reconstituted with a 1:1 mixture of GITR+/+ Thy1.2 CD45.1: GITR−/− Thy1.2 CD45.2 bone marrow cells. Chimeric mice were rested for 90 days and the level of chimerism checked in the blood from each mouse, before i.n. infection with influenza A/HKx31 followed by CD4 T cell analysis at day 6 and 10 p.i. (B) At day 6 p.i., OX40 expression was evaluated in donor GITR+/+ or GITR-/- cells in CD44hi Ly6Chi or CD44hi Ly6Clo CD4 T cell compartments in the lung, mLN, and spleen with representative histograms shown in (C). At day 6 (D) or day 10 (E) p.i., CD127 expression was evaluated in donor GITR+/+ or GITR-/- cells in CD44hi Ly6Chi or CD44hi Ly6Clo CD4 T cell compartments in the lung, mLN, and spleen with representative histograms shown in (F). (G) At day 10 p.i., the frequency of CD127+ cells was evaluated in donor GITR+/+ or GITR-/- cells in CD44hi Ly6Chi or CD44hi Ly6Clo CD4 T cell compartments in the lung with representative flow cytometry plots shown in (H). (I) At day 6 p.i., CXCR6 expression was evaluated in donor GITR+/+ or GITR-/- cells in CD44hi Ly6Chi or CD44hi Ly6Clo CD4 T cell compartments in the lung, mLN, and spleen with representative histograms shown in (J). dMFI refers to the MFI for the specific antibody stain minus the FMO control. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from 8 individual chimeric mice from two independent experiments.

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Lung day 7 p.i. Gated: live CD45.1+ CD4+ OT-II

FMO GITR+/+

GITR-/-

CX3CR1 Figure 3.8. Similar CX3CR1 expression between GITR+/+ and GITR-/- OT-II cells following influenza infection. WT CD45.2 C57BL/6 mice received a 1:1 mixture of 1 x 105 each of GITR+/+ and GITR-/- CD45.1 OT-II isolated from littermates and were infected i.n. the following day with influenza A/HKx31-OVAII. Representative flow cytometry plot depicting the expression of CX3CR1 in GITR+/+ or GITR-/- OT-II cells isolated from the lung at day 7 p.i. Data are representative of 3 independent experiments with 4 mice per experiment.

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3.4.6 Upon adoptive transfer, the Ly6Clo CD4 T cells preferentially enter the lung parenchyma

To gain insight into which effector CD4 T cell subset gives rise to lung CD4 Trm after influenza infection, we infected naïve C57BL/6 mice with influenza A/HKx31 and sorted CD44hi Ly6Chi and CD44hi Ly6Clo effector CD4 T cells from the lung at day 7 p.i. Sorted cells were adoptively transferred in equal number into separate infection matched CD45.1 congenic mice. Infection matched recipients were used so that the transferred effector T cells could encounter the same inflammatory environment before and after isolation. The Trm population among the donor CD4 T cells was analyzed at 24 days post transfer (day 31 p.i.) (Figure 3.9A-C). Host CD45.1+ CD4+ T cells contained both CD44hi and CD44lo cells whereas CD45.2+ donor CD4 T cells were exclusively CD44hi reflecting their prior antigen exposure (Figure 3.9C). At day 31 p.i., recipient mice that had received a transfer of the Ly6Chi subset had a higher frequency of lung vascular donor CD45.2+ CD4 T cells, whereas recipient mice that had received the Ly6Clo subset had a higher frequency of donor CD45.2+ CD4 T cells in the lung parenchyma (Figure 3.9D, E). Recipient mice that had received the Ly6Clo subset had a trend toward a higher total number of CD4 Trm in the lung when compared to mice that received the Ly6Chi subset (Figure 3.9F). These results suggest that both the Ly6Chi and Ly6Clo effector CD4 T cells can give rise to lung CD4 Trm after influenza infection. However, the Ly6Clo subset preferentially accumulates in the lung parenchyma and this is associated with a trend towards increased Trm formation in the lung.

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Figure 3.9. Compared to Ly6Chi effector CD4 T cells, Ly6Clo effector CD4 T cells preferentially home to the lung parenchyma and have a higher potential to form CD4 Trm. (A) WT C57BL/6 mice were i.n. infected with influenza A/HKx31. At day 7 p.i., CD44hi Ly6Chi (red) or CD44hi Ly6Clo (blue) effector CD4 T cells were sorted from the lung before adoptively transferred into separate infection matched CD45.1 mice. 24 days post transfer (day 31 p.i.), recipient mice were sacrificed for CD4 Trm analysis. (B) Flow cytometry analysis of Ly6C expression on effector CD44hi CD4 T cells pre- and post-sort. (C) Top row: A sequential gating strategy used to identify donor CD45.2+ CD69+ CD11ahi CD4 Trm in the lung parenchyma. Bottom row: Flow cytometry analysis of CD44 expression on recipient or donor CD4 T cells. (D) At day 31 p.i., proportions of donor CD45.2+ CD4 T cells in the lung vasculature or lung parenchyma from each transferred effector T cell subset were evaluated with representative staining shown in (E). (F) At day 31 p.i., total number of lung donor CD45.2+ CD69+ CD11ahi CD4 Trm recovered from each transferred effector T cell subset was evaluated. Statistical analyses were performed using the Mann-Whitney test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two independent experiments with 5-9 mice per group.

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3.4.7 Ly6C and CX3CR1 expression defines three subsets of effector CD8 T cells in the lung

Next, we investigated the heterogeneity of lung effector CD8 T cells by infecting C57BL/6 mice with influenza A/HKx31 followed by co-staining with Ly6C and CX3CR1. Lung effector CD8 T cells can be subdivided into three subsets based on differential Ly6C and CX3CR1 expression (Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo, and Ly6Clo CX3CR1lo subsets) (Figure 3.10A). Among the three subsets, the Ly6Chi CX3CR1hi subset exhibited the highest level of T-bet and KLRG1 expression (Figure 3.10B). Although the Ly6Chi CX3CR1lo and Ly6Clo CX3CR1lo subsets had similar levels of T-bet, the Ly6Chi CX3CR1lo subset had a slightly higher level of KLRG1 (Figure 3.10B). Runx3, a transcription factor known to be critical in the formation of Trm in non-lymphoid tissues (146), was expressed at a lower level in the Ly6Chi CX3CR1hi subset when compared to the other two subsets, which had similar levels of Runx3 protein (Figure 3.10B). Similar to the endogenous influenza-specific CD8 T cells, following adoptive transfer and infection, OT-I cells isolated from influenza A/HKx31-OVA infected lungs at day 7 p.i. could be subdivided into Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo, and Ly6Clo CX3CR1lo subsets (Figure 3.10C). Moreover, the Ly6Chi CX3CR1hi OT-I expressed highest levels of T-bet and KLRG1 but the lowest amount of Runx3 among the three different subsets (Figure 3.10D). Together, these data show that during influenza infection both OT-I transgenic and endogenous CD8 effector T cells can be subdivided into three populations. The Ly6Chi CX3CR1hi subset, which expresses the most T-bet and KLRG1, likely represents the most differentiated effector T cell subset.

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Figure 3.10. Lung effector CD8 T cells can be divided into three subsets by Ly6C and CX3CR1. (A) WT C57BL/6 mice were infected i.n. with influenza A/HKx31. At day 7 p.i., Ly6Chi CX3CR1hi (red), Ly6Chi CX3CR1lo (green), and Ly6Clo CX3CR1lo (blue) subpopulations were identified from lung CD44hi CD8 T cells. (B) T-bet, KLRG1, and Runx3 expression in endogenous Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo, or Ly6Clo CX3CR1lo CD44hi CD8 T cells isolated from the lung of C57BL/6 mice 7 days p.i. with influenza A/HKx31. Representative histograms shown below each summary plot. (C) Flow cytometry plot depicting Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo and Ly6Clo CX3CR1lo subpopulations from OT-I cells in the lung 7 days after influenza A/HKx31-OVA infection. (D) At day 7 p.i. with influenza A/HKx31-OVA, T-bet, KLRG1, and Runx3 expression were evaluated in OT-I cells in the Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo or Ly6Clo CX3CR1lo compartments in the lung. Representative histograms shown below each summary plot. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Grey filled histograms indicate FMO controls. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data from (B) are pooled from two independent experiments with 7 mice. Data from (D) are pooled from two independent experiments with 8 mice.

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3.4.8 The Ly6Clo CX3CR1lo CD8 T cell subset is the most dependent on GITR costimulation

To examine the effect of GITR on the three different subsets of effector CD8 T cells, we adoptively transferred a 1:1 mix of CD45.2 GITR+/+:GITR-/- transgenic OT-I CD8 T cells into naïve CD45.1 mice followed by infection with influenza A/HKx31-OVA virus and analysis at day 7 and 10 p.i. (Figure 3.11A). GITR expression was used to distinguish GITR+/+ and GITR-/- OT-I cells within the same host pre- and post-infection (Figure 3.11B). Among the three different subsets, the Ly6Clo CX3CR1lo OT-I cells were most affected by lack of GITR at day 7 p.i., and was the only subset affected by the lack of GITR at day 10 p.i. (Figure 3.11B-E).

To rule out that these findings were an artifact of the OT-I cell transfer model, we also analyzed the effect of GITR on the three CD8 subsets using analysis of endogenous T cells in mixed bone marrow chimeras (Figure 3.12A, B). Similar to the results from the OT-I transgenic system, the Ly6Clo CX3CR1lo CD8 subset was the most dependent on GITR. The GITR+/+ Ly6Clo CX3CR1lo subset had a 3-fold accumulation advantage over their GITR-/- counterparts compared to a 2-fold and a 1.5-fold advantage in the Ly6Chi CX3CR1lo and Ly6Chi CX3CR1hi subsets, respectively (Figure 3.12C, D).

Overall, these data show, using both a TCR transgenic OT-I adoptive transfer model as well as analysis of the endogenous CD8 T cell responses to influenza virus in mixed bone marrow chimeras, that GITR affects accumulation of all three effector CD8 T cell subpopulations in the lung, with the Ly6Clo CX3CR1lo CD8 T cell subset showing the most dependence on GITR.

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Figure 3.11. In the absence of GITR, there is a greater loss of Ly6Clo CX3CR1lo OT-I CD8 T cells in the lung during influenza infection. (A) Naïve CD45.1 mice received a 1:1 mixture of 1 x 104 each of GITR+/+ and GITR-/- CD45.2 OT-I isolated from littermates and were infected i.n. the following day with influenza A/HKx31-OVA, followed by analysis at day 7 and 10 p.i. (B) Representative flow cytometry plots showing the initial input ratio and the post-infection GITR+/+:GITR-/- ratio in Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo or Ly6Clo CX3CR1lo OT-I compartments in the lung day 7 p.i. At day 7 (C) or day 10 (D) p.i., the proportions (left) and ratio (right) of GITR+/+ and GITR-/- OT-I cells were evaluated in Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo or Ly6Clo CX3CR1lo compartments in the lung. (E) The total number of Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo or Ly6Clo CX3CR1lo OT-I cells in the GITR+/+ or GITR-/- compartments was evaluated in the lung day 7 and 10 p.i. Ratios were calculated by dividing the proportion of GITR+/+ cells by the proportion of GITR-/- cells in each mouse within each specific cell compartment. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from two independent experiments with 8 mice.

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Figure 3.12. In the absence of GITR, there is a greater loss of endogenous Ly6Clo CX3CR1lo effector CD8 T cells in the lung during influenza infection. (A) Schematic indicating that lethally irradiated Thy1.1 CD45.2 recipients were reconstituted with a 1:1 mixture of GITR+/+ Thy1.2 CD45.1: GITR−/− Thy1.2 CD45.2 bone marrow cells. Chimeric mice were rested for 90 days and the degree of chimerism checked in the blood from each mouse, before i.n. infection with influenza A/HK-X31 followed by CD8 T cell analysis at day 10 p.i. (B) Gating strategy to identify GITR+/+ and GITR-/- cells from Ly6Chi CX3CR1hi, Ly6Chi CX3CR1lo or Ly6Clo CX3CR1lo subpopulations within the donor CD44hi CD8 T cell compartments. (C) The normalized GITR+/+:GITR−/− ratio in CD44hi Ly6Chi CX3CR1hi, CD44hi Ly6Chi CX3CR1lo, or CD44hi Ly6Clo CX3CRlo CD8 T cell compartments was evaluated in the lung day 10 p.i. Normalization was done by dividing the post-infection ratio in each mouse in the lung by the pre-infection ratio of CD8 T cells in blood from the same mouse. (D) Representative flow cytometry plots showing proportions of GITR+/+:GITR−/− of blood CD8 T cells before infection and of lung CD44hi Ly6Chi CX3CR1hi, CD44hi Ly6Chi CX3CR1lo, or CD44hi Ly6Clo CX3CRlo CD8 T cells day 10 p.i. Statistical analyses were performed using the Wilcoxon test comparing pre- and post- infection ratios. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from 8 individual chimeric mice from two independent experiments.

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3.4.9 Effect of GITR on OX40 induction on CD8 T cells during influenza infection

Previous results, as well as the results shown in figure 3.6 and 3.7 of this chapter, demonstrated that GITR regulates the pro-survival TNFR family member OX40 on CD4 T cells (258). Here we asked whether GITR also regulates OX40 on CD8 T cells during influenza virus infection by assessing OX40 expression in GITR+/+ and GITR-/- cells in each effector T cell subset using mixed bone marrow chimeras (Figure 3.13A). Similar to naïve CD4 T cells, OX40 was absent on naïve CD8 T cells (Figure 3.14). Furthermore, in the lung, mLN, and spleen, OX40 expression was minimal in all three effector CD8 T cell subsets at day 6 p.i. (Figure 3.13B), consistent with a previous study showing substantially higher OX40 expression on CD4 T cells than CD8 T cells (203). However, there was a small but significant effect of GITR on OX40 levels in the Ly6Chi CX3CR1lo CD8 population in the mLN of influenza infected mice (Figure 3.13B).

3.4.10 GITR regulates CXCR6 expression in Ly6Chi CX3CR1lo and Ly6Clo CX3CR1lo effector CD8 T cells during influenza infection

GITR is required for maximal formation of lung CD8 Trm following influenza virus infection (322). As CXCR6 has been implicated in CD8 Trm formation in the skin (335) , we used GITR+/+: GITR-/- mixed bone marrow chimeras to ask whether GITR also regulates CXCR6 expression in CD8 T cells during influenza infection (Figure 3.13A). Similar to naïve CD4 T cells, we found that CXCR6 was expressed at a low level on naïve CD8 T cells and the expression was not different between GITR+/+ and GITR-/- naïve CD8 T cells (Figure 3.14). However, at day 6 p.i., lung GITR+/+ cells among the Ly6Chi CX3CR1lo and Ly6Clo CX3CR1lo but not the Ly6Chi CX3CR1hi CD8 subsets had a higher level of CXCR6 expression than GITR-/- cells (Figure 3.13C). These data show that GITR selectively increases the level of CXCR6 on the CX3CR1lo CD8 effector subsets in the lung after influenza infection.

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Figure 3.13. Regulation of OX40 and CXCR6 expression by GITR in endogenous effector CD8 T cells during influenza infection. (A) Schematic indicating that lethally irradiated Thy1.1 CD45.2 recipients were reconstituted with a 1:1 mixture of GITR+/+ Thy1.2 CD45.1: GITR−/− Thy1.2 CD45.2 bone marrow cells. Chimeric mice were rested for 90 days and chimerism checked in the blood from each mouse, before i.n. infection with influenza A/HKx31 followed by CD8 T cell analysis at day 6 p.i. (B) At day 6 p.i., OX40 expression was evaluated in donor GITR+/+ or GITR-/- cells in CD44hi Ly6Chi CX3CR1hi, CD44hi Ly6Chi CX3CR1lo, or CD44hi Ly6Clo CX3CR1lo CD8 T cell compartments in the lung, mLN, and spleen with representative histograms shown below summary plots. (C) At day 6 p.i., CXCR6 expression was evaluated in donor GITR+/+ or GITR-/- cells in CD44hi Ly6Chi CX3CR1hi, CD44hi Ly6Chi CX3CR1lo, or CD44hi Ly6Clo CX3CR1lo CD8 T cell compartments in the lung, mLN, and spleen with representative histograms shown below summary plots. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from 8 individual chimeric mice from two independent experiments.

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Naive mLN Gated: live CD45.2+ CD8+ OT-I

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OX40 CXCR6 Figure 3.14. OX40 and CXCR6 expression on naïve OT-I CD8 T cells. CD45.1 mice received a 1:1 mixture of 5 x 105 each of GITR+/+ and GITR-/- CD45.2 OT-I isolated from littermates. One day post transfer, expression of OX40 and CXCR6 was evaluated in GITR+/+ or GITR-/- OT-I cells isolated from the mLN. dMFI refers to the MFI for the specific antibody stain minus the FMO control. Statistical analyses were performed using the Wilcoxon test. Each symbol represents an individual mouse, with bars indicating mean ± SEM. Data are pooled from a single experiment with 4 mice.

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3.5 Discussion

During an immune response, the responding effector T cells are heterogeneous; some have a more differentiated phenotype and are short-lived, while others have a higher potential to give rise to long-lived memory cells. Previous studies have used markers such as CD127, KLRG1, Ly6C, and CX3CR1 to subdivide effector T cells into subsets with different potential to form long-lived memory cells (214, 323-326, 338). However, the signals that regulate the population size and survival of different effector CD4 and CD8 T cell subsets have not been fully elucidated. Here, we show that during influenza infection, Ly6C defines two subsets of effector CD4 T cells while a combination of Ly6C and CX3CR1 defines three subsets of effector CD8 T cells. We further show that the Ly6Chi CD4 subset and the Ly6Chi CX3CR1hi CD8 T cell subsets exhibit a phenotype consistent with a more differentiated state. The Ly6Clo CD4 subset preferentially localizes to the lung parenchyma and shows a trend towards increased Trm formation after influenza infection, suggesting that this population is more enriched in Trm precursors than the Ly6Chi population.

Here we have provided evidence that GITR differentially affects effector T cell subpopulations during influenza infection. GITR showed similar effects on the accumulation of both CD4 effector T cell subsets but had differential effects on CD127 expression on the two subsets, maintaining higher levels of CD127 on the Ly6Clo compared to the Ly6Chi T cells. We also showed that GITR is required for the optimal accumulation of all three effector CD8 T cell subsets, but with a greater effect on the Ly6Clo CX3CR1lo subset. Moreover, GITR showed a selective effect on the upregulation of CXCR6 on the less differentiated CX3CR1lo CD8 T cell subsets.

T-bet, a transcription factor that induces IFNγ production and Th1/Tc1 differentiation, is more highly expressed in terminally differentiated effector T cells (324, 325). Here, we show that during influenza infection Ly6Chi effector CD4 T cells had higher expression of T-bet than the Ly6Clo subset, suggesting that the Ly6Chi subset is more differentiated or contains more terminally differentiated effector cells (Figure 3.1). In addition, we show that the Ly6Chi subset produced more IFNγ and IL-2 on a per cell basis (Figure 3.1). These results are consistent with previous reports showing higher T-bet and IFNγ expression in Ly6Chi effector CD4 T cells compared to their Ly6Clo counterparts in LCMV as well as in γ-herpesvirus infections (325,

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326). Thus, the notion that Ly6C expression positively correlates with the differentiation state of effector T cells and the ability to produce cytokines may be generalizable to several viral infections. In comparison to the Ly6Chi CD4+ subset, we show that the Ly6Clo subset expresses a lower level of T-bet, preferentially enters the lung parenchyma and has an increased tendency to form lung CD4 Trm. This finding is in line with a previous report showing enhanced memory cell potential in Ly6Clo effector CD4 T cells during LCMV infection (325).

A previous study showed that during LCMV or VSV infections, CX3CR1 defines three effector CD8 T cell subpopulations with the CX3CR1hi subset expressing the highest levels of T- bet and KLRG1, a phenotype characteristic of terminally differentiated effector cells (214). In the same study, it was shown that CX3CR1lo cells give rise to CD8 Trm (214). In line with the findings of Gerlach et al. (214), here we show that during influenza infection, the Ly6Chi CX3CR1hi CD8+ subset exhibits a high level of T-bet and KLRG1, suggesting a more differentiated phenotype (Figure 3.10). In contrast, the Ly6Clo CX3CR1lo CD8+ subset expresses the least amount of KLRG1, suggesting that this subset is the least differentiated among the three subsets. Although we did not address the memory potential of the three subsets, recent work has shown that CD8 T cells that previously expressed KLRG1 can give rise to exKLRG1 cells that become memory T cells (338). Thus, KLRG1 expression does not rule out memory potential. Rather, CD127 expression seems to be a better marker of memory potential (338).

CD127 (IL-7Rα) is known to be expressed by naïve T cells, but downregulated upon T cell activation and re-expressed as cells transition to memory T cells (332, 333). Moreover, during acute LCMV infection, the CD127hi KLRG1lo population of effector cells has a greater propensity to give rise to memory T cells (323). Consistently, CD127 plays an important role in the homeostasis of both naïve and memory T cells (332, 333). Trm are also thought to arise from CD127+ memory precursor cells (146, 339, 340). Furthermore, treatment with IL-7 leads to increased accumulation of CD4 Trm-like cells in the lung after influenza infection and blocking CD127 leads to a reduction in the number of airway resident CD4 T cells in a Th2 allergy model (341, 342). Here, we show that GITR regulates CD127 expression in Ly6Clo effector CD4 T cells (Figure 3.6 and 3.7). Although the effects of GITR on CD127 expression are small, it is likely that those cells with highest CD127 are the ones most likely to persist to the memory phase. Thus, we speculate that one of the ways GITR contributes to optimal lung CD4 Trm accumulation could be through maintaining CD127 expression in Ly6Clo CD4 T cells. 126

The chemokine receptor CXCR6 has been implicated in the formation or maintenance of Trm in different tissue sites (70, 335, 343, 344). Zaid et al. showed that CXCR6 is required for the formation and local survival of skin CD8 Trm (335). Moreover, CXCR6 is expressed by liver CD8 Trm and is required for their maintenance (343, 344). A recent report showed that CXCR6 is required for the accumulation of CD8 Trm in the lung airways following influenza infection (70). We have shown that GITR is required for the optimal formation of lung CD4 and CD8 Trm after influenza infection (322). Here, using mixed bone marrow chimeras, we show that GITR regulates CXCR6 expression in both CD4 and CD8 T cells after influenza infection (Figure 3.7 and 3.13). It remains to be determined if the small increase in CXCR6 expression induced by GITR signaling contributes to the effect of GITR on Trm formation.

In this study, we showed that both the Ly6Chi and Ly6Clo CD4+ subsets are equally dependent on GITR costimulation for their accumulation when the entire population of CD44hi cells is examined (Figure 3.2 and 3.3). In contrast, when we focused on cytokine producing cells, we found that the cytokine producing Ly6Chi CD4 T cells are more dependent on GITR costimulation than their Ly6Clo counterparts (Figure 3.4). Cytokine producing Ly6Chi CD4 T cells produce more IFNγ and IL-2 than their Ly6Clo counterparts ((326) and this chapter). Thus, cytokine producing Ly6Chi CD4 T cells may need higher demands on protein translation machinery for cytokine production. GITR regulates the Akt-mTOR-pS6 pathway (244), an important determinant of cell size and protein translation (345). Thus, we speculate that cytokine producing Ly6Chi CD4 T cells are more dependent on GITR than their Ly6Clo counterparts due to their higher demand for protein translation.

Effector T cells are critical for initial control of infection, whereas memory T cells are critical for protection against reinfection. Previously, using GITR as a prototype, it was shown that TNFR family members provide a post-priming signal 4 for T cell accumulation (258). GITR is critical for viral control by CD8 T cells (243, 244). Moreover, GITR costimulation in the lung is important for optimal formation of lung CD4 and CD8 Trm after influenza infection (322). Here we have shown that GITR is important to allow both effector and memory precursor populations to persist. It makes sense that GITR might have differential effects on the effector T cell sub-populations. In the case of CD4 T cells, we saw that cytokine producing cells had a higher requirement for GITR, perhaps because of the greater demands on the mTOR pathway due to high level cytokine production. In contrast, in CD4 T cell populations that were enriched 127

in memory precursor potential, we saw a greater effect of GITR on CD127 expression. In the case of CD8 T cells, we saw the greatest effect of GITR on accumulation and CXCR6 expression on the less differentiated effector T cells, consistent with an important role for GITR in Trm formation (322). Thus, GITR plays a crucial role in sustaining both highly differentiated effector cells as well as precursors of Trm, with differential effects on effector subpopulations.

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Chapter 4

Summary and Future Directions

129

4 Summary and Future Directions

The T cell costimulatory molecule GITR contributes to optimal T cell responses against both acute and chronic viral infections. During acute influenza infection, GITR contributes to maximal CD8 T cell responses and protects mice against lethal infection (243). In chronic LCMV infection, GITR delivers a post-priming signal to virus specific CD4 T cells in the spleen, allowing for increased T cell accumulation and enhanced viral control (258). In this thesis, we provided evidence that GITR costimulation can take place both in the secondary lymphoid tissues as well as in a non-lymphoid tissue, the lung (Figure 4.1). This signal is necessary for the maximal accumulation of effector CD4 and CD8 T cells as well as optimal formation of lung CD4 and CD8 Trm during influenza infection (Figure 4.1). We showed that during influenza infection, lung effector CD4 and CD8 T cells can be subdivided into subpopulations with different memory potential (Figure 4.2). We further showed that GITR is required for sustaining both highly differentiated effector cells as well as precursors of Trm, but with differential effects on effector T cell subpopulations (Figure 4.2).

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Figure 4.1. Model of GITR costimulation. During influenza infection, T cells likely receive GITR costimulation (signal 4) first in the mLN by engaging GITRL expressing InfAPC. Then, T cells migrate to the lung tissue and receive GITR costimulation again in the lung tissue. GITR costimulation leads to enhanced accumulation of effector T cells and optimal Trm formation in the lung after influenza infection. This figure was modified from the manuscript “Monocyte-derived cells in tissue resident memory T cell formation” which was accepted for publication at the Journal of Immunology.

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Figure 4.2. Model of effect of GITR on effector T cell subpopulations. During influenza infection, CD4 and CD8 T cells likely receive GITR costimulation (signal 4) by interacting with inflammatory APC in the lung. GITR equally affects the accumulation of the two effector CD4 T cell subsets and regulates OX40 and CXCR6 expression in both subsets, but selectively regulates CD127 expression in the Ly6Clo subset. In CD8 T cells, GITR affects the accumulation of all three subsets with a predominant effect on the Ly6Clo CX3CR1lo subset. GITR selectively upregulates CXCR6 in both CX3CR1lo CD8 subsets. This figure was modified from the graphical abstract of the manuscript “GITR differentially affects lung effector T cell subpopulations during influenza virus infection” which was submitted to the Journal of Leukocyte Biology.

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By measuring the amount of tetramer binding and performing high throughput deep sequencing for the TCR repertoire, Van Gisbergen and colleagues have shown that the TNFR family member CD27 maintains a clonally diverse CD8 T cell repertoire by sustaining low affinity influenza NP-specific CD8 T cells (198). They further showed that by maintaining an overall low-affinity but diverse T cell repertoire, mice were better protected from secondary challenge with a heterologous influenza virus (198). In this thesis, by using the level of tetramer binding as a surrogate for TCR affinity, we show that the TNFR family member GITR can also rescue low affinity influenza NP-specific CD8 T cells (Figure 2.4). The results from the mixed bone marrow chimeras showed that this effect is due to GITR directly acting on CD8 T cells rather than indirectly through CD4 T cells or other immune cell types. Although it remains to be verified experimentally, it is plausible to speculate that GITR-mediated costimulation may augment protection against heterologous viruses by maintaining a low affinity but clonally diverse CD8 T cell repertoire. Furthermore, it will be of interest to examine if other members of the TNFR family members are involved in maintaining low affinity influenza-specific CD8 T cells, especially 4-1BB since it is known to be a potent stimulator of CD8 T cells (201, 208, 346).

The GITRL conditional knockout mice (GITRLexon2fl/fl mice) used in this thesis were designed to delete exon2 and cause a frame shift mutation, such that exon 3 would be put out of frame. However, we discovered that exon 1 was expressed from a different reading frame such that exon 3 was brought back into its normal reading frame, producing a truncated GITRL protein that can still bind to the anti-GITRL antibody (258). Further analysis showed that this truncated GITRL protein has reduced stability compared to the WT protein, but still retains some b function (258). In chapter 2, we observe an increase in the overall affinity of D /NP366-374- specific CD8 T cells in GITRLexon2fl/fl mice when compared to WT mice (Figure 2.4), but this effect was reduced when compared to GITR-/- mice, likely due to the residual function of GITRLΔexon2. Our laboratory has generated a new GITRL conditional knockout mice (GITRLexon3fl/fl mice) in which the extracellular domain of GITRL will be deleted by deleting exon 3. By crossing GITRLexon3fl/fl mice to inducible Cre-ERT2 mice, GITRL deletion can be controlled temporally using tamoxifen delivery. Timed deletion of GITRL allows the assessment to see if GITR costimulation is only needed at the effector stage or also plays a role in the maintenance of lung Trm after influenza infection.

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b OT-I T cells are transgenic CD8 T cells specific for OVA257-264 in the context of H-2K b while OT-II T cells are transgenic CD4 T cells specific for OVA323-339 in the context of I-A (347, 348). OT-I and OT-II mice are widely used to study many aspects of T cell biology such as T cell activation and thymic selection. However, results generated using OT-I mice may not reflect the endogenous immune responses since OT-I cells have a very high TCR affinity towards OVA antigen when compared to endogenous T cells. In this thesis, we show that GITR plays a more predominant role in CD8 T cell responses against influenza when compared to CD4 T cell responses (Figure 2.7). However, GITR+/+ OT-II from the lung had a much higher level of pS6 than GITR-/- OT-II from the same mice whereas GITR+/+ OT-I from the lung had a statistically significant but small increase in the level of pS6 than GITR-/- OT-I isolated from the same lung (Figure 2.15). It is plausible that the high affinity TCR receptor of OT-I cells makes the detection of differences in pS6 signals between GITR+/+ and GITR-/- cells more difficult. In contrast, the TCR affinity is weaker in OT-II cells when compared to OT-I cells, hence it is easier to detect differences in pS6 signals between GITR+/+ and GITR-/- OT-II cells (349). It will +/+ -/- b b be of interest to measure the level of pS6 in endogenous GITR and GITR D -NP366-374- or D -

PA224-233-specific CD8 T cells using mixed bone marrow chimeras. In chapter 3, to be more physiologically relevant, we compliment the results from transgenic OT-I and OT-II systems with the results from endogenous T cell responses using mixed bone marrow chimeras.

It is known that during chronic LCMV infection, GITR regulates the expression of CD25, CD127, and OX40 in LCMV-specific CD4 T cells (258). Similar to the LCMV model, we show that GITR regulates CD127 and OX40 expression in CD4 T cells during influenza infection in both transgenic models as well as endogenous responses (Figure 3.6 and 3.7). Interestingly, GITR regulates OX40 expression in both Ly6Chi and Ly6Clo effector CD4 T cells whereas GITR influences CD127 expression only in Ly6Clo but not Ly6Chi effector CD4 T cells. In contrast to CD4 T cells, OX40 is expressed at a substantially lower level on CD8 T cells and GITR has a small effect on OX40 levels in the Ly6Chi CX3CR1lo CD8 T cell population in the mLN but has no effect on other CD8 T cell subpopulations (Figure 3.13). To fully elucidate what GITR does to T cells at the transcriptome level, it would require a more in depth analysis with single cell approaches. One such approach is to co-transfer GITR+/+ and GITR-/- OT-II into the same recipient mice, followed by sorting GITR+/+ and GITR-/- OT-II cells within Ly6Chi and Ly6Clo

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effector CD4 T cell compartment after influenza infection, followed by single cell RNA sequencing.

Trm in the lung can be found in two distinct compartments, some localize in the lung airway, while others reside in the lung parenchyma (350). Lung airway Trm and lung parenchymal Trm have different properties. Even though lung airway Trm do not possess a high cytolytic capacity, they rapidly produce IFNγ in response to antigen re-exposure (118). In contrast, lung parenchymal Trm have a high cytolytic ability and rapidly divide upon antigen reactivation (118). Lung airway Trm can be isolated by a technique known as bronchoalveolar lavage (118). One limitation of our lung Trm studies is that we did not specifically isolate and analyze lung airway Trm through bronchoalveolar lavage. Instead, we analyzed the effect of GITR on Trm isolated from total lung tissue. Thus, the lung Trm population that we analyzed was likely a mixture of both lung airway and lung parenchymal Trm. Further experiments are required to assess the effect of GITR on lung airway Trm versus lung parenchymal Trm.

Unlike the stable Trm populations in the skin, the Trm populations in the lung are relatively shorted lived (113, 351). For instance: the number of influenza-specific Trm in the lung decreases over time reaching undetectable levels 7 months post influenza infection, resulting in the loss of protection against influenza virus (113). Why do lung Trm decay to undetectable levels overtime? This may be explained from a physiological standpoint. The lung is a delicate structure involved in respiration, a vital function of all living organisms. It is crucial that the immune responses in the lung be tightly regulated in order to clear the infections successfully with minimal collateral damage. The lung has evolved multiple mechanisms to prevent potential damage caused by excessive inflammatory immune responses. One such mechanism is the production of anti-inflammatory cytokine IL-10 by lung CD8 T cells after influenza infection (118, 352). Even though Trm are highly protective against respiratory pathogens, too many of these potentially harmful protectors residing in the lung increases the likelihood of immunopathology (353). Thus, the lung may have evolved mechanisms to slowly eliminate Trm from the tissue (353). One potential mechanism is through reduced Bcl-2 expression in lung Trm, rendering Trm more susceptible to apoptosis (339, 354). Takamura and colleagues showed that lung Trm reside in areas of active tissue regeneration after injury (known as RAMD), suggesting that these Trm cells may be involved in protecting damaged lung tissues (129). Since the decay of lung Trm coincides with tissue repair, the authors proposed that 135

maintaining a large number of lung Trm would not be needed once the damage tissues are repaired (129). However, our lab found that supraphysiological stimulation of 4-1BB in mice through delivery of replication defective adenovirus carrying 4-1BBL and influenza NP dramatically increased the number of lung CD8 Trm after influenza infection (208). This robust enlargement of the lung CD8 Trm population protected mice against secondary challenge without causing lung pathology (208). Thus, it may be desirable to explore vaccination strategies that induce lung Trm through enhancement of signal 4 ligands, such as 4-1BBL or GITRL.

In chapter 3, I show that GITR sustains both highly differentiated effector T cells as well as Trm precursors during influenza infection. Furthermore, in the absence of GITR, there is a defective accumulation of lung CD4 and CD8 Trm (Figure 2.5). These results have implications for inducing GITRL as a vaccine strategy to enhance lung Trm. Targeting GITR/GITRL pathway seems to be a safe choice since treatment of mice with agonist anti-GITR antibody did not cause splenomegaly or liver pathology (224). As shown in Figure 2.14, GITRL induction in the lung is partly mediated by IFN-I during influenza infection. Thus, one of the strategies to induce GITRL using vaccines is to include agonists or adjuvants that induce IFN-I production. Perhaps, this could be done through the inclusion of STING agonist since activation of STING leads to robust IFN-I production (355).

The initial activation of T cells requires 3 signals: antigen, costimulation by B7 molecules, and cytokines (295, 296). Early studies suggest that naïve T cells need only a brief interaction with APC to become fully activated and differentiate into effector and memory T cells (297, 298). However, later studies suggest that multiple encounters with APC are required for optimal T cell responses as well as formation of Trm (152, 299, 318). Recently, using GITR as a prototype, it was shown that costimulation through TNFR family members can be physically and temporally segregated from initial T cell priming with monocyte-derived InfAPC providing the signals during chronic LCMV infection (258). These signals are referred to as “signal 4” since they take place after initial T cell priming and are provided by monocyte-derived InfAPC instead of cDC (258). In chapter 2, I show that signal 4 coming from GITR can take place both in the secondary lymphoid tissues as well as in the lung tissue and is important for effector T cell and Trm accumulation after influenza infection. Why is signal 4 needed in the lung? Perhaps it serves as a post-priming checkpoint to see if T cells need to be maintained longer to clear infection in the local tissues. In support of this view, Lin and colleagues reported that signal 4 136

from 4-1BB is dispensable for CD8 T cell responses against mild influenza but is critical for CD8 T cell responses against severe influenza infection (201). With respect to signal 4, one key outstanding question remains: is antigen presentation necessary for TNFR family signals to T cells? One possible experiment to test this hypothesis is to cross LysM-Cre mice to MHC-IIfl/fl mice to delete MHC-II molecules on monocyte-derived InfAPC, the main providers of signal 4 to T cells, and assess the effect of signal 4 on T cell responses. In conclusion, this thesis explored different aspects of GITR/GITRL costimulation during influenza infection including its expression, timing, location, and effects on T cell subpopulations. We provided evidence that in addition to secondary lymphoid organs, GITR costimulation can occur in non-lymphoid tissues. This signal is important for the accumulation of both effector CD4 and CD8 T cells as well as optimal formation of lung CD4 and CD8 Trm after influenza infection. Furthermore, GITR is required for the accumulation of both highly differentiated effector cells and Trm precursors, but differentially affects different effector T cell subpopulations. Further work would be required to determine whether GITR plays a role in the maintenance of lung Trm after influenza infection. Understanding where and when GITRL signals are critical to T cells and which APC provide the GITRL signals has important implications for design of vaccines or immunotherapeutics. Targeting vaccines to the correct APC to induce GITRL and ensure its engagement in the immune response could lead to a much more lasting and effective immune response.

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Chapter 5

References

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