The Pennsylvania State University. The Graduate School. Department of Biochemistry, Microbiology and Molecular Biology

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1 The Pennsylvania State University The Graduate School Department of Biochemistry, Microbiology and Molecular Biology REGULATION OF T HELPER CELL RESPONSES AND ALLERGIC ASTHMA BY TEC KINASES, ITK AND TXK A Dissertation in Biochemistry, Microbiology and Molecular Biology by Nisebita Sahu 2008 Nisebita Sahu Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy May 2008

2 The dissertation of Nisebita Sahu was reviewed and approved* by the following: Avery August Associate Professor of Immunology Thesis Adviser Chair of Committee Andrea M. Mastro Professor of Microbiology and Cell Biology Robert F. Paulson Associate Professor of Veterinary Science Wendy Hanna-Rose Assistant Professor of Biochemistry and Molecular Biology Pamela A. Hankey Associate Professor of Immunology Biao He Associate Professor of Veterinary Science Nadine B. Smith Associate Professor of Bioengineering Richard J. Frisque Professor of Molecular Virology Head of the Department of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School

3 iii ABSTRACT The Tec family of non-receptor kinases has recently emerged as key players in intracellular signaling pathways of T lymphocytes. Tec kinases associate with and activate numerous proteins and serve as amplifiers of the signals generated downstream of the TCR signaling pathway. Lack of Tec kinase Itk reduces the development of life threatening T H 2 responses during allergic asthma in murine models. Hence, the pathways regulated by Itk that control the development of T H 2 cells or that regulate their cytokine production could represent good targets for controlling this disease. Therefore, in this work, we attempted to get better insight on the role that Itk plays in T H 2 responses during allergic asthma. Using a murine model of allergic asthma, we show here, that in the absence of Itk, there are decreased airway hyperresponsiveness and tracheal responses in addition to decreased airway inflammation and mucous production. We also observed decreased production of effector T H 2 specific cytokines in the lungs of these mice. These studies demonstrate that Itk plays an important role in developing all the various symptoms associated with allergic asthma. Allergic asthma is dependent on chemokine mediated T H 2 cell migration and T H 2 cytokine secretion into the lungs. Itk regulates the production of T H 2 cytokines as well as migration in response to chemokine gradients. However, the role of kinase activity of Itk in the development of this disease is unclear. In addition, whether distinct Itk-derived signals lead to T cell migration and secretion of T H 2 cytokines is also unknown. Using

4 iv transgenic mice specifically lacking Itk kinase activity, we show that active kinase signaling is required for control of T H 2 responses and development of allergic asthma. Moreover, dominant suppression of Itk kinase activity led to normal T H 2 responses, but significantly reduced chemokine mediated migration, resulting in prevention of allergic asthma. These observations indicate that signals required for T H 2 responses and migration are differentially sensitive to Itk kinase activity. The Tec kinases, Txk and Itk are believed to play an important role in regulating the differentiation of T helper cells. Itk is expressed in both T H 1 and T H 2 cells. By contrast, Txk is preferentially expressed in T H 1 cells. Although Itk is required for T H 2 responses in vivo and Txk is believed to regulate IFN-γ expression and T H 1 responses, it remains unclear whether these kinases have distinct roles in T H cell differentiation and/or function. To evaluate this question, we examined T H 2 responses in an allergic asthma model system using Itk-null mice overexpressing a Txk transgene. We found that the Txk transgene rescued all symptoms of allergic asthma without notable enhancement of IFN-γ expression. These results argue that Txk is not a specific regulator for T H 1 responses. Importantly, they suggest that Itk and Txk exert their effects on T H cell differentiation/function at the level of expression. To determine if the effect of Itk in the development of allergic asthma was evident in another model of asthma, we tested these mice using the common allergen, house dust mite. Our results show that the absence of Itk also prevented the development of the disease to various doses of this allergen. These data indicate that Itk mediates T H 2 responses to common allergens. The data also suggest that Itk regulates T H 2 responses

5 v via specific signaling mechanisms which cannot be overcome by increasing the amount of antigenic exposure. It has been suggested that cytokines IL-4, IL-5 and IL-13 play central roles in the development of allergic asthma, and that Itk regulates the production of these cytokines during the development of the disease. However, Itk is also believed to play an important role in mediating chemokine specific migration. In order to investigate the various roles of Itk, we determined if exogenously administered IL-4 or IL-13, or chemokine CCL-11 into the lungs of Itk null mice during the development of asthma bypassed the potential defects. Our results show that exogenous administration of IL-4 or IL-13 could rescue the development of allergic asthma in these mice suggesting that generation of these effector cytokines is the key function of Itk during induction of allergic asthma. Furthermore, we also show that addition of chemokine CCL-11 alone is able to partially rescue the symptoms associated with allergic asthma via eosinophil mediated IL-4 secretion. These data suggest that chemokines also contribute to the development of the disease by not only facilitating leukocyte migration but also by inducing effector cytokine production through eosinophils. In summary, these studies demonstrate that Itk plays a crucial role in the development of various symptoms associated with T H 2 specific allergic asthma by producing T H 2 cytokines and regulating chemokine-mediated migration in a kinase activity dependent manner. Hence, manipulation of Itk s kinase activity can provide a new strategy to treat allergic asthma by differentially affecting migration of T cells into the lungs, leaving T H 2 responses intact.

6 vi TABLE OF CONTENTS LIST OF FIGURES...x LIST OF TABLES...xiii ABBREVIATIONS...xiv ACKNOWLEDGEMENTS...xix Chapter 1 Literature Review Introduction Role of TCR in T cell activation T cell receptor signaling pathway Role of Src kinases in T cell activation Role of Syk kinases in T cell activation Role of Adapter proteins in T cell activation Role of intracellular calcium increase in T cell activation Role of Tec-family kinases in T cell activation Structure of Tec kinases Pleckstrin-Homology (PH) Domain Tec-Homology (TH) domain SRC homology 2 (SH2) domain SRC homology 3 (SH3) domain Kinase domain Factors regulating the activation of Tec family kinases Specific functions of Tec kinases in T cell activation Role of Transcription factors in T cell activation T helper (T H ) cells Factors affecting T helper cell lineage commitment Type of APC and concentration of antigen Strength of the signal TCR and costimulator mediated signaling pathways Cytokine milieu Transcription factors Specific Role of Tec kinases in T helper cell differentiation Possible Mechanisms for T helper cell lineage commitment Instructive Mechanism Stochastic/Selective Mechanism...38

7 1.3.4 Function of T helper 1 cells in diseases Function of T helper 2 cells in diseases Allergic Asthma Role of T cells and T H 2 cytokines in Allergic Asthma Role of other cell types in allergic asthma Role of Chemokines in allergic asthma Concluding Remarks...46 Chapter 2 Materials and Methods Mice Induction of Experimental Allergic Asthma Analysis of AHR Analysis of tracheal responses Adoptive transfer of antigen specific cell populations Histology Quantitative RT-PCR analysis Analysis of leukocyte population in the lungs and Intracellular staining ELISA Analysis of lymphocyte proliferation and cytokine secretion Analysis of IgE levels CCL11 mediated actin polymerization assay CCL-11 mediated migration assay NFAT nuclear localization analysis Dendritic cell isolation and Flow cytometry Data analysis...64 Chapter 3 Reduced airway hyperresponsiveness and tracheal responses during allergic asthma in mice lacking tyrosine kinase inducible T-cell kinase Introduction Results Itk null mice exhibit reduced Airway Hyperresponsiveness during allergic asthma induction Itk null mice exhibit reduced tracheal contractile responses to Acetylcholine, Carbachol and KCl during allergic asthma induction Reduced T H 2 cytokines in lungs of Itk null mice during allergic asthma induction Rescue of AHR responses in Itk null mice by transfer of antigen specific WT CD4 + T cells Discussion...72 Chapter 4 Differential sensitivity to Itk kinase signals for T helper 2 cytokine production and chemokine mediated migration...81 vii

8 4.1 Introduction Results The kinase domain of Itk is required for development of AHR and airway inflammation The kinase domain of Itk is required for the expression of T H 2 cytokines in the lung during allergic airway inflammation Role of the kinase domain of Itk in modulating the ability of T cells to proliferate and induce T H 2 responses to OVA The kinase domain of Itk is required for T cell recruitment into the lung and chemokine induced migration in vitro Discussion...87 Chapter 5 Selective expression rather than specific function of Txk and Itk regulate T H 1 and T H 2 responses Introduction Results The CD2 promoter driven Txk transgene is expressed in T H 2 cells Rescue of allergic airway inflammation and airway hyperresponsiveness (AHR) in mice lacking Itk by expression of Txk transgene Expression of Txk transgene in Itk-null mice enhances production of T H 2 cytokines in response to allergic inflammation Rescue of CD4 + T cell recruitment in Itk-null mice expressing Txk transgene Discussion Chapter 6 Absence of ITK prevents the development of allergic airway disease irrespective of the level of allergen exposure Introduction Results Exposure to varying doses of HDM extract does not lead to the development AHR in mice lacking Itk WT but not Itk null mice develop lung pathology in response to HDM extract Dose dependent effects of HDM extract exposure on T H 2 cytokine and chemokine production in WT but not Itk null mice No difference in dendritic cell mediated allergen uptake between WT and Itk null mice Discussion Chapter 7 Eotaxin/CCL-11 rescues airway hyperresponsiveness and airway inflammation in mice lacking Tec kinase, Itk viii

9 7.1 Introduction Results Effect of IL-4 and IL-13 cytokine addition on Itk null mice in response to OVA Introduction of exogenous Eotaxin/CCL-11 into the lungs of Itk -/- mice induces AHR and leukocyte recruitment Eotaxin/CCL-11 partially rescues effector cytokines IL-4, IL-5 and IL-13 and chemokine CCL-7 production in the lungs of Itk -/- mice Eotaxin/CCL-11 triggers eosinophil mediated IL-4 production in lungs of OVA-challenged Itk -/- mice In vivo exposure to Eotaxin/CCL-11 rescues in vitro proliferation and cytokine production of Itk -/- T lymphocytes in response to OVA Effect of CCL-11 on expression of Tec in T H 2 cells from WT and Itk -/- mice CCL-11 mediates increased IL-4 production in in vitro differentiated WT T H 2 cells but not Itk -/- T H 2 cells Discussion Chapter 8 Conclusions and Discussion Bibliography ix

10 LIST OF FIGURES x Figure 1.1: TCR signaling pathway...48 Figure 1.2: Structure of Tec kinases...49 Figure 1.3: T H 1 signaling pathway...50 Figure 1.4: T H 2 signaling pathway...51 Figure 1.5: Instructive Mechanism of T helper cell differentiation...52 Figure 1.6: Stochastic / Selective Mechanism of T helper cell differentiation...53 Figure 3.1: Reduced basal and OVA induced AHR in mice lacking Itk following induction of Allergic Asthma...75 Figure 3.2: Reduced tracheal responses to cholinergic stimulation in mice lacking Itk...76 Figure 3.3: Reduced tracheal responses to KCl stimulation in mice lacking Itk...77 Figure 3.4: Reduced T H 2 cytokine message in lungs of mice lacking Itk during allergic asthma induction...78 Figure 3.5: Rescue of AHR in mice lacking Itk by transfer of CD4 + T cells...79 Figure 3.6: Rescue of airway inflammation in mice lacking Itk by transfer of CD4 + T cells...80 Figure 4.1: The kinase domain of Itk is required for the induction of AHR and allergic inflammation...92 Figure 4.2: The kinase domain of Itk is required for the production of T H 2 cytokines in the lung during the development of allergic asthma...93 Figure 4.3: Role of the kinase domain of Itk in T cell responses and T H 2 development in response to OVA immunization...94 Figure 4.4: Reduced Itk signals specifically affect chemokine mediated migration in Itk Kin Tg /WT T cells...95 Figure 5.1: Rescue of AHR in Itk-null mice by expression of the related kinase Txk...107

11 xi Figure 5.2: Txk-mediated rescue of cytokine production in vitro and in vivo Figure 5.3: Rescue of CD4 + T cell recruitment into the lungs of Itk-null mice by expression of the related kinase Txk Figure 6.1: Reduced AHR to varying doses of HDM in mice lacking Itk following induction of Allergic Asthma Figure 6.2: Reduced airway inflammation in mice lacking Itk after exposure to different doses of HDM Figure 6.3: Reduced mucous production in mice lacking Itk after exposure to different doses of HDM Figure 6.4: Dose dependent effects of HDM extract exposure on T H 2 cytokine production in WT but not Itk null mice Figure 6.5: Dose dependent effects of HDM extract exposure on chemokine production in WT but not Itk null mice Figure 6.6: No difference in dendritic cell mediated allergen uptake between WT and Itk null mice Figure 7.1: CCR3 chemokine mediated signaling pathway Figure 7.2: Rescue of AHR by addition of IL-4 and IL-13 cytokine in Itk null mice in response to OVA Figure 7.3: Rescue of airway inflammation and mucous production by addition of IL-4 and IL-13 cytokines in Itk null mice in response to OVA Figure 7.4: Rescue of T H 2 cytokines and chemokines in the lung of Itk -/- mice during the development of allergic asthma by addition of IL Figure 7.5: Rescue of chemokines in the lung of Itk -/- mice during the development of allergic asthma by addition of IL Figure 7.6: Rescue of CD4 + T cell and eosinophil recruitment into the lungs of Itk-null mice by addition of IL Figure 7.7: Rescue of AHR and leukocyte recruitment in the lungs by addition of CCL-11 with or without IL-13 cytokine in Itk null mice in response to OVA...146

12 Figure 7.8: Rescue of airway inflammation and mucous production by addition of CCL-11 with or without IL-13 cytokine in Itk null mice in response to OVA Figure 7.9: Rescue of T H 2 cytokines and chemokines in the lung of Itk -/- mice during the development of allergic asthma by addition of CCL-11 or CCL-11 and IL Figure 7.10: CCL-11 mediated rescue of cytokine production in BAL fluid Figure 7.11: CCL-11 mediated rescue of proliferation in vitro Figure 7.12: CCL-11 mediated rescue of cytokine production in vitro Figure 7.13: CCL-11 rescues IL-4 production Figure 8.1: Functions of Itk and Txk in T helper cell differentiation Figure 8.2: Differential sensitivity to signal strength for T H 2 cytokine production and chemokine mediated migration Figure 8.3: Proposed model for role of Tec kinases Txk and Itk in T helper cell signaling Figure 8.4: Proposed model for the role of Itk in the development of allergic asthma xii

13 LIST OF TABLES xiii Table 1.1: Summary of proteins downstream of TCR that contribute to T helper cell differentiation...54

14 ABBREVIATIONS xiv Ach Acetylcholine ADAP Adhesion and Degranulation promoting adapter protein AHR Airway Hyperresponsiveness AP-1 Activator Protein-1 APC Antigen presenting cell ATF Activating transcription factor BAL Bronchioalveolar Lavage BALF Bronchioalveolar Lavage Fluid BMX Bone-marrow tyrosine kinase gene on chromosome X BTK Bruton s Tyrosine Kinase Ca 2+ Cas-L Cbl CBP CCh CCL CCR Cdc42 CRAC CREB CsA CXCL CXCR DAG Dll-4 Calcium Crk-associated substrate lymphocyte type Casitase b-lineage Lymphoma CREB binding protein Carbachol Chemokine (C-C motif) ligand Chemokine (C-C motif) receptor Cell division control protein-42 Ca 2+ release activated Ca 2+ channel c-amp response element binding protein Cyclosporin A Chemokine (C-X-C motif) ligand Chemokine (C-X-C motif) receptor Diacylglycerol Delta like ligand EBI3 Epstein Barr virus induced gene 3 ECP Eosinophilic cationic protein EGFP Enhanced Green Fluorescence protein

15 ELISA Enzyme Linked ImmunoAssay EMT Expressed in Mast cells and T lymphocytes ER Endoplasmic Reticulum ERK Extracellular Response Kinase Eta-1 Early T lymphocyte activation-1 FBI Fluorescent bead immunoassay FOG Friend of GATA-1 GADD Growth Arrest and DNA damage G-CSF Granulocyte colony stimulating factor GDP Guanosine diphosphate GEF Guanine exchange factor GEM Glycolipid enriched membrane GMCSF Granulocyte-macrophage colony stimulating factor Grb-2 Growth factor receptor bound protein 2 GTP Guanosine triphosphate H&E Hematoxylin and Eosin HDM House Dust Mite HRP Horseradish Peroxidase ICAM Intercellular Adhesion Molecule ICOS Inducible Costimulator IEL Intraepithelial lymphocytes IFN-γ Interferon-γ IFN-γR Interferon-γ Receptor IgE Immunoglobulin E IgG2a Immunoglobulin G2a IL Interleukin IL-12R Interleukin - 12 Receptor IL-4R Interleukin- 4 Receptor inkt invariant Natural Killer T cell xv

16 IP3 IRAK IRF-1 IRS-2 ITAM ITK IқB JAK JNK KCl LAT LCK LFA-1 LT MAPK MBP MCP-1 MDC MEK MHC mrna NFAT NF-ҚB NIP45 NK OVA PAS PBS PCR Inositol Triphosphate IL-1R associated kinase Interferon regulatory factor-1 Insulin receptor substrate-2 Immunoreceptor tyrosine based activation motifs Interleukin-2-inducible T-cell Kinase Inhibitory protein қb Janus Kinase Jun N-terminal Kinase Potassium Chloride Linker of activated T cells Lymphocyte specific protein tyrosine kinase Lymphocyte Function-associated Antigen-1 Leukotriene Mitogen Activated Protein Kinase Major Basic Protein Monocyte Chemoattractant Protein-1 Macrophage derived chemokine Mitogen activated ERK-activating Kinase Major Histocompatibility Complex messenger Ribonucleic Acid Nuclear Factor of Activated T cells Nuclear Factor kappa B NFAT interacting protein Natural Killer Ovalbumin Periodic acid-schiff Phosphate Buffered Saline Polymerase Chain Reaction xvi

17 xvii PH Pleckstrin Homology PI3K Phosphotidylinositol 3-Kinase PIP2 Phosphoinositol biphosphate PIP3 Phosphoinositol triphosphate PIP5K Phosphatidylinositol-3-phosphate phosphatidylinositol 5-kinase PKC Protein Kinase C PLCγ Phospholipase C gamma 1 PMA Phorbol Myristoyl Acetate PRR Proline rich region PTB Phosphotyrosine binding pocket PTEN Phosphatase and Tensin homolog PTK Protein tyrosine kinase QRT-PCR Quantitative Reverse transcription-pcr RIBP Rlk and Itk binding protein RLK Resting Lymphocyte kinase ROG Repressor of GATA ROR Retinoic orphan receptor SDF1-α Stromal cell derived factor 1-α SH2 Src Homology domain 2 SH3 Src Homology domain 3 Shc SH2 adapter protein SHIP Src-Homology 2 domain-containing Inositol Phosphatase SHP-1 Src-Homology domain-containing tyrosine Phosphatase SLAP-130 SLP-76 associated phosphoprotein SLAT SWAP-70 like adapter of T cells SLP-76 SH2 domain-containing Leukocyte Phophoprotein of 76kDa SOCS Suppressor of cytokine signaling SOS Son of Sevenless SRF Serum responsive factor

18 STAT TCCR TCR TEC TH TNFα TRAF TSK VCAM WASP WT XID XLA ZAP70 Signal transducer and activators of transcription T cell cytokine receptor T cell receptor Tyrosine kinase Expressed in hepatocellular Carcinoma T Helper Tumor Necrosis Factor TNF receptor associated factor T cell specific kinase Vascular cell adhesion molecule Wiskott-Aldrich syndrome protein Wild type X-linked immunodeficiency X-linked agammaglobulinaemia Zeta chain Associated Protein xviii

19 ACKNOWLEDGEMENTS xix I would first like to thank my adviser Dr. Avery August for his immense support and cooperation over the years. He has helped and guided me at every step of the way and has been extremely understanding and helpful both at a professional as well as personal level. It was a tremendous experience working with him and has taught me valuable lessons and has helped me grow in every aspect. I truly appreciate all the faith and confidence he had in me and for being extremely patient and giving me the freedom to grow scientifically. I would like to sincerely thank my committee members: Dr. Robert Paulson, Dr. Andrea Mastro, Dr. Pamela Hankey, Dr. Wendy Hanna-Rose, Dr. Biao He and Dr. Nadine Smith for their constant support, valuable suggestions and constructive criticism. I would also like to thank all the past and present members of the August lab for all their help and support. It was a great experience to work in such a friendly and amicable atmosphere and definitely helped me get through the rough times of research. I would like to thank Cynthia Mueller for training me initially. I would also like to specially thank Meg Potter for always going the extra mile to help me in every way possible and for doing a great job taking care of our innumerous mouse strains without which none of my research would have been possible. I thank Elaine Kunze, Susan Magargee and Nicole Bem at the Pennstate s Centre for Quantitative Cell Analysis facility for all their patience and help. I would also like to thank Dr. Deborah Grove at Pennstate s Nucleic Acid Facility for her extensive help with my quantitative RT-PCR analysis and Roberta Horner at Animal Diagnostic Laboratory

20 for her help with tissue sectioning and histology. I would also like to thank all the xx members of CMIID and BMB department for their help and cooperation. I would like to sincerely thank my best friends, for believing in me tremendously and for always being there for me during all my good and bad times. Above all, I extend my deepest and heartfelt gratitude to my parents for everything that they have done for me. To my dad for all those sleepless nights at the hospital taking care of mom, for taking care of all the responsibilities at home as well as office, for all his love, affection and devotion for the family and for always doing everything possible in the world to give us all the comfort and happiness amidst all our adversities. To my mom, for always being very optimistic, strong and encouraging. Even with her disabilities, she has always had a great attitude towards life and has always been a great fighter. She has taught me to make the most out of even the worst situations and to never give up under any circumstance. I consider myself extremely blessed to have such great personalities as my mom and dad. And I am immensely thankful to them for always believing in me and helping me accomplish my dreams. I would also like to thank all the rest of my family members for their help and support. I wish to dedicate this work to my parents and my best friends for their endless love and support without which none of this would have been possible. I hope I have made all of you proud.

21 1 Chapter 1 Literature Review 1.0 Introduction T cells are lymphocytes that belong to agranulocyte group of white blood cells and play a central role in cell-mediated immunity. T cells are one of the first hematopoietic cells to be discovered that are generated in the thymus instead of the bone marrow. This unique feature led to the name T cells, where the abbreviation T stands for thymus. They also differ from other lymphocytes such as B cells and NK cells by having a specific receptor complex on the cell surface called the T cell receptor (TCR) complex via which T cells receive specific responses. T cell receptors belong to the immunoglobulin supergene family and are most commonly composed of α and β polypeptide chains, which play a critical role in the ability of T cells to recognize specific antigens, leading to their activation. T cells usually get activated upon exposure to antigens that are presented as antigen-mhc complexes on antigen presenting cells (APC s). Upon antigen exposure, some of these naïve T cells transform into memory T cells which are antigen specific; and as the name suggests, provide memory for more rapid response to the specific antigen by persisting long term after an infection and expanding to large numbers of effector T cells upon re-exposure to the antigen. There are several different subsets of T cells including Helper T cells (T H cells), Cytotoxic T cells (T C cells), Regulatory T cells (T reg cells), Natural Killer T cells (NKT

22 cells) and γδ T cells, each having a specific function. T H cells, as the name suggests, 2 help in regulating the immune response by secreting small proteins called cytokines. Naïve T H (T H 0) cells can differentiate into various subsets of T helper cells such as T H 1, T H 2 and T H 17 depending on the kind of cytokine signals received. T H cells express CD4 glycoprotein which helps these cells to bind to antigen expressed by class II MHC molecules on antigen presenting cells and get activated. T C cells express CD8 glycoprotein which binds to class I MHC molecules. These cells are involved in destroying virally infected cells and tumor cells. They also play a role in transplant rejection. Treg cells, also known as suppressor T cells help in the maintenance of immunological tolerance. NKT cells are involved in innate immune response by recognizing glycolipid antigens presented by CD1d. γδ T cells are T cells whose TCR is comprised of γ-and δ-chains instead of α- and β-tcr chains. They only represent about 5% of the total T cell population, are mainly present in gut mucosa and are part of intraepithelial lymphocytes (IEL). In some cases these T cells have been shown to be able to recognize whole proteins and to not require antigen presentation by MHC molecules. 1.1 Role of TCR in T cell activation The T-cell antigen receptor is responsible for the intitation of T cell activation in response to foreign antigens that eventually lead to clonal expansion, differentiation, cytotoxic responses or programmed cell death. The TCR determines the specificity of the T cell response. However, it is dependent on coreceptors such as CD2, CD4, CD8 and co-

23 3 stimulators such as CD28 to induce downstream signaling pathways and to modulate the signal threshold. Structure of TCR The TCR is a heterodimer of the immunoglobulin supergene family and is composed of α and β polypeptide chains linked by disulphide bonds (1). However, the TCR lacks a cytoplasmic tail, and is incapable of inducing any downstream signaling on its own. Instead the TCR is associated with proteins of the CD3 complex that transduce downstream signals by recruiting various families of cytoplasmic tyrosine kinases to the membrane. The proteins of the CD3 complex are the γ, δ and ε chains, a ζ-ζ homodimer or a ζ-η heterodimer (2, 3). The γ, δ and ε chains have single immunoreceptor tyrosine based activation motifs (ITAMs), while the ζ chain has three ITAM motifs (4). These tyrosine residues in the ITAM motifs are phosphorylated by Src kinases, Lck and Fyn upon TCR activation and eventually provide a platform for the recruitment of the Syk kinase, ZAP-70 to the TCR-CD3 complex which is essential for further downstream signaling (5-7) (Figure 1.1). T cell activation is initiated upon binding of antigen by TCR in the MHC-peptide complexes on the antigen presenting cells (APC s). Upon binding to the APC s, the T cells polarize towards the APC s, the TCR/CD3 complex enter into lipid rafts, and recruitment and polymerization of F-actin and other signaling molecules to the site of TCR stimulation is initiated (8). The CD4 or CD8 coreceptors and costimulators such as CD28 or ICOS then cluster at the site of interaction by binding to their ligands CD80 and CD86 (for CD28) and ICOS-L (for ICOS-ligand) expressed on the APC (9-12). This leads to the formation of a highly ordered membrane-associated complex, the

24 immunological synapse. The central zone of immunological synapse is composed of 4 TCR/CD3 complexes and other intracellular signaling proteins, while the outer zone consists of adhesion molecules such as LFA-1 which stabilizes the interaction between the T cell and APC and mediates sustained T cell activation (13). 1.2 T cell receptor signaling pathway TCR binding to antigen-bearing APC leads to the activation of CD3-associated tyrosine kinases, calcium mobilization, activation of protein kinase C (PKC), initiation of the Ras signaling cascade, which finally leads to the production of cytokines such as IL-2 and cell proliferation (Figure 1.1) Role of Src kinases in T cell activation The two Src kinases that have been shown to be involved in TCR mediated signaling are Lck and Fyn. These proteins are the key proximal kinases downstream of the TCR and play a critical role in transmitting intracellular signals. Both of these proteins have N-terminal attachment sites for saturated fatty acid addition, a unique region, a SH3 domain, a SH2 domain, a tyrosine kinase domain and a C-terminal regulatory domain. The N-terminal attachment sites allow myristoylation of these proteins which facilitates these proteins to insert into the inner leaflet of the plasma membrane. The unique region includes a di-cysteine motif which mediates Lck interaction with coreceptors CD4 and CD8 via their cysteine residues in the cytoplasmic

25 5 domain. The SH2 and SH3 domains help Lck and Fyn to interact with other proteins, and can regulate this kinase by undergoing conformational changes, thus rendering the kinase domain accessible (14). The kinase activity of these proteins is regulated by the phosphorylation status of their tyrosine residue (Y 505 in Lck and Y 508 in Fyn). In an inactive state, this tyrosine residue is maintained in the phosphorylated state via the Csk kinase. CD45, a transmembrane phosphatase, dephosphorylates this residue, allowing the kinase domain to autophosphorylate and become active (15). Upon TCR activation, Lck is recruited to the vicinity of TCR/CD3 complex along with those Lck molecules associated with coreceptors CD4 and CD8 (16). The kinase activity of Lck is then increased, allowing it to phosphorylate the ITAM motifs of the CD3 complex followed by the recruitment and activation of ZAP-70 (17) Role of Syk kinases in T cell activation Syk kinases, Syk and ZAP-70 have been shown to play an important role in activating the proximal events downstream of Src kinases in TCR signaling. Although ZAP-70 has been proven to be critical, the role that Syk plays still remains controversial as it is expressed at very low levels in T cells. ZAP-70 is composed of two tandem amino-terminal SH2 domains, an extended interdomain consisting of interdomain A and B, and the kinase domain (18, 19). The two tandem SH2 domains interact in a cooperative manner in order to form the complete phosphotyrosine-binding (PTB) pocket. The PTB pocket mediates the recruitment of ZAP-70 to the plasma membrane via interaction with phosphorylated ITAM motifs of CD3 complex. The interdomain A plays

26 6 a crucial role in stabilizing this interaction between the two SH2 domain via its tyrosine residue Y 126, which upon phosphorylation induces a conformational change required for the interaction (17, 20). The tyrosine residues Y 315, Y 319 and Y 292 in the interdomain B of ZAP-70 help in regulating its kinase activity and mediating its interactions with other proteins. Upon phosphorylation, Y 315 acts as the docking site for Vav, via its SH2 domain (21). Y 319 phosphorylation helps in the recruitment of SH2 domains of Lck as well as PLCγ-1 (22, 23). Once phosphorylated, Y 292 acts as a docking site for inhibitory protein Cbl and helps in the negative regulation of ZAP-70 function (24). The tyrosine site, Y 493 in the activation loop of the kinase domain of ZAP-70, is the site which is phosphorylated by Lck and induces its kinase activity (25, 26) that leads to phosphorylation of adapter proteins, SLP-76 and LAT. Y 492 has been shown to have negative regulatory effects for kinase activity of ZAP-70 (26) Role of Adapter proteins in T cell activation Adapter proteins are intracellular molecules that lack any enzymatic activity but consist of modular domains such as SH2, SH3, PTB, tyrosine and/or proline rich domains which help in the recruitment of other cytoplasmic proteins to assemble into multiprotein signaling complexes (27). Thus, adapter proteins help in providing a platform for enabling the formation of multimolecular complexes that are required for efficient downstream TCR mediated signaling. There are adapter proteins such as SLP-76, LAT, Grb2 and Shc which help in the positive regulation of TCR signaling. However, other

27 adapter proteins such as Cbl, Crk, Cas-L and SLAP-130 negatively regulate TCR 7 signaling by inhibiting Ras signaling and SLP-76 mediated NFAT activation (27). LAT consists of only a putative transmembrane domain that helps it to localize in the glycolipid- enriched membrane (GEM) fraction of the plasma membrane and numerous tyrosine residues that can act as docking sites after phosphorylation. Upon TCR activation, ZAP-70 mediates the phosphorylation of the tyrosine sites on LAT which then enables the recruitment of Grb2, PLCγ-1, p85 subunit of PI3K, NCK, ADAP and Sos to the GEMS thereby forming a multimolecular complex. This eventually leads to signal amplification, formation of second messengers and activation of Ras. Phosphatase CD148 dephosphorylates LAT and PLCγ-1 and regulates their activities. SLP-76 consists of an acidic domain, a tyrosine rich region, a proline rich region and a SH2 domain. Upon TCR activation, there is rapid tyrosine phosphorylation of SLP- 76 via ZAP-70, which is immediately followed by binding of SH2 domain of Vav to its acidic domain. SLP-76 also associates with Grb2. Vav helps in the activation of Rac-1, while SLP-76 mediates PLC-γ activation, increase in calcium flux, ERK activation and NFAT activation. ITK has also been reported to bind and phosphorylate SLP-76 which in turn maintains ITK in an active state (28, 29). Grb-2 consists of two SH3 domains and one SH2 domain. Upon TCR activation, Grb2 binds to phosphotyrosine residues on the ζ-chain ITAMs, resulting in the recruitment of Sos to the plasma membrane. The Grb-2-Sos complex also associates with LAT, SLP-76 and also mediates transient activation of Ras.

28 1.2.4 Role of intracellular calcium increase in T cell activation 8 Increases in intracellular calcium lead to the activation of many signaling pathways. Upon TCR activation, PLCγ activation induces production of second messenger, IP 3. IP3 binds to IP3 receptors present on the membrane of endoplasmic reticulum (ER) and causes the opening of Ca2 + channels of the ER (30). This increase in cytoplasmic levels of Ca2 + further mediates the opening of the store-operated Ca2 + channel, the CRAC channel, on the plasma membrane leading to a large influx of Ca2 + into the cells (31). These calcium signals are required for stabilizing the interaction between the T cell and APC carrying the correct antigen by preventing the crawling of T cells (32, 33). Calcium signals also function in regulating the reorientation of the actin cytoskeleton and in bringing coreceptors to the contact zone. Calcium signaling is required for activation of transcription factors such as NF-қB, AP-1 and NFAT. Spikes of intracellular Ca 2+ increases are sufficient for inducing NF-қB and AP-1 activation because they have low sensitivity to Ca 2+ and their activity persists for a longer time. However, transient to repetitive oscillations of Ca 2+ influx that eventually generate a low plateau is required for the induction NFAT activation, because NFAT has higher Ca 2+ sensitivity and its activity is highly reversible. These low oscillations also help T cells detect weak stimuli and low doses of antigens, thereby maximizing the sensitivity of T cells (34, 35). The calcium in the cytosol binds to calmodulin, which in turn activates the phosphatase, calcineurin. Upon activation, calcineurin dephosphorylates serine residues present on NFAT, allowing for its activation and nuclear translocation. Calcineurin also migrates with NFAT into the nucleus and maintains NFAT in a transcriptionally active

29 state which is necessary for normal function of NFAT to induce cytokine production (36, 37) Role of Tec-family kinases in T cell activation Tec kinases have been shown to be important mediators for actin reorganization, activation of PLCγ, mobilization of calcium, activation of MAP kinases and IL-2 production during T cell signaling. The Tec family of kinases consists of five members, Tec, Btk, Itk, Rlk/Txk and Bmx (38). They are mainly expressed by hematopoietic cells. They lack the extracellular ligand binding domain and hence belong to the non-receptor or cytoplasmic group of Tyrosine kinases. They are involved in signal transduction required for activation of cells for proliferation, differentiation and development. Among the five Tec-family kinases, Tec (Tyrosine kinase Expressed in hepatocellular Carcinoma) was the first to be discovered from a mouse liver cdna library. It was also found to be expressed in T cells (39). Btk (Bruton s Tyrosine Kinase) was the second Tec-family kinase to be discovered in 1993 as the tyrosine kinase which when mutated causes severe B-cell immunodeficiency X-linked agammaglobulinaemia (XLA) in humans and X-linked immunodeficiency (XID) in mice. It was not until this discovery that the importance of Tec family of kinases was realized (40, 41). Itk (Interleukin-2-inducible T-cell Kinase; also known as Emt or Tsk) was later discovered during cloning of T-cell specific tyrosine kinases using degenerate PCR, as a tyrosine kinase which was dependent on IL-2 for induction (42-46). Itk was later shown to be

30 10 expressed at high levels in patients with atopic dermatitis suggesting a regulatory role in this disease (47). Itk has also been shown to form Itk-Syk fusion proteins via chromosomal translocation in peripheral T cell lymphomas (48). Rlk (Resting Lymphocyte Kinase; also known as Txk) was also found during similar degenerate PCR screening for novel T-cell specific tyrosine kinases (49-52). Rlk was reported to be expressed in resting mature T cells and later specifically in T H 1 cells (51, 53-55). Increased levels of Rlk were also reported in patients with Behcet s disease (56). Bmx (Bone-marrow tyrosine kinase gene on chromosome X; also known as Etk) was discovered only in hematopoietic cells, but not T cells. Hence while Btk and Tec were found to be expressed in B cells, only three of the Tec family kinases namely, Tec, Itk and Rlk/Txk were found to be expressed in T cells. Of these, Itk has been shown to be the main Tec kinase that is expressed in naïve mouse T cells at the mrna level. In comparison, Txk is expressed at 10 fold lower levels while Tec is expressed at 100-fold lower levels than Itk in naïve T cells (57, 58). In activated T cells, expression of Itk is further increased, especially in T H 2 cells, while expression of Txk rapidly decreases initially but later increases specifically in T H 1 cells (59). In contrast, Tec is upregulated in activated T cells only after several days (39). In addition, Tec kinases have also been shown to be expressed in various myeloid lineages such as mast cells, platelets, macrophages, neutrophils and dendritic cells. Btk, Itk and Tec are expressed in mast cells, Btk and Tec in platelets, Bmx, Btk and Tec in macrophages and neutrophils and Btk in dendritic cells (60).

31 Structure of Tec kinases 11 These five tyrosine kinases share the following domain organization: an aminoterminal Pleckstrin-Homology (PH) Domain, followed by a Tec-Homology (TH) domain, a SRC homology 3 (SH3) domain, a SRC homology 2 (SH2) domain and finally the Kinase domain at the C-terminus (Figure 1.2). Except for the PH domain, which is absent in Rlk/Txk, all of these domains have similar functions in all the Tec-family kinases. Rlk contains a cysteine string motif instead of the PH domain and is always localized at the plasma membrane after myristoylation (Figure 1.2). Due to these unique structural characteristics, especially the PH domain, all of these five tyrosine kinases were later subgrouped as Tec-family of kinases Pleckstrin-Homology (PH) Domain The PH domain is required by the Tec-family kinases to be recruited to the plasma membrane via binding to the Phosphatidylinositol-3,4,5-triphosphates (PtdIns(3,4,5)P 3 ) present on the plasma membrane. Among the Tec family of kinases, Txk/Rlk is an exception in not having a PH domain. Instead, Txk exists in two isoforms. The longer isoform consists of a cysteine string motif which upon palmitoylation gets recruited to the plasma membrane. The shorter isoform lacks the cysteine string motif and is always localized in the nucleus (61).

32 Tec-Homology (TH) domain 12 It is suggested that TH domain is involved in regulating conformational changes in the Tec-family kinases in response to stimulation and also in inducing interactions between the Tec-family kinases and various other proteins. It has been found that these functions of the TH domain are due to the presence of Proline rich regions (PRRs) which are capable of binding to the other proteins via their SH3 domains. Since the Tec-family kinases also contain SH3 domains, the PRRs can therefore either bind to itself as seen in Itk and induce intramolecular interactions or can also induce intermolecular interactions between Tec-family kinases and SH3 domain bearing other proteins (62-64). Under conditions of low concentrations of Itk, the SH3 domain and PRR region on the same molecule has been suggested to form an intramolecular interaction. But under high concentrations, Itk is suggested to form an intermolecular dimer by undergoing reciprocal interactions between SH2 domain of one molecule and SH3 domain of the other molecule. The intramolecular interactions are believed to lead to closed conformation, rendering the Tec-family kinases inactive by preventing them from binding to other proteins. The intermolecular interactions on the other hand leads to the activation of various other proteins thereby leading to the activation of multiple pathways downstream required for the activation of the cells. Tec also can undergo both intra and intermolecular interactions via its SH3 and PRR regions; however, Tec is believed to exist preferentially as an intermolecular dimer. Txk/Rlk on the other hand is predicted to undergo only intermolecular interactions between SH3 and PRR regions and may exist as a homodimer (65).

33 SRC homology 2 (SH2) domain 13 The SH2 domain helps the Tec-family kinases to interact with other proteins by binding to tyrosine residues present on those proteins. The interaction between the SH2 domain and the SH3 domain on the same molecule may also regulate Tec-family kinasemediated activation of other proteins. The SH2 domain of Itk also contains a proline residue that can undergo cis-trans isomerization mediating various conformational changes in the Itk structure and facilitating interactions with other proteins depending on the state of the conformation. In the cis conformation, the SH2 domain of Itk preferentially interacts with the SH3 domain of the Itk. But in the trans conformation, the SH2 domain mediates interaction of Itk with phosphotyrosine containing proteins such as SLP-76 which also simultaneously interacts with SH3 domain (65) SRC homology 3 (SH3) domain The SH3 domain is capable of binding to proline residues of other proteins thereby enabling the Tec-family kinases to interact with and activate other proteins downstream of the signaling pathways required for T cell activation (65). In addition, it can also regulate the interaction of Tec-family kinases with other proteins by binding to the SH2 domains on the same Tec-family kinase molecule thereby preventing the Tecfamily kinases from binding to other proteins (65, 66). However, most or all of these findings have been obtained by using isolated domains in vitro. Thus they could be different from interactions that occur in full length proteins in cells.

34 Kinase domain 14 The kinase domain can activate other proteins by tyrosine phosphorylation. Once an intermolecular interaction is established between Tec-family kinases and other proteins through their SH3 or SH2 domains, the kinase domain is able to phosphorylate other proteins (65) Factors regulating the activation of Tec family kinases In order for Tec family kinases, Tec and Itk to get activated, they first must be recruited to the plasma membrane in close vicinity to the activated TCR. This is accomplished by Vav1-mediated activation of PI3K after TCR stimulation which then phosphorylates inositol lipids at the D3 position and increases the plasma membrane concentration of phosphatidylinositol 3, 4, 5-triphosphate [PtdIns(3,4,5)P 3 ] that generates a high affinity binding site for the PH region of the Itk (67). Phosphatases such as PTEN (phosphatase and tensin homologue) and SHIP (SH2-domain-containing inositol-5-phosphatase) breakdown PtdIns(3,4,5)P 3 thereby regulating the membrane localization and activation of TEC-family of kinases (68, 69). A similar mechanism is used in B cells for the recruitment of Btk (70). In addition, Btk is also capable of inducing the production of more PI (4,5) P2 in the plasma membrane via a positive feedback loop by the recruitment of PIP5 kinase, which converts PI (4) P to PI (4, 5) P2 (71). Inositol phosphatases such as SHIP and PTEN have been shown to hydrolyze PIP3 to PIP2 and downregulate the recruitment of Tec kinases to the plasma membrane (72, 73). In mast cells, PKCβ has been shown to assist in the recruitment of Btk and Itk to the membrane

35 15 by phosphorylating their PH domains (74, 75). In contrast, Txk lacks the PH domain but is constitutively localized in the plasma membrane via its palmitoylated cysteine string motif (61). Upon recruitment to the plasma membrane, the Tec kinases interact with other adapter proteins to form a multimolecular complex that is crucial for T cell activation. This is accomplished by first colocalizing Tec kinases via their SH2 domain to SLP-76 (63, 65). The Tec kinases then bind to adapter protein LAT either directly after binding to SLP-76 or indirectly via Grb-2, which acts as a bridge by binding to PRRs of Tec kinases on one side and tyrosine phosphorylated LAT on the other side. The Itk/SLP76/LAT complex further recruits other hematopoetic specific proteins such as Vav1, as well as PLC-γ, Cdc42, WASP, GADS, ADAP, NCK and RIBP, thereby forming the multimolecular complex. The Tec kinases are then activated by transphosphorylation of conserved tyrosine residue (Y 551 in Btk, Y 511 in Itk and Y 420 in Rlk/Txk) in the activation loop of the kinase domain via Src kinases such as Lyn (for Btk), Lck (for Itk) and Fyn (for Rlk/Txk). Tec kinases Btk and Itk have then been shown to induce autophosphorylation of conserved tyrosine residue (Y 223 in Btk and Y 180 in Itk) in their substrate binding surface of SH3 domain. It has been suggested that this autophosophorylation is required for interactions with other proteins such as WASP rather than the enzymatic activity of Tec kinase, Btk (76). Also the disruption of intramolecular interactions between PRR and SH3 domains in Btk, Tec and Itk and reciprocal intermolecular interactions between SH3 and PRR regions in Btk, Tec and Txk and between SH2 and SH3 domains of Itk is required for enabling the Tec kinases to bind to other proteins and to induce their enzymatic activity. In addition, biochemical studies

36 16 with different domains of Itk have suggested that deletion of PH domain and part of TH domain of Itk decreases the kinase activity of Itk by 10 fold while the mutation of proline residues in the PRR region reduces the kinase activity by 100 fold. Itk can also get activated by other receptors such as CD28 and CD2 in a Src dependent but ZAP70 independent manner. Itk is directly associated with CD28 and is rapidly phosphorylated upon CD28 ligation Specific functions of Tec kinases in T cell activation Biochemical studies in response to TCR stimulation have indicated that Tec kinases play a crucial role in activating PLC-γ via direct phosphorylation when it comes in close proximity with Tec kinases after the formation of the multimolecular complex. T cells from Itk -/- mice have reduced phosphorylation of tyrosine residue Y 783 on PLCγ1 which leads to decreased levels of second messengers IP3 and DAG production and reduced Ca 2+ flux (77, 78). The calcium mobilization is affected more severely at the late stage in these cells because Itk -/- cells have normal internal stores of calcium but have defects in mediating influx of calcium from extracellular stores via CRAC channels (79, 80). This has been shown to decrease the activation of transcription factor NFAT which is dependent on sustained levels of calcium for its activation (81). In addition, due to the decreased levels of DAG generated, there is decreased activation of DAG-binding proteins PKCθ and Ras-GRP which are in turn required for the activation of MAP kinases, ERK1, ERK2 and JNK and transcription factors AP-1 and NF-қB (82). All these defects have been shown to be exacerbated in T cells from Rlk -/- Itk -/- mice. However, all

37 17 the initial phosphorylation of proteins upstream of Itk such as ZAP-70, Vav, SLP-76, Cbl and WASP as well as activation of p38 was normal in these mice (65). Overexpression studies have suggested that Itk also helps in maintaining the integrity of the signaling complexes by inducing phosphorylation of LAT on Y 171 and Y 191, which is required for the recruitment of Vav and Grb2. In addition, overexpression of Tec has been shown to activate NFAT activity in Jurkat cells independently of LAT and SLP-76 via a different pathway involving Dok1 and Dok2. In addition, Itk and Tec have been shown to have different sensitivities to inhibitors SHIP and PTEN; and thus suggest a difference in their function and regulation (69). Mature T cells from Itk -/- single knockout mice also show severely decreased but not complete absence of proliferation, IL-2 production and expression of effector cytokines in response to TCR stimulation. Similar defects were observed in Rlk -/- Itk -/- double knockout mice, although slightly more severe than the Itk single knockout mice. In contrast these defects were not observed in Rlk -/- or Tec -/- mice (65). These results also suggested that Itk plays a more important role than Rlk or Tec in TCR signaling. All of these defects were rescued upon addition of phorbol myristate acetate (PMA) and calcium ionophore ionomycin, indicating that the Tec kinases play a crucial role in TCR signaling at a relatively proximal stage. Itk has been shown to be required for actin polarization during immunological synapse formation after TCR stimulation (83). Experiments using T cells from Itk -/- and Rlk -/- Itk -/- mice as well as Jurkat cell lines where Itk expression is suppressed using sirna have demonstrated that Itk s adapter function allows the formation of Itk/SLP76/LAT complex which is essential for the recruitment of Vav1 that binds to LAT (82, 84). In the absence of Itk, Vav1 is not present, and thus activation of Rho GTPase

38 Cdc42 does not occur, which in turn reduces actin reorganization. Itk has also been 18 shown to regulate PIP5K which mediates generation of PIP2 that possibly mediate the binding of WASP to the plasma membrane. Using Jurkat cell lines, it has been shown that Itk is also required for PI3K mediated β1 integrin adhesion and calcium dependent activation of calpain which is required for conjugate formation between T cells and APC s. Itk -/- and Rlk -/- Itk -/- cells have also shown defects in LFA-1 clustering which is dependent on recruitment of PKCθ to the site of synapse which is also defective in these cells as it occurs downstream of PLCγ-1. Itk -/- mice have also shown defects in T cell development and in generation of conventional CD4 + and CD8 + T cell lineages. They have reduced number of mature T cells, decreased ratios of CD4:CD8 single positive cells, as well as defects in positive selection (78, 85, 86). Rlk -/- Itk -/- mice have more severe defects in both positive and negative selection and both Rlk -/- Itk -/- and Itk -/- mice generate populations of non conventional innate-like CD8 + T cells expressing markers of memory T cells (87-89). Lack of Itk has been shown to result in defects in T cell migration and adhesion in response to chemokines such as SDF-1α (90, 91). These defects were worse in T cells lacking both Itk and Rlk. Biochemical studies using Jurkat cells expressing kinase inactive form of Itk have shown that Itk is required for chemokine SDF-1α mediated actin polarization and activation of Rac and Cdc42. A kinase inactive form of Itk has also been shown to be able to rescue the activation of Btk-deficient DT-40 cells in response to serum response factor (SRF) which is dependent on Rho family of GTPases (92).

39 1.2.6 Role of Transcription factors in T cell activation 19 Transcription factors NFAT, NF-қB and AP-1 play a crucial role in activating the genes required during TCR activation. NFAT binds directly to promoters of genes such as IL-2 and induces their transcription. NFAT also forms quaternary complexes with AP- 1 (Activation Protein-1) which consist of dimers of Fos and Jun. The NFAT-AP1 complex help in integrating the calcium signaling and MAPK pathways and induce the activation of different set of genes to generate a specific pattern of gene expression (93). Downstream of the TCR, IқB proteins get degraded, which mediates the release of active form of NF-қB, which migrates into the nucleus and induces the transcription of various genes (94). AP-1 transcription factors are activated via the JNK family of MAP kinases, translocate into the nucleus and associate with NFAT transcription factor to induce the activation of IL-2 gene which is a crucial outcome of TCR mediated signaling (95, 96) Role of Co-stimulators in T cell activation Co-stimulators play an important role in T cell activation, preventing the induction of T cell anergy. Co-stimulators such as CD28 and ICOS help in inducing sustained signaling by participating in the immunological synapse via interaction with their corresponding ligands on the APC s. CD28 interacts with ligands B7-1 and B7-2 on the APC s, is phosphorylated by Lck, and recruits PI3K, as well as a variety of cellular proteins such asvav-1, Itk/Tec, Akt and p62 Dok to the plasma membrane. Itk is then believed to mediate the phosphorylation of tyrosine residues in the CD28 cytoplasmic tail

40 and regulate their signaling (58, 77, 97-99). CD28 stimulation is also accompanied by 20 increases in Ca 2+ levels and PLCγ1 but does not lead to increased Zap-70 and LAT phosphorylation ( ). CD28 also interacts with Grb2/Sos complex and activates Ras pathway that leads to the activation of MAPK s, inducing the production of cytokines IL-2, GMCSF, IFN-γ and TNFα ( ). ICOS on the other hand, is weakly expressed on naïve T cells and more highly expressed on memory and effector T cells (compared to CD28). ICOS binds to its ligand B7h/B7-RP1/B7-H2/GL50 on the APC s and upon activation leads to induction of intracellular signaling molecules, PI3- kinase/akt, p38 kinase and the Erk signaling cascade but is incapable of activating Jnk. ICOS activation leads to the production of effector cytokines like IFN-γ, TNF- α, IL-4, IL-5, IL-10 and IL-13 but not IL-2 (11, 12, 107). 1.3 T helper (T H ) cells T helper cells were first discovered in 1960 s and were categorized as a special group of T cells on the basis of their ability to help B cells upon antigenic stimulation, which played a crucial role in adaptive immunity. The existence of two distinct subsets of CD4 + T helper lymphocytes, namely T H 1 and T H 2 cells was later discovered by Mossman, Coffman and colleagues in This discovery eventually led to the establishment of T H 1-T H 2 paradigm (108). They observed that upon antigenic stimulus, naïve CD4 + T helper cells could differentiate into two distinct subsets, T H 1 and T H 2 with unique cytokine profile and function. T H 1 cells produced Interferon-γ (IFN-γ),

41 Interleukin-2 (IL-2), Tumor necrosis factor (TNF) and Lymphotoxin-β (LT-β) and 21 induced cell mediated and inflammatory immune responses providing protection against intracellular microbes (Type 1 immunity). On the other hand, T H 2 cells secreted IL-4, IL- 5, IL-6, IL-10, IL-13 and IL-19 and provided protection against extracellular parasites such as helminths (Type 2 Immunity) in part by mediating humoral immune response. Also, while T H 1 cells mediates isotype switching to immunoglobulin G2a (IgG2a), T H 2 cells mediates isotype switching to IgG1 and IgE (109). Upon restimulation, differentiated T H 1 or T H 2 cells are capable of producing large amounts of the specific cytokines, thereby fighting infections at a much faster rate. A third new lineage of T helper cells, T H 17, has recently been discovered that secrete cytokines such as Interleukin 17A (IL-17A), IL-6, G-CSF, GMSCF and TNFα and appear to be involved in providing enhanced protection against extracellular bacteria, some fungi and other microbes. However, not much is known about these cells and further studies are needed to better understand the functions of the T H 17 cells (110) Factors affecting T helper cell lineage commitment Naïve CD4 + T helper cells usually originate in the thymus after differentiation and later migrate and reside in the secondary lymphoid organs. These cells are functionally immature and can only secrete IL-2 and act as precursors for differentiating into effector T H cells. The differentiation process is initiated in naïve T helper cells upon activation of TCR by cognate antigen presented by APC s such as dendritic cells, followed by the activation of co-stimulatory pathways. However, numerous factors influence the

42 22 progression of T helper cell differentiation towards T H 1 vs. T H 2 lineage such as type of APC, concentration of antigen, duration and strength of the signal, ligation of costimulatory molecules and the cytokine milieu surrounding the naïve T helper cells. Differentiation of T H 17 cells has been shown to be induced by TGF-β and IL-6 which later upregulate the expression of retinoic orphan receptor (ROR)γt, the master regulator of T H 17 cells. (ROR)γt later upregulates the expression of IL-23R and facilitates IL-23 mediated signaling that helps in further differentiation of T H 17 cells and secretion of effector T H 17 cytokines IL-17A, IL-17F, IL-6 and G-CSF (110) Type of APC and concentration of antigen The presentation of antigen to naïve T helper cells is the first step required for the initiation of differentiation. In addition, the type of APC presenting the antigen can regulate T H 1 vs. T H 2 differentiation because each subset of APC s has distinct functions. While CD8α + lymphoid-like dendritic cells produce IL-12 and IFN-γ and preferentially stimulate T H 1 differentiation, CD8α - myeloid-like dendritic cells activate T H 2 differentiation through an unknown mechanism (111). In addition, certain T-cell secreted factors such as Eta1/osteopontin can also mediate T H 1 differentiation by inducing IL-12 and inhibiting IL-10 secretion from APC s (112). Chemokines such as monocyte chemoattractant protein-1 (MCP-1) have also been implicated in inducing T H 2 differentiation by regulating APC function (113). APC s have also been reported to regulate T helper cell differentiation by expressing Jagged and Delta-like ligands which induce the Notch signaling pathway (114). In response to T H 2 stimuli, APC s have been

43 shown to express Jagged2 and induce T H 2 differentiation. While in response to T H 1 23 stimulus, APC s secrete Delta-like 4 (Dll-4) ligands and induce T H 1 differentiation. However, the signaling pathways that are activated through these ligands in T helper cells are still not known Strength of the signal The structure and dose of the antigen can also influence the avidity of the TCR and thus play a crucial role in regulating T helper differentiation. They can affect the strength of the signal generated via the TCR by regulating the duration and magnitude of Ca + fluxes (115, 116). In addition, the location of TCR components play an important role as they ultimately define the nature and integrity of cluster formation between TCR and MHC that dictates transient vs. sustained calcium mobilization in T cells. Only T H 1 cell populations have been observed to specifically recruit TCR components to the lipid raft region upon stimulation, enabling these cells to respond to both low and high affinity antigens due to sustained calcium mobilization. In contrast, T H 2 cells were able to respond only to the high affinity antigens and high antigen dose due to transient calcium mobilization (117) TCR and costimulator mediated signaling pathways Signaling pathways initiated downstream of the TCR via Src kinases, Tec kinases and MAP kinases and transcription factors such as NFAT, AP-1 and NF-қB can also

44 regulate T H 1 vs. T H 2 differentiation. The specific roles of these molecules in T helper cell differentiation are summarized in Table Cytokine milieu The nature of the cytokines that surround the naïve T helper cells greatly influence the type of differentiation that occurs. The set of cytokines that induce T H 1 differentiation are different from those that induce T H 2 diifferentiation. For T H 1 cell differentiation Cytokines IFN-γ, IL-12, IL-18, IL-23 and IL-27 play a crucial role in inducing T H 1 cell differentiation (118). The presence of these cytokines in the cytokine milieu surrounding the naïve T helper cells can orchestrate the differentiation of the naïve cells towards T H 1 cells. Each of these cytokines has specific functions and the coordination between these various cytokines is important for efficient T H 1 differentiation (Figure 1.3). Role of IFN-γ IFN-γ is mainly secreted by CD4 + T H 1 cells, CD8 + cells, and NK cells. IFN-γ mediates IL-12 mediated T H 1 differentiation via STAT4 activation by stabilizing the expression of IL-12Rβ2 chain which is usually downregulated in the absence of IFN-γ in T H 2 cells (119). IFN-γ also regulates T H 1 responses by inducing activation of STAT1 via tyrosine kinases Jak1 and Jak2. However, detailed mechanism by which IFN-γ regulates

45 25 T H 1 differentiation is still unclear and controversial and thus further studies are needed in this direction (120). Role of IL-12 IL-12 is a heterodimer consisting of two subunits, p35 and p40 and is usually secreted by antigen presenting cells such as macrophages, monocytes and dendritic cells. IL-12 plays an essential role in T H 1 cell development. Naïve T helper cells usually do not express receptors for IL-12; and hence, do not respond to them initially. However, IL- 12Rβ1 and β2 chains, which form the IL-12R complex, are actively expressed in the T helper cells upon activation via TCR (119). IL -12 also mediates activation of Jak2 and Tyk2 tyrosine kinases via JAK/STAT pathway and ultimately leads to the activation of STAT1, STAT3 and STAT4 (121). IL-12 is believed to play a more important role in regulating optimum levels of IFN-γ in T H 1 cells in later stages than in initiating the T H 1 responses (122). Role of IL-18 IL-18 belongs to IL-1 family and is mainly produced by macrophages and dendritic cells. IL-18 functions in association with IL-12 in mediating T H 1 development and IFN-γ production. But unlike IL-12, IL-18 is not crucial for the T H 1 development (118). The IL-18 receptor is also absent in naïve T helper cells until they are activated by the TCR. Upon binding to its receptor, IL-18 induces the activation of kinases such as IRAK that further lead to activation of TRAF6 and nuclear translocation of NF-қB. IL-18 also mediates activation of AP-1 via JNK pathway. IL-18 plays an important role in mediating IL-12 dependent T H 1 development and in optimal production of IFN-γ.

46 Role of IL IL-23 is functionally very similar to IL-12, and is a heterodimer of the p40 subunit of IL-12 and p19 subunits, and is secreted by activated dendritic cells (118). The receptor complex for IL-23 consists of IL-12Rβ1 chain and a novel IL-23R chain, expressed on memory CD4 cells but not naïve T cells. Similar to IL12, IL-23 also induces activation of tyrosine kinases Jak2 and Tyk2 via JAK/STAT pathway leading to predominant activation of STAT3. IL-23 is not involved in the initiation of T H 1 cell differentiation. Instead, IL-23 helps in the maintenance of optimum levels of IFN-γ in the later stages of T H 1 cell development. Role of IL-27 IL-27 is a recently discovered cytokine shown to play an important role in T H 1 differentiation (118). It is a heterodimer consisting of EBI3 (Epstein Barr virus induced gene 3) and p28. It is also expressed by activated macrophages, monocytes and myeloid derived dendritic cells. The receptor for IL-27 is WSX-1/TCCR and is expressed in naïve T cells and can thus regulate initial development of T H 1 cells along with the IFN-γR. In addition, IL-27 can also synergize with IL-12 for optimal production of IFN-γ. However, not much is known yet about the signaling pathways downstream of TCCR which mediate these effects.

47 For T H 2 cell differentiation 27 Cytokines IL-4 and IL-2 play an important role in mediating T H 2 differentiation. Of these, IL-4 is the hallmark cytokine that plays the most important and crucial role for the T H 2 cell development (Figure 1.4). Role of IL-4 IL-4 is usually produced by T H 2 cells, mast cells, eosinophils, basophils and NKT cells. There are two types of IL-4 receptors, Type I and Type II (123). Type I IL-4 receptor consist of the IL-4Rα and γ c chains and is expressed on naive T helper cells and thus mediate the development of T H 2 cells. The Type II IL-4 receptor consist of IL-4Rα and IL-13α1 chains and are mainly expressed on smooth muscle cells, gut and lung epithelial cells and is thus involved in later effector responses of T H 2 cells (124). IL-4R can mediate both positive and negative feedback loops thereby regulating T H 2 differentiation. Upon binding to IL4-R, IL-4 induces positive feedback loop by initiating signaling via JAK/STAT pathway leading to the activation of tyrosine kinases, Jak1 and Jak3 which in turn mediate activation and nuclear translocation of transcription factor STAT6 (125). STAT6 plays a crucial role in activation of IL-4 gene by binding to its promoter and 3' enhancer and inducing more production of IL-4. STAT6 also helps in the activation of T H 2 cell specific transcription factor, GATA3. IL-4 induces activation of another transcription factor, STAT5 via calcineurin dependent TCR signaling. STAT5 also plays an important role in the expansion of T H 2 cells in an IL-4 dependent manner. IL-4 can also induce phopshorylation of adapter protein, IRS-2, which mediates activation of Akt signaling pathway via PI3K. IRS2 also regulates the production of other T H 2 cytokines, IL-5 which is required for the survival of T H 2 cells. Shp-1 protein has also

48 28 been shown to be activated via IL4-R mediated signaling and is involved in promoting normal IL-4 mediated signaling. SOCS5, a member of the SOCS family of proteins, can bind to the IL-4R thus inhibiting its signaling and T H 2 differentiation. Another member of SOCS family, SOCS1, has also been shown to regulate T H 2 differentiation by negatively regulating IFN-γ production while maintaining survival and proliferation of T H 2 cells in response to IL-2 and IL-4. Role of IL-2 IL-2 is usually expressed in naïve T helper cells and is necessary for the normal proliferation of T cells. However, while IL-2 is later secreted in T H 1 cells only, during the initial stages of T helper cell differentiation, IL-2 is expressed in both T H 1 and T H 2 cells in order to mediate normal levels of proliferation and cytokine production (126). The receptor complex for IL-2 consist of IL-2Rα (CD25), IL-2Rβ (CD122) and γ c cytokine receptor subunits (127). During early stages of T H 2 differentiation, T H 2 cells maintain high levels of IL-2R expression via c-maf transcription factor (125). Upon binding to IL- 2R, IL-2 induces activation of STAT5a and b proteins via JAK/STAT pathway using tyrosine kinases, Jak1 and 3. STAT5a helps in the induction of IL-4 production by directly binding to the DNAse hypersensitive sites of IL-4 gene.

49 Transcription factors 29 Similar to cytokines, distinct set of transcription factors also exist that regulate the differentiation pattern of T helper cells. For T H 1 differentiation Numerous transcription factors downstream of TCR, IL-12R and IFN-γR such as NF-қB, NFAT, AP-1 and STAT have been shown to regulate IFN-γ production and thus are believed to be involved in T H 1 development. However, none of these transcription factors are solely capable of orienting naïve T cells towards T H 1 differentiation, and are incapable of mediating tissue-specific expression of IFN-γ. Detailed studies have revealed the existence of transcription factors T-bet, Hlx, STAT4, IRF1 and ERM that play a central role in mediating tissue-specific T H 1 differentiation (Figure 1.3). Role of T-bet T-bet, also known as Tbx-21, belongs to the T-Box family of transcription factors as it contains the highly conserved DNA binding domain, T-box (128). T-bet is specifically expressed in T H 1 cells and is considered to be the master regulator of T H 1 cell differentiation. Upon exposure to IFN-γ, naïve T helper cells induce the activation of IFN-γR that leads to STAT1 activation which further upregulates the expression of T-bet (129, 130). Autoactivation of T-bet has also been suggested which is independent of IFNγ (131). T-bet regulates T H 1 differentiation by upregulating IFN-γ production and repressing T H 2 specific IL-4 and IL-5 cytokine production (132). T-bet induces a positive feedback loop for high levels of secretion of IFN-γ, by initiating chromatin remodeling of IFN-γ gene thereby exposing its hypersensitive sites in naïve T cells which then mediates

50 30 T H 1 cell differentiation (133). T-bet also initiates IL-12/STAT4 mediated upregulation of IFN-γ by inducing IL-12Rβ2 chain expression in naïve T cells (118). T-bet also mediates simultaneous repression of cytokines IL-4 and IL-5 that can help in promoting T H 2 differentiation. Recent studies have suggested that T-bet mediates this repression by directly binding to phosphorylated form of GATA-3 and preventing it from binding to the T H 2 cytokine gene locus (134). Tec kinase, Itk has been shown to be involved in this process by phosphorylating T-bet and enabling its interaction with GATA3. However, there is a lot of speculation about this phenomenon and further studies are necessary to understand the mechanism by which T-bet suppresses T H 2 cell development and function. Role of Hlx Hlx is a homeobox gene and is also specifically expressed in T H 1 cells. Transcription factor T-bet has been shown to induce the expression of Hlx, few days after initial stimulation. Hlx in turn helps in the activation of T-bet and also upregulates IL- 12Rβ2 expression in an IFN-γ independent manner through a yet unknown mechanism. Hlx works in close association with T-bet in helping the upregulation of levels of IFN-γ production as well as number of IFN-γ producing cells (118). Role of STAT4 STAT4 enhances the production of IFN-γ in each cell via the IL-12 signaling pathway by binding to IFN-γ gene promoter probably by association with transcriptional coactivator p300/cbp (118). However, actual contribution of STAT4 in the initial production of IFN-γ during T H 1 cell differentiation is still controversial.

51 31 Role of IRF-1 and ERM IRF-1 belongs to IRF family while ERM belongs to ETS family. Although not much is known about the functions of these transcription factors, initial studies have revealed that both these transcription factors are induced via the IL-12/STAT4 signaling pathway. IRF-1 in turn has been shown to regulate the expression of IL-12 (118). Both IRF-1 and ERM are believed to mediate T H 1 differentiation by regulating IFN-γ production. However, further studies are necessary to confirm the function of these transcription factors. Role of NFAT and NF-қB Single knockout studies have shown that NFATc-2 and NFATc-3 are involved in inducing T H 1 development and cytokine production ( ). Members of NF-қB family, c-rel and Rel-B help in the induction of IFN-γ and T H 1 differentiation (141, 142). For T H 2 differentiation T H 2 cell differentiation is also dependent on transcription factors that are specifically expressed in T H 2 cells namely GATA3, c-maf and STAT6. Additional help is provided by the transcription factors downstream of the TCR such as NFAT, NF-қB and AP-1 (Figure 1.4). Role of GATA-3 GATA-3 belongs to GATA family of zinc finger proteins and is considered to be the master regulator of T H 2 cell differentiation. It mediates the activation of T H 2 cell differentiation while simultaneously suppressing the T H 1 cell differentiation. Upon

52 32 exposure to IL-4, GATA-3 expression is upregulated in the naïve T cells via IL4/STAT6 signaling pathway (143). GATA-3 eventually mediates its own upregulation by inducing a autoactivation loop that is independent of STAT6. The T H 2 cytokine gene locus consists of IL-4, IL-5 and IL-13, which lie in the same chromosomal location, separated by only a couple of kilobases. GATA-3 directly binds to the promoters of this T H 2 cell specific cytokine locus and exposes their hypersensitive sites thereby inducing the production of IL-4 and IL-13 in a STAT-6 independent manner (144). GATA-3 also suppresses the activation of T H 1 specific genes, especially STAT4 and IL-12Rβ2 gene in a IL-4 independent manner (125). GATA-3 has also been reported to be postranslationally modified by p38 MAPK via phosphorylation of GATA-3. This modification is suggested to be required for normal transactivation function of GATA-3. However, the expression of GATA-3 is suggested to be negatively regulated by various proteins such as Runx1, Friend of GATA-1 (FOG) and Repressor of GATA (ROG) that can affect the impact of GATA-3 during the later stages of T H 2 development. Hence, further studies are required to analyze this. Role of c-maf c-maf is a basic leucine zipper transcription factor that belongs to the subfamily of AP-1/cAMP responsive binding (CREB)/activating transcription factors (ATF). It is usually induced via TCR and ICOS rather than cytokines in T H 2 cells (125). It mediates T H 2 cell differentiation via induction of IL-4 production by directly binding to a MARE site located 5' of TATA box in the IL-4 promoter. c-maf also helps in the induction of positive feedback loop for IL-4 production. However, c-maf is incapable of inducing the production of other T H 2 cytokines such as IL-5, IL-13 and IL-10. c-maf is also believed

53 33 to suppress IFN-γ production by directly binding to IFN-γ promoter. But further studies are needed to confirm the function of c-maf. Role of STAT6 STAT6 belongs to the STAT family of transcription factors. STAT6 is not required during the initial stages of T H 2 differentiation which is mainly regulated by GATA-3. However, STAT6 is essential for the stability of the T H 2 lineage (125). Upon binding to IL-4R, IL-4 mediates phosphorylation and nuclear translocation of STAT6 which then mediates the initiation of positive feedback loop for IL-4 production by binding to the IL-4 promoter. STAT6 also helps the induction of transcription activators GATA-3 and c-maf and other proteins such as growth factor independent-1 (Gfi-1) which help in driving T H 2 differentiation. STAT6 function is usually regulated by a transcriptional repressor, Bcl-6 by competing for DNA-binding sites with STAT6. Role of AP-1, NFAT and NFқB Members of AP-1 transcription factor family, JunB and c-fos are involved in T H 2 differentiation and upregulation of T H 2 cytokines, IL-4 and IL-5, and are expressed at high levels in T H 2 cells (145, 146). Certain E3 ligases such as Itch, have also been shown to negatively regulate T H 2 development via inhibition of JunB (147). Members of NF-қB family such as c-rel is believed to be capable of regulating T H 2 development by regulating the expression of IFN regulatory factor 4 (IRF4), which inhibits T H 1 cytokine production and enhances T H 2 cytokine production (148, 149). Members of NFAT transcription family, NFATc-1, NFATc-2 and NFATc-3 have been shown to play an important role in regulating T helper cell differentiation. NFATc-1 has been shown to be important for IL-4 production (150). NFATc-2 has also been shown to directly bind to 3'

54 34 enhancer region of IL4 gene which is CsA sensitive and induce IL-4 production (151). NFAT also dimerizes with other nuclear proteins such as AP-1 and IRF-4 on the IL-4 promoter and induce IL-4 production thereby mediating T H 2 differentiation (118, 125). NFAT also associates with NIP45 and c-maf to form a transcriptional activator that induces IL-4 production and prevents the inhibitory effects of TNF receptor associated factor 2 (TRAF-2) Specific Role of Tec kinases in T helper cell differentiation Tec kinases Itk and Rlk were first believed to play a role in T helper cell differentiation after the discovery that Rlk is selectively expressed in T H 1 cells and not in the T H 2 cells (51, 53, 54). Overexpression of Rlk in Jurkat cells results in increased production of IFN-γ but not IL-2 or IL-4, while suppression of Rlk using antisense oligonucleotides leads to decrease in IFN-γ without affecting IL-2 or IL-4 production (152, 153). One isoform of Rlk has also been reported to be constitutively located in the nucleus and has been shown to bind directly to IFN-γ promoter and induce IFN-γ production (153, 154). In addition, intravenous administration of a Rlk encoding plasmid into normal mice led to increased production of IFN-γ without affecting IL-2 or IL-4 production (155, 156). However, the specificity of this experiment is unclear as overexpression of other Tec kinases, especially Itk using similar approach was not attempted. Nevertheless, in vivo studies using Rlk -/- mice showed that these mice have normal T H 1 cytokine production and were able to induce a normal T H 1 response against Toxoplasma gondii (156, 157). These conflicting results thereby imply that further

55 studies are needed to determine the actual role of Rlk in T H 1 cell differentiation and 35 function. In contrast, Itk has been shown to be upregulated in T H 2 cells compared to naïve cells (158). In addition, the Itk promoter has multiple binding sites for the T H 2 master regulator, GATA3 (53). In vivo studies using Itk -/- mice have shown that lack of Itk only affected IL-4 production without affecting the IFN-γ levels and did not succumb to infection caused by T H 1 cell inducing pathogen Toxoplasma gondii (77). Itk -/- T cells differentiated under T H 2 differentiating conditions also express high levels of T-bet (158). When exposed to T H 2 cell inducing pathogens Schistosoma manasoni, Nippostrongylus brasiliensis, Leishmania major, these mice also elicited weak T H 2 responses (99, 159), and had attenuated responses in a T H 2 cell mediated model of allergic asthma (160, 161). Retroviral expression of Itk in T cells lacking Itk restored the IL-4 production suggesting a specific role of Itk in inducing IL-4 production (99). Since the lack of Itk has been shown to affect NFATc1 activation, crucial for the induction of IL-4 production, it is suggested that Itk might regulate T H 2 differentiation and function via regulation of NFATc1 production which can influence the production of IL-4 both directly and indirectly via GATA3 and c-maf. However, recent reports suggest that Itk is involved in phosphorylating the tyrosine residue Y 525 on T-bet which then facilitates the interaction of T-bet with GATA-3 and prevent GATA-3 from binding to the T H 2 cytokine locus thereby inhibiting T H 2 cytokine production and T H 2 cell function (162). But this theory contradicts the phenotype observed in the Itk -/- mice because according to this theory, lack of Itk should prevent T-bet from binding to GATA-3 and thus should be able to induce a robust T H 2 response. But Itk -/- mice have been shown to have defective T H 2

56 responses. Thus, further studies are needed to understand the role of Itk in T H 2 cell 36 function. However, T cells in Rlk -/- Itk -/- mice had impaired T H 1 responses to T. gondii but induced normal T H 2 responses to S. manasoni although they had defective NFAT activation (159, 163). In addition, effective downregulation of GATA3 was lacking on these cells and is thus suggested to be responsible for inducing a T H 2 response. Thus, research done so far propose that Itk and Txk/Rlk, signaling downstream of TCR signaling, can also regulate T helper differentiation as they directly regulate calcium flux in T cells via direct activation of PLCγ. The current notion is that Txk regulates T H 1 differentiation by mediating IFN-γ production via direct interaction with the IFN-γ promoter as well as the adapter protein, RIBP (164). On the other hand, Itk is suggested to regulate IL-4 production by mediating nuclear translocation of NFATc1 (99). Although, this evidence sheds some light on the role Tec kinases in T helper differentiation, the exact mechanism by which they mediate T H 1 vs. T H 2 differentiation is still not known. Hence, further studies are needed to better understand the contribution of Tec kinases in T helper cell differentiation Possible Mechanisms for T helper cell lineage commitment Based on evidence obtained so far, two possible mechanisms, instructive and stochastic/selective, have been proposed to explain the process of T helper cell differentiation and lineage commitment (118). While the Instructive Mechanism suggests that cytokines provide the instructions required for T helper differentiation, the

57 Stochastic Mechanism proposes that cytokines only promote the growth of cells that are already committed to specific lineage type through a stochastic process Instructive Mechanism The Instructive Mechanism is suggested to occur in two stages the cytokine independent stage and the cytokine dependent stage (Figure 1.5). Cytokine Independent Stage This stage begins immediately after the activation of naïve T cells via the TCR/CD28 stimulation. Naïve T helper cells induce the production of low levels of both IL-4 and IFN-γ even in the absence of IL-4 and IFN-γ in the surrounding cytokine milieu. This low level of transcription of both IL-4 and IFN-γ is believed to occur by basal levels of GATA-3 and T-bet transcription factors respectively which are capable of inducing chromatin remodeling of these genes. Transcription factors that are downstream of TCR such as NFAT, AP-1 and NF-қB have also been suggested to mediate the initial transcription of IL-4 and IFN-γ. These low levels of secreted cytokines are thought to induce the next stage which is cytokine dependent. Cytokine dependent stage This stage begins couple of hours after TCR/CD28 stimulation of naïve T cells; and master regulators T-bet and GATA-3 play a crucial role during this stage. Depending on the nature of cytokines that are secreted, lineage specific genes are activated. If high level of IFN-γ is present, it induces enhanced expression of its master regulator T-bet which further augments IFN-γ and IL-12 production, while suppressing the expression of

58 38 IL-4R and GATA-3 expression in these cells and silencing the gene loci for T H 2 specific cytokines. When IL-4 is present at high levels, it activates the expression of GATA-3 which induces the transcription of IL-4, IL5, IL-13 and IL-10. GATA-3 also suppresses the expression of IFN-γ and IL-12R in the cells and silences the T H 1 specific gene loci Stochastic/Selective Mechanism This mechanism proposes that upon TCR/CD28 stimulation, naïve T cells produce a heterogeneous population of both T H 1 and T H 2 cytokine producing cells (165). The cytokines that are secreted during activation then act as growth factors for the proliferation of specific type of T helper cells. This mechanism thus suggests that even at high levels of IL-4, a small populations of T H 1 cells can also be generated and vice versa. This mechanism also suggests that a single cell is incapable of producing all the lineage specific cytokines. Thus the various cytokines that are suggested to be produced by a specific T helper lineage comes from a mixture of cells, each producing a different cytokine that is regulated independently in a stochastic manner. Indeed, further evidence shows that most of the cytokine genes are expressed in a monoallelic manner and only under high signal strength, are they capable of biallelic expression (Figure 1.6). However, further studies are needed to decide which of these two mechanisms actually mimic the mechanism that occurs in vivo.

59 1.3.4 Function of T helper 1 cells in diseases 39 T helper 1 cells have been shown to play both protective as well as pathologic roles in the development of various diseases. T H 1 specific cytokines are usually secreted in order to fight infections caused by intracellular pathogens. However, in some cases, induction of T H 1 responses has been shown to cause exacerbation of the diseases such as Crohn s disease, inflammatory bowel disease, multiple sclerosis, diabetes, autoimmune thyroid disease, and lupus (118) Function of T helper 2 cells in diseases T H 2 responses are usually generated to mainly combat extracellular parasites. However, T H 2 responses also induce pathological responses against foreign allergens which consequently lead to allergic asthma. T H 2 responses sometimes also hinder the clearance of certain infections such as those caused by Leishmania major. Protective role of T H 2 responses T H 2 mediated immune responses play a central role in clearing extracellular parasitic infections caused by Nippostrongylus brasiliensis, Trichuris muris, Heligomosoides polypgyrus, Trichinella spiralis, Schistosoma manasoni (166). T H 2 cytokines induce the activation of various cell types to exert its effects (167). They act on mast cells, and induce mastocytosis and IgE production to eliminate T. spiralis. They help in the expulsion of N. brasiliensis via induction of mucous production by goblet cells. T H 2 cytokines can also induce gut muscle hypercontractivity and eosinophilia to eliminate these parasites.

60 Pathologic role of T H 2 responses 40 T H 2 cells have also been known to be responsible for mounting unwanted immune response that leads to severe disease conditions such as allergic asthma that could sometimes be fatal. In case of infection with L. major, a T H 2 response is sometimes mounted instead of a T H 1 response that is actually necessary for the clearing of the infection. However due to increased T H 2 response, the induction of a T H 1 response is suppressed thereby preventing the clearing of infection. 1.4 Allergic Asthma Allergic asthma is a chronic inflammatory disorder of the lungs that occurs due to the induction of unwanted immune response upon exposure to commonly encountered allergens including those from house dust mite, pollen, cockroach, Alternaria, cats and dogs (168). Allergic asthma is more prevalent in the industrialized countries; the incidence of this disease has almost doubled in the past two decades with about six billion US dollars being spent every year for patient care. This disease usually starts in childhood and environmental factors such as allergens, viruses and occupational exposure can regulate the evolution of the disease. The Hygiene Hypothesis poses that this increased prevalence in the industrialized countries is due to reduced exposure to infections that stimulate the immune system in the early childhood which can mediate allergen sensitization (169). Allergic asthmatic response usually occurs in two phases early and late (170). During the early phase, there is an immediate response to the allergen or antigenic stimuli

61 41 via mast cell activation and release of mediators causing an acute asthmatic attack which resolves soon. The late phase occurs a couple of hours after the initial response due to constant exposure to the allergens that mediate infiltration of inflammatory cells such as T H 2 lymphocytes and eosinophils into the lungs which release cytokines and chemokines and cause severe airway damage. Allergic asthma is characterized by airway hyperresponsiveness, bronchoconstriction, airway smooth muscle spasm, airway edema, mucous hypersecretion and airway remodeling that ultimately leads to intermittent airway obstruction (171). Various cellular elements that play an important role in mediating allergic asthma are T lymphocytes, mast cells, eosinophils, neutrophils and epithelial cells (168). The resident cells in the airways are mainly mast cells, alveolar macrophages, airway epithelium and endothelium as well as migrating inflammatory cells such as eosinophils, lymphocytes, neutrophils, basophils and platelets. These cells can secrete a variety of mediators, such as histamine, the cysteinyl leukotrienes (LTC4, LTD4, and LTE4), prostaglandin D2, platelet-activating factor and various inflammatory mediators which lead to bronchoconstriction and mucosal edema. Neuroregulatory factors such as acetylcholine also facilitate the induction of bronchial muscle contraction. Airway inflammation occurs as a consequence of denudation of airway epithelium, mucous plugging of the bronchioles, collagen deposition in the subepithelial basement membrane, edema of the submucosa, infiltration of inflammatory cells especially T H 2 lymphocytes and eosinophils, and smooth muscle hypertrophy/hyperplasia. T H 2 lymphocytes mediate the secretion of proinflammatory cytokines such as IL-4, IL-5, and IL-13 and chemokines such as RANTES and eotaxin/ccl-11 in association with epithelial cells and mediate the recruitment and activation of eosinophils. Endothelial adhesion proteins, such as ICAM-1

62 42 and VCAM-1 also contribute in the process of airway inflammation by interacting with specific receptors on the lymphocytes and eosinophils and reducing their flow in the vessel and directing these cells to the airway. Under chronic conditions, airway inflammation transitions into airway remodeling by structural changes that includes airway smooth muscle hypertrophy, goblet cell hyperplasia, vascular hyperplasia, and collagen deposition in the airway Role of T cells and T H 2 cytokines in Allergic Asthma T H 2 cells that secrete cytokines such as IL-4, IL-5, IL-9, and IL-13 and induce the generation of IgE have been shown to play a crucial role in the development of allergic asthma. T H 2 cells and T H 2 specific cytokines are usually found in the BAL and airway biopsies from patients with asthma (172, 173). In animal models also it has been shown that transfer of only T H 2 cells into naïve mice before antigen challenge is sufficient for inducing airway eosinophilia, mucous production and airway hyperresponsiveness indicating that T H 2 cells alone can induce allergic asthma (174, 175). Transgenic mice overexpressing T H 2 cytokines also exhibit similar symptoms as seen in allergic asthma further emphasizing the role of T H 2 cell specific cytokines in allergic asthma. The primary role of IL-4 in allergic asthma is to modulate the differentiation of T H 2 cells ( ). IL-4 also induces the expression of vascular cell adhesion molecule 1 (VCAM1) on endothelial cells that enhances adhesiveness of the endothelium for T cells, eosinophils, basophils, and monocytes and directs them to remain in the airways (168, 180, 181). However, overexpression studies have shown that IL-4 is not required

63 for inducing airway hyperresponsiveness in response to allergens but it causes 43 lymphocytic and eosinophilic inflammation (182). IL-4 has also been shown to mediate isotype class switch from IgM to IgE, but this function is not believed to be extended to airway hyperactivity ( ). IL-13, which is closely related to IL-4, has been shown to be an important mediator of allergic asthma. Overexpression of IL-13 in the lungs induces inflammation, mucus hypersecretion, subepithelial fibrosis, and eotaxin/ccl-11 production (185). Administration of IL-13 intratracheally in mice also induces airway hyperreactivity, eosinophilia, and increased IgE production. Blocking of IL-13 function using soluble IL- 13 receptor (IL-13R), reduced AHR and mucus production without affecting IgE production or eosinophilia (186, 187). IL-13 has been suggested to mediate these effects through direct or indirect alterations in epithelial cells or smooth muscle function that express IL-13 receptors (188). IL-5 plays a crucial role in regulating the development and function of eosinophils and in long term airway remodeling during allergic asthma (189, 190). Transgenic mice constitutively expressing IL-5 in all T cells have been shown to have lifelong eosinophilia, with large numbers of eosinophils in the blood, spleen, and bone marrow (191). Mice with inactive IL-5 gene show the absence of lung eosinophilia with very little inflammation and lung damage upon induction of allergic asthma but still developed AHR (192). This indicates that airway eosinophilia is not required for allergen induced airway hyperresponsiveness but is crucial for long term airway remodeling. Another T H 2 cytokine whose involvement in allergic asthma has recently been discovered is IL-9. Transgenic mice overexpressing IL-9 have shown that this cytokine

64 44 may be able to induce AHR, mast cell activation, and mucous production. IL-2 has been identified as the major mediator of IL-9 expression by inducing the production of IL-4 and IL-10 which together induce IL-9 production (193). IL-9 has been shown to be capable of being both an early and late effector of T H 2 cell response. IL-9 has been shown to promote the proliferation of eosinophil progenitors and indirectly induce their migration into the lungs via upregulation of eotaxin and other chemokines by lung epithelial cells (194). IL-9 has also been suggested to function synergistically with IL-4 to produce IgE (195). However, further studies are needed to understand the role of IL-9 in association with other T H 2 cytokines in allergic asthma. Recent reports have suggested that other T cell types such as CD4 + invariant NKT (inkt) cells that secrete T H 2 cytokines also play a crucial role in development of allergen induced AHR in mouse models (196) Role of other cell types in allergic asthma Mast cells have been shown to play an important role during the initial acute phase of allergic asthma in response to high levels of IgE that is generated during the disease and are responsible for inducing bronchial hyperresponsiveness (197). Fc RI receptors present on the mast cells have been shown to interact with IgE and trigger the release of preformed vasoactive mediators such as histamine, induce the production of prostaglandins and leukotrienes, and mediate the transcription of cytokines. These mediators cause immediate hypersensitivity reactions and lead to mucosal edema, mucus production, and smooth muscle constriction in the bronchial mucosa. Mast cells are also

65 45 believed to play a role in the late phase of allergic response by secreting cytokines and chemokines required for the recruitment of T H 2 cells and eosinophils (198). Eosinophils have been shown to play an important role during late phase of allergic asthma (199). Their recruitment to the lungs is mediated by IL-4 and IL-5 cytokines and chemokine eotaxin/ccl-11 which are secreted by the T H 2 cells and mast cells and VCAM1 secreted by lung epithelial cells after allergic challenge. Eosinophils release cytotoxic proteins such as eosinophil cationic protein (ECP) and the major basic protein (MBP), which mediate cell lysis and are responsible for the detrimental effects of the disease. Airway epithelial cells also contribute to the airway disease by secreting a wide variety of cytokines and chemokines such as monocyte chemotactic protein 1 (MCP-1) and RANTES, that mediate the recruitment and activation of inflammatory cells into the lungs. Structural alterations of airway smooth muscle due to increase in airway smooth muscle, edema, infiltration of inflammatory cells, glandular hypertrophy and connective tissue deposition lead to thickening of airway walls that eventually lead to airway constriction and airway remodeling (200) Role of Chemokines in allergic asthma Chemokines are small secreted proteins which play a crucial role in regulating leukocyte migration and activation, especially eosinophils. During allergic asthma, several chemokines have been identified in the lungs such as Eotaxin/CCL11, RANTES/CCL5, MCP-3/CCL7, and MCP-4/CCL13 (201, 202). Chemokines CCL3,

66 CCL7 and CCL22 are highly expressed by interstitial macrophages and mediate the 46 emigration of transmigrating eosinophils from the vascular supply into the interstitium (203). Chemokines CCL5, CCL11 and CCL13 secreted by airway epithelial cells then mediate the migration of these eosinophils from the interstitium into the airways where they are specifically degranulated by CCL11 to release leukotriene C, which severely damage the epithelium and exacerbate the asthmatic symptoms (204, 205). T H 2 cells also express chemokine receptors CCR3,CCR4, CXCR4 and CCR8 whose ligands CCL11, CCL17, CCL22, CXCL12 and CCL1 can also mediate chemotactic migration and activation of T H 2 cells in the lungs (206). T H 2 cell mediated secretion of cytokines IL-4, IL-5 and IL-13 in turn induce the secretion of more CCL11, CCL13, CCL22 and CCL1 thereby continuing the cycle ( ). These chemokines, especially CCL1, can further mediate smooth muscle cell activation and proliferation that can contribute to airway reactivity. Another ligand for CCR4, MDC, has been shown to play a role in retaining the leukocytes in the lungs after their migration into the airways (207). 1.5 Concluding Remarks Numerous studies conducted in T cells revealed that Tec kinases Itk and Txk are responsible for inducing calcium mobilization via direct phosphorylation and activation of PLCγ, and thereby regulate signaling pathways downstream of the TCR that are sensititve to calcium levels. In addition, Itk can also function as adapter proteins to regulate actin reorganization. There are therefore potentially both kinase dependent and independent pathways that are regulated by these kinases. Further analysis indicated that

67 47 Itk and Txk have differential expression patterns in T helper cells, suggesting a specific functions in these cell types. Our laboratory has observed attenuated T H 2 responses in response to allergic asthma in mice lacking Itk. However, the mechanisms by which these kinases potentially regulate T H 1 and T H 2 responses remain unknown. Various contradicting models have been proposed to explain the specific roles of Itk and Txk/Rlk. One model suggests that Txk/Rlk is exclusively expressed in T H 1 cells in order to induce IFN-γ production and mediate T H 1 differentiation. Another model proposes that Itk inhibits GATA-3 mediated T H 2 differentiation and function by activating T-bet via direct phosphorylation. Itk has also been proposed to inhibit T-bet expression, thus enhancing T H 2 responses. All these observations suggest that further investigations are necessary to shed further light into the specific functions of these kinases in T cells. We have therefore attempted to address these controversial observations here and investigated the mechanisms by which these kinases regulate T helper cell function. The experiments presented in this thesis test the hypothesis that Itk regulates the T H 2 cell signaling pathways during allergic asthma by means of its catalytic activity and suppressing T-bet activity, and that Txk and Itk perform distinct functions in T H 1 and T H 2 cells due to their selective expression, not specific function.

68 48 Figure 1.1: TCR signaling pathway. Sequence of events occurring during T cell activation via TCR. Both the bold and dotted arrows represent signaling that occurs downstream of TCR and CD28. Refer to text for more details.

69 Figure 1.2: Structure of Tec kinases. Tec kinases, Btk, Itk, Bmx and Tec have the unique PH domain, in addition to TH, SH2, SH3 and Kinase domain. Tec kinase, Txk/Rlk has all the domains as the other Tec kinases, with an exception in having cysteine rich motifs in the N-terminal instead of the PH domain. Refer to text for more details. 49

70 Figure 1.3: T H 1 signaling pathway. Role of IL-12, IFN-γ, STAT1, STAT4 and various signaling molecules downstream of the TCR in inducing T H 1 differentiation. Refer to text for more details. 50

71 Figure 1.4: T H 2 signaling pathway. Role of IL-2, IL-4, STAT5, STAT6 and various signaling molecules downstream of the TCR in inducing T H 2 differentiation. Refer to text for more details. 51

72 Figure 1.5: Instructive Mechanism of T helper cell differentiation. The cytokine independent phase (downstream of TCR activation) and cytokine dependent phase (regulated by GATA-3 and T-bet) of commitment during T helper cell differentiation. Refer to text for more details. (Adapted from Susanne J. Szabo and Laurie H. Glimcher, Molecular Mechanisms Regulating TH1 Immune Responses, Annual Reviews of Immunology, 2003). 52

73 53 Figure 1.6: Stochastic/Selective Mechanism of T helper cell differentiation. Cytokines IFN-γ and IL-4 produced after TCR activation act only as growth factors to generate a heterogeneous population of T H 1 and T H 2 cells. Refer to text for more details.

74 Table 1.1: Summary of proteins downstream of TCR that contribute to T helper cell differentiation. 54 Genes (downstream of TCR) Lck Fyn ZAP-70 CD28 OX-40 CTLA-4 ICOS Rac2 Ras and Erk p38 Jnk1 Jnk2 GADD45 β and γ Role in T helper cell differentiation Induces T H 2 differentiation via formation of distinct signaling complexes. Induces T H 1 differentiation. Induces T H 2 differentiation at low phosphorylation levels which is achieved via SLAT. Induces T H 2 differentiation via induction of NFATc1 nuclear translocation and GATA-3 expression. Involved in T H 2 cell development. Involved in T H 1 cell development. Induces T H 2 development if activated at an earlier time point. Induces T H 1 development if activated at a later time point. Induces T H 1 differentiation by regulating IFN-γ production via p38 and NF- қb. Induce T H 2 differentiation by mediating activation of IL-4Rα, Jak1 and STAT6. Induces T H 1 differentiation via IL-12 mediated IFN-γ production that is independent of STAT4. Inhibits T H 2 differentiation by phosphorylating NFATc1 and preventing its activation by calcineurin and nuclear translocation. Induces T H 1 differentiation by regulating IFN-γ production and IL- 12Rβ2 expression. Induces T H 1 differentiation via p38 and JNK pathways and activates IL-12 driven IFN-γ production.

75 55 Chapter 2 Materials and Methods 2.1 Mice C57Bl/6 (WT) and Itk null mice (85) between 6-12 weeks old were used for all these studies. We also generated mice carrying a kinase deleted mutant of Itk either on a WT background (Tg(Lckpr-Itk Kin)WT) or on an Itk null background (Tg(Lckpr- Itk Kin)/Itk -/- ) which is expressed at about 25-30% of endogenous Itk. The Tg(Lckpr- Itk Kin) was generated by cloning a mutant Itk with the kinase domain replaced with EGFP (212), into a transgenic expression cassette driven by the Lck proximal promoter and CD2 enhancer (kind gift of Dr. Anuradha Ray, University of Pittsburgh) (213). All mice were backcrossed to C57Bl/6 background for at least 10 generations. For some experiments we also used Tg(CD2-Txk)Itk -/- mice (214). All the experiments were approved by the Office of Research Protection s Institutional Animal Care and Use Committee at Pennsylvania State University. 2.2 Induction of Experimental Allergic Asthma Mice were injected intraperitoneally with 50 µg of OVA complexed with Alum (8.8 mg, Pierce, Rockford, IL) in a total volume of 220 µl PBS on days 0 and 5. On days 12 through 15, mice were challenged intranasally with either 40 µg of OVA in 50 µl

76 56 sterile PBS or PBS alone once a day for a total of 4 challenges. For cytokine addback experiments, 2µg of murine IL-4 or IL-13 was delivered along with OVA intranasally daily from day 12 to 15. For chemokine addback experiments, 750ng of murine CCL-11 with or without 2µg of IL-13 was similarly delivered intranasally from day 12 through 15. On day 16, mice were analyzed for airways hyperresponsiveness, then sacrificed and tracheal samples, BALF and RNA were obtained from lungs as detailed below. For the experiments using HDM, we induced allergic airway disease by exposing the mice intranasally to 5, 25, 100 or 200 µg of whole body HDM extract (Greer Laboratories, Lenoir, NC) resuspended at 2mg/ml concentration in PBS. Mice were exposed daily for 10 consecutive days, and 24 hours after the final HDM exposure, were analyzed for AHR in response to increasing doses of methacholine. 2.3 Analysis of AHR AHR was analyzed using either a single chamber Buxco whole body plethysmograph model PLY3211 coupled to a MAX1320 modular accessory unit, PLY1040 bias flow regulator, and data handled using the BioSystem XA software for Windows (Wilmington, NC) or a flexivent apparatus. With plethysmograph model, twenty-four hours after the final OVA challenge, mice were analyzed for responses to PBS or escalating doses of aerosolized methacholine from 3.25 mg/ml to 25 mg/ml. Data were collected using Drorbaugh and Fenn calculations for 5 minutes, logging a line of data every 25 good breaths, and Penh (enhanced pause) values plotted vs. methacholine concentration.

77 57 With flexivent model, twenty-four hours after the final ovalbumin challenge, mice were analyzed for airway hyperresponsiveness using the mechanical ventilator apparatus in response to PBS or increasing doses of aerosolized methacholine from 1 mg/ml to 100 mg/ml. Mice were first anesthetized using pentobarbital, and then a 20-guage steel canula was placed in the trachea which was secured using suture. The respiratory system resistance was measured by calculating the pressure difference between peak-inflation and end-inflation plateau and dividing that by steady-state flow obtained just prior to occlusion ( ). Respiratory system resistance (Rrs) values (cm/h 2 0/ml) were plotted vs. methacholine concentration. 2.4 Analysis of tracheal responses Tracheae were isolated following CO 2 asphyxiation of mice. The trachea was exposed and removed using blunt incisions below the larynx and above the carina. Tracheae were placed in an organ bath containing a modified Krebs-Henseleit (K-H) solution at 37 C, and bubbled with a 95% Oxygen/5% Carbon dioxide mixture. Modified K-H solution had the following makeup: 118 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl 2, 1.2 mm MgSO 4, 1.2 mm KH 2 PO 4, 11 mm D-glucose, buffered with 25 mm NaHCO 3 to maintain the ph at 7.4 at 37 o C when bubbled with a mixture of 95% O 2 and 5% CO 2. Isometric force measurements were determined on a whole trachea mounted on triangle-shaped tissue supports and slipped onto a 0.5 mm stainless-steel pin that was fixed to L-shaped glass rod. The triangle-shaped tissue supports were then connected to an isometric force transducer (Radnoti Glass Technology, Model TRN001). A transducer

78 58 positioner was used to stretch each trachea to its optimum resting tension before any measurements were obtained. Use of two identical force transducers and organ baths allowed for paired experiments using one WT and one ITK null trachea. To determine the gram force of the contraction or resting tension, the two force transducers were calibrated with known weights, and derived equations from the resulting calibration curves were used to convert volts into grams for each particular transducer. Contractile responses were determined following a 1-hour equilibration of trachea at the optimum resting tension of 1.25 grams at the start of the experiment and allowed to equilibrate for one hour in the organ baths. After equilibration, the responses of both WT and ITK null tracheae to the different agonists were tested. Cumulative concentrationresponse curves (CCRC) in the tracheal smooth muscles to CCh ( M), ACh ( M), or KCl ( M) were analyzed, the tracheae weighed at the end of each experiment and contractile responses expressed as isometric tension developed per gram-wet weight of tissue (g/g). 2.5 Adoptive transfer of antigen specific cell populations Mice were immunized by intraperitoneal injection with 50 µg of OVA complexed with Alum (8.8 mg, Pierce, Rockford, IL) in a total volume of 220 µl PBS on days 0 and 5. Animals were then sacrificed on day 12 (7 days after the antigen boost), and splenocytes isolated and cultured at 5 X 10 6 /ml with 2 mg/ml OVA for 72 hours. Cells were then isolated and either whole splenocytes, CD4 + or CD4 - cells (1 X 10 7 cells) transferred to naïve WT or ITK null mice. Forty-eight hours after transfer, the mice were

79 59 challenged intranasally with 40 µg of OVA in 50 µl sterile saline once a day for a total of 4 challenges. Twenty-four hours after the final challenge (7 days after transfer of cells), mice were analyzed for airways hyperresponsiveness, then sacrificed and lungs collected for histology. 2.6 Histology After AHR analysis, lungs were fixed with 4% formaldehyde and embedded in paraffin wax and cut into thin sections. The sections were then fixed onto slides and stained with Hematoxylin and Eosin (H&E) that specifically stain leukocytes and help in examining the extent of leukocyte infiltration. We analyzed the level of mucous production by using periodic acid-schiff (PAS) stain that specifically binds to mucous produced by goblet cells. 2.7 Quantitative RT-PCR analysis Twenty-four hours after the last OVA challenge, total RNA was isolated from lung tissue by using Trizol reagent (Invitrogen Life Technologies). Total RNA (5 µg) was reverse transcribed to cdna with random primers using the Ready-To-Go You- Prime First Strand Beads following manufacturer s instructions (Amersham Biosciences, Piscataway, NJ). PCR was performed in triplicate on an Applied Biosystems ABI PRISM 7300 Sequence Detection System using Taqman Universal PCR master mix with commercially available primers and FAM labeled probes (Assays on Demand TM, Applied

80 60 Biosystems). Relative quantification was performed using the Comparative C T (threshold cycle) method which uses the arithmetic formula 2 - CT to represent the amount of target gene, normalized to an endogenous reference (GAPDH, in our case) and relative to a calibrator sample (WT, in our case). The C T value was determined by subtracting the average GAPDH C T value from the average target gene C T value. The C T value was then calculated by subtracting the C T value of the chosen calibrator sample from the C T value of the target gene. The relative gene expression levels were then determined from the equation 2 - CT. 2.8 Analysis of leukocyte population in the lungs and Intracellular staining After the last ovalbumin challenge on day 16, lungs were minced into small pieces and then incubated with 1 mg Collagenase A for 45 minutes in order to obtain a single cell suspension. The cells were stained with specific antibodies for CD4 and eosinophils (Gr-1 and CCR3); and cell populations were analyzed using flow cytometry. For intracellular staining, the cells obtained from lungs were stimulated with 100ng/ml PMA (Sigma) and 0.5µM Ionomycin (Sigma) in the presence of 1mg/ml Brefeldin A (Sigma) for 8 hours. The cells were then fixed using 4% paraformaldehyde and first stained for CD4 (using CD4 antibody) or eosinophils (using Gr-1 and CCR3 antibodies). The cells were then permeabilized using 0.1% saponin buffer; and intracellular IL-4 was measured using specific antibodies and analyzed by flow cytometry.

81 2.9 ELISA 61 BALF was collected from the mice immunized and challenged with OVA as described above. Cells were removed by centrifugation and BALF samples concentrated 3 fold using Centricon concentrators (Millipore, Bedford, MA). For some experiments, IL-13 was analyzed using an ELISA kit from R&D Systems following manufacturer s instructions (sensitivity, 1.5 pg/ml). IL-4 and IFN-γ were analyzed using a Bender MedSystems FlowCytomix Multiplex bead assay kit (sensitivity, IL-4: 7.9 pg/ml, IFN-γ: 1.7 pg/ml, kind gift of Bender MedSystems, Vienna, Austria). For other experiments, the samples were analyzed using the Bioplex System following the manufacturer s instructions (sensitivity, 1.5 pg/ml, BioRad) Analysis of lymphocyte proliferation and cytokine secretion Purified splenocytes and lymph node cells from the indicated mice were cultured with 10 or 100 µg/ml of OVA (2 X 10 5 cells/well in 200 µl per well in 96-well round bottom plates). After 72 hours of culture, cells were pulsed with [ 3 H]-thymidine for 18 hours. The cultures were then harvested using a cell harvester and incorporated radioactivity determined by scintillation counting using a Betaplate Liquid Scinitillation Counter. Cytokine analysis was performed by stimulating these cells in a similar fashion to proliferation assay for 96 hours, followed by harvesting of supernatants and analysis by cytokine specific ELISA for IFN-γ, IL-4, IL-5 and IL-13 using either the ELISA kit

82 from R&D systems following manufacturer s instructions or by using the Bioplex System. (sensitivity, 1.5 pg/ml, BioRad) Analysis of IgE levels Following priming and challenge, mice were sacrificed and serum was obtained. Dilutions of sera were analyzed for OVA-specific IgE by coating OVA onto the ELISA wells (20 mg/ml) and testing dilutions of sera ELISA using anti-murine IgE (2 µg/ml, 1/250) as capture Abs and HRP-conjugated anti-murine IgE (1/250) as detection reagents (Southern Biotechnology Associates, Birmingham, AL) CCL11 mediated actin polymerization assay CD4 + T cells were purified from splenocytes and lymph nodes of WT and Tg(Lckpr-Itk Kin)/WT mice using a T-cell negative selection column (Miltenyl Biotec, Auburn, CA). Purified CD4 + T cells were differentiated into T H 2 cells by stimulating with 1 µg/ml anti-cd3 and CD28 (BDBiosciences), 10 ng/ml ril-4 (Peprotech) and 10 µg/ml anti-ifn-γ (clone XMG1.2) for 7 days. Following analysis of these differentiated T H 2 cells for the expression of CCR3, T H 2 cells were then stimulated with 100 ng/ml CCL11 or PBS as a control for 5 minutes at 37 o C, fixed with 4% paraformaldehyde, washed twice with FACS buffer, then stained with Alexa fluor-568 phalloidin (Invitrogen, Carlsbad, CA) for 30 min on ice. The cells were then washed and analyzed for F-actin content by confocal imaging. The mean fluorescence intensity of individual cells was

83 determined using NIH Image software and the fold increase in fluorescence intensity over PBS treated cells was plotted CCL-11 mediated migration assay The bottom wells of a 96-well ChemoTx Chamber (Corning Incorporated) with 5 µm pore size was loaded with 100 ng/ml of CCL11 or PBS suspended in migration medium (RPMI 1640 medium containing 0.5% BSA and 20 mm HEPES). In vitro differentiated WT or Tg(Lckpr-Itk Kin)/WT T H 2 cells (2 x 10 5 /well) were applied to the upper wells and incubated for 2 hrs at 37 o C in 5% CO 2. The migrated cells were collected from the bottom chamber after two hours and the number of cells migrated was determined and expressed as fold over those that migrated in response to carrier (PBS) NFAT nuclear localization analysis Purified CD4 + T cells from WT and Tg(Lckpr-Itk Kin)/WT mice were stimulated with anti-cd3 for 45 minutes, fixed, permeabilized and stained for NFAT-c1 using a specific antibody (Santa Cruz Biotechnology). The cells were analyzed for nuclear versus cytoplasmic location using confocal microscopy. Images were analyzed using ImagePro software (Media Cybernetics, Inc, Bethesda, MD).

84 2.15 Dendritic cell isolation and Flow cytometry 64 HDM was conjugated to Alexa-750 flurophore by following manufacturer s instructions (Molecular Probes). Twenty five µg of HDM-Alexa-750 was then delivered intranasally to WT an ITK -/- mice and lungs were collected after 48 hours. The lungs were minced into small pieces and then incubated with 1 mg Collagenase A for 45 minutes. The cells were then treated with ACK lysis buffer (0.15M NH 4 Cl, 10M KHCO 3, 0.1mM Na 2 EDTA) in order to lyse the RBC s. The single cell suspension was then stained with CD11b-ECD, CD11c-APC, MHCII-FITC and Gr1-PE and incubated for 30 minutes on ice, followed by analysis using flow cytometry for dendritic cells Data analysis Statistical evaluation was conducted by using Student s t test or ANOVA where appropriate, with a probability value of P 0.05 considered statistically significant.

85 65 Chapter 3 Reduced airway hyperresponsiveness and tracheal responses during allergic asthma in mice lacking tyrosine kinase inducible T-cell kinase This chapter is a partial reprint of a paper originally published in the Journal of Allergy and Clinical Immunology 117(4):780-6 (2006). The authors are Sahu N*, Ferrara TJ*, Mueller C*, Ben-Jebria A and August A. (* co-first authors) Copyright permission for the use of the materials has been approved by Elsevier Limited, Copyright Introduction The prevalence of Asthma has steadily increased, resulting in morbidity and in some cases mortality due to symptoms of this disease. Current views of this disease hold that the T cell response is critical for its development. In particular, the development of allergen specific T H 2 cells and their resultant cytokine production, IL-4, IL-5 and IL-13, which have effects on cells of the lung. These effects include smooth muscle proliferation, goblet cell hyperplasia and mucous production, which usually accompany the development of airway hyperresponsiveness (AHR) (218). Cytokines secreted by T H 2 cells seem to play a critical role in the development of AHR as transfer of allergen specific T H 2 cells alone lead to AHR in mice (219). In addition, IL-4 or IL-13 alone can induce AHR in mice, and blocking these cytokines prevent the development of AHR in

86 66 mice and humans (186, 187, 220, 221). Current treatments for asthma include bronchodilators; short and long acting β-agonists, and anti-cholinergics, corticosteroids, anti-leukotrienes, anti-mast cell agents and anti-ige monoclonal antibody (222). Targeting the activation and/or development of T H 2 cells has the potential to lead to more effective therapies for treating subjects with asthma. GATA-3 is a master regulatory transcription factor for T H 2 cytokines, while T-bet is a master regulatory transcription factor for regulating T H 1 cytokines (223). Dominant negative forms of GATA-3 can inhibit airway inflammation observed in murine experimental allergic asthma, while mice lacking T-bet spontaneously develop immunological symptoms of asthma, and children with asthma carrying a particular allele of T-bet have improved responses to corticosteroids (213, ). Early signals that control T cell activation are promising targets, and we have previously shown that the non-receptor Tec family tyrosine kinase, Itk, is involved in regulating the development of airway inflammation in murine experimental allergic asthma (215). We have also shown that tracheal responses in non-manipulated Itk null mice is reduced compared to WT mice (228). In this report, we show that Itk null mice exhibit reduced tracheal response both prior to and following airway allergen challenges. We also show that Itk null mice exhibit reduced airways hyperresponsiveness in response to airway allergen challenge, accompanied by reduced expression of T H 2 cytokines in the lung. The reduced AHR observed could be rescued by transfer of antigen specific WT CD4 + T cells. Together, these data indicate that pharmaceutical targeting of Itk may be efficacious in treating asthma.

87 3.2 Results Itk null mice exhibit reduced Airway Hyperresponsiveness during allergic asthma induction. Using a murine model of allergic asthma, we have previously shown that mice lacking Itk exhibit reduced airway inflammation, airway eosinophilia and mucous production compared to WT mice (215). In order to determine if this reduced immunological response correlated with reduced airway hyperresponsiveness, we performed experiments to analyze the response of WT and Itk null mice to methacholine challenge using a whole body plethysmograph. Control mice exhibited the expected changes in airway responses as a function of increased methacholine challenge (Figure 3.1a). However, mice lacking ITK exhibited significantly reduced airway responses compared to WT mice, in the absence of intranasal challenge with OVA. By contrast, the response of the mice to PBS was not significantly different. Similarly, when the mice were challenged with OVA, WT mice exhibited increased responses, which were again significantly higher than those observed in Itk null mice (Figure 3.1 b). These data indicate that in addition to reduced immunological symptoms and airway inflammation in response to allergic asthma induction, mice lacking Itk also exhibited reduced AHR responses to allergen challenge.

88 3.2.2 Itk null mice exhibit reduced tracheal contractile responses to Acetylcholine, Carbachol and KCl during allergic asthma induction. 68 To determine if this reduced response observed in Itk null mice could also be observed in the isolated tracheae, we analyzed the contractile response of isolated tracheae from these mice to cholinergic stimulation using ACh and CCh (228). Tracheae from WT and Itk null mice treated as described above were isolated and CCRCs to these agonists were generated from contractile measurements. Similar to what was observed using the whole body plethysmograph, Itk null tracheae exhibited reduced contractile responses to stimulation with ACh as well as CCh compared to WT mice under conditions where they were challenged with PBS (Figure 3.2a and b). However, while the EC 50 for the two mice and two agonists were similar, the contractile response at the E max was significantly higher in WT tracheae than those from Itk null mice (Figure 3.2a and b). The data indicate that under non-allergic asthma conditions, i.e. control conditions, trachea from mice lacking Itk exhibit significantly reduced responses to cholinergic stimulation than WT mice. We next performed similar experiments to determine the tracheal responses to cholinergic stimulation from mice that had been induced to develop allergic asthma. Similar to what was observed using the whole body plethysmograph, tracheae from WT mice induced to develop allergic asthma exhibited significantly increased tracheal responses to ACh and CCh, with similar EC 50, but larger E max than those from Itk null mice (data not shown). However, the contractile response at the E max was significantly higher in WT tracheae than those from Itk null mice, excepting the CCh stimulated

89 69 trachea, which was higher in WT, but the difference did not reach statistical significance (Figure 3.2 c, d). To determine if the responses we observed were specific to cholinergic stimulation, we used a depolarizing agent, KCl. Similar to what was observed in the whole body plethysmograph in response to methacholine, as well as the in vitro response to ACh and CCh, WT tracheae stimulated with KCl had significantly greater contractile responses at E max to KCl stimulation compared to Itk null mice (data not shown). However, the EC 50 values were similar, regardless of whether or not the mice had been induced to develop allergic asthma (Figure 3.3a, b). Thus the absence of Itk results in reduced basal responses to methacholine as measured by whole body plethysmography, as well as reduced responses to ACh, CCh and KCl stimulation as measured using isolated tracheae from these mice. In addition, as previously observed for immunological symptoms and airway inflammation, in mice induced to develop allergic asthma, those lacking Itk exhibited reduced AHR responses to methacholine, as well as reduced tracheal responses to ACh, CCh and KCl in vitro. We should note that Itk is not expressed in the trachea (data not shown), and thus these responses may represent the effects of the immunological environment in the Itk null mice Reduced T H 2 cytokines in lungs of Itk null mice during allergic asthma induction The observed reduced physiological responses as well as the previously observed reduced immunological symptoms and airway inflammation in Itk null mice compared to WT mice, prompted us to examine the expression of cytokines that may be critical in

90 70 controlling the physiological responses in these two mice. For these experiments, mrna from the lung tissue of WT and Itk null mice that had been induced to develop allergic asthma were analyzed by quantitative RT-PCR for T H 1 and T H 2 cytokines shown to be important in regulating AHR, particularly IFN-γ, IL-4 and IL-13. In addition, since we had previously observed reduced T H 2 immunological responses in Itk null mice in this model, we also determined the expression of T-bet and GATA-3, two critical transcription factors for the induction of T H 1 and T H 2 responses respectively. Our analysis indicate that lungs from Itk null mice had significantly reduced expression of T H 2 cytokines IL-4, and IL-13, whereas IFN-γ expression was similar (Figure 3.4 a). We confirmed the differential expression of IL-4, IL-13 and IFN-γ from BALF of the mice using ELISA or FBI (Figure 3.4 b). Interestingly, while expression of T-bet was not affected by the absence of Itk, GATA-3 was significantly reduced, suggesting that the difference in cytokine expression in the lungs of Itk null mice may be due to differential expression of these transcription factors (Figure 3.4 a) Rescue of AHR responses in Itk null mice by transfer of antigen specific WT CD4 + T cells. We have previously shown that Itk null T cells secrete less T H 2 cytokines during allergic asthma induction compared to WT T cells in vitro (215). To determine if WT T cells could rescue AHR in Itk null mice, we transferred various populations of cells from OVA sensitized WT or Itk null mice into naïve WT or Itk null mice, followed by

91 71 intranasal challenges. In these experiments, WT or Itk null mice were sensitized with OVA plus Alum, followed by sacrifice and the splenocytes purified. These cells were then incubated in vitro with OVA, washed, and directly transferred into naive Itk null mice. While transfer of splenocytes from sensitized Itk null mice into naïve WT mice did not result in the development of AHR, transfer of splenocytes from sensitized WT mice into naïve Itk null mice led to the development of AHR in the recipient mice (Figure 3.5 a). To determine if WT CD4 + T cells could transfer AHR to naïve Itk null mice, similar experiments were performed, except that we separated the splenocytes into CD4 + cells, and CD4 - cells (including CD8 + T cells), followed by transfer into naïve Itk null mice. Analysis of these recipient mice indicated that while transfer of the CD4 - population could not rescue the induction of AHR in naïve Itk null mice, transfer of the CD4 + population was able to rescue AHR (Figure 3.5b). As a control, transfer of splenocytes from sensitized Itk null mice did not induce AHR in naïve Itk null recipients (Figure 3.5b). Analysis of histological sections indicated that these transferred T cells (and splenocytes, (data not shown)) also lead to increased airway inflammation in the naïve Itk null lungs following OVA challenge (Figure 3.6). All together, these data suggest that Itk null mice have reduced airways response to allergic asthma induction, which could be rescued by transfer of WT CD4 + cells. They also indicate that Itk null trachea has reduced responses to cholinergic stimulation in the absence or presence of conditions that lead to allergic asthma.

92 3.3 Discussion 72 The Tec family kinase Itk is expressed in T cells, and has been shown to regulate the production of a number of T cell derived cytokines, in particular, IL-2, and the T H 2 cytokines IL-4, -5 and 13 (59, 160, 229, 230). Although the lack of Itk also affects T cell production of IFN-γ, this effect does not seem as significant as that seen for IL-4 (59, 160, 230). T H 2 cytokines have been demonstrated to be important for the development of allergic asthma (218, ). We demonstrated in this report that the lungs of Itk null mice have preferential reduced expression of T H 2 cytokines, along with reduced AHR during allergic asthma induction. We also demonstrated that the isolated trachea from Itk null mice have reduced contractile response to both cholinergic agonists as well as depolarizing agents. This difference was observed both in control PBS challenged mice as well as OVA challenged mice, suggesting that Itk may indirectly regulate the tone of the tracheal responses in these mice, resulting in reduced AHR to allergic asthma induction. Our finding of reduced tracheal responses to acetylcholine, carbachol as well as KCl in the trachea from Itk null mice, in the absence of challenge was surprising. This difference was not in the potency of the stimulating agents, but in their efficacy, suggesting that smooth muscle cells from Itk null mice exhibit altered responses to stimulation. As Itk is not expressed in smooth muscle cells or other cells in the trachea (data not shown), our data would suggest that Itk may control the tone of the smooth muscle cells that line the trachea via mediators such as cytokines made by T cells and regulated by Itk. Indeed, IL-13 can affect the contractile response of smooth muscle

93 73 cells. We observed reduced IL-13 message and protein in lungs from Itk null mice (234). We have also shown that Itk null T cells secrete reduced levels of IL-13 upon rechallenge following immunization with OVA (215). We speculate that reduced exposure to such cytokines may alter the response of Itk null trachea, and perhaps lungs, resulting in reduced responses upon stimulation (235). The same situation may apply under conditions of allergic asthma, where WT mice are exposed to large amounts of these cytokines, while Itk null mice may not be, leading to altered AHR and tracheal responses. This view is supported by the fact that we can rescue the development of AHR in naïve Itk null mice by transfer of CD4 + T cells from sensitized WT mice. This finding suggests that these cells and their cytokines are critical for the development of AHR in Itk null mice, similar to what has been reported by other investigators in WT mice (219). Thus cytokines such as IL-5 and IL-13 alone can induce AHR if recombinant proteins are delivered to the lungs of WT mice, or if mice carry a transgene for these cytokines, and there is interplay amongst these cytokines in regulating AHR (220, 232, 236). These cytokines can then act on the smooth muscle cells of the lungs to induce AHR. Of interest is the fact that children with asthma were found to have better responses to inhaled corticosteroids if they carried a particular allele of T-bet (227). Transfer of WT CD4 + cells into Itk null mice may recover the production of these critical cytokines resulting in recovery of AHR in the Itk null mice. The observed reduction in T H 2 cytokines in the lungs of Itk null mice, may be the result of reduced T cell differentiation to the T H 2 lineage, because we observed little difference in the expression of T-bet, there was a significant reduction in expression of GATA-3. Recently, data supporting a significant role for GATA-3 in the development

94 74 and maintenance of T H 2 cells was reported, and it is possible that mice lacking Itk have reduced GATA-3 expression, and thus reduced T H 2 cytokine expression ( ). Other investigators have also suggested that Itk may regulate either the expression of T- bet, or its tyrosine phosphorylation in T H 2 cells, therefore regulating T H 2 cytokine production (59, 134). Our data would suggest that in the absence of Itk, the expression of T-bet is not affected in lung, which could be the result of other cells expressing this factor, or lack of an effect of Itk on the expression of this transcription factor. Another role for Itk in regulating tracheal responses could be in regulating mast cell function via regulating signaling through the high affinity Fc receptor for IgE on mast cells (FcεR). Mast cells can be found interacting with smooth muscle cells in the trachea and lungs, and Itk is expressed in mast cells (74, 241). Indeed, Itk null mast cells have recently been reported to have some defects in degranulation (A. Iyer and A. August, in preparation, (242)). Thus Itk null mast cells may release reduced amounts of pharmacological agents that could affect smooth muscle function in the airways of these mice (243). These results, taken together with our previously published work and that of others, suggest that inhibitors to the tyrosine kinase Itk being developed may represent a good target for developing drugs to treat allergic asthma, a view supported by recent efforts in the area where inhibitors of Itk have been shown to be efficacious in a murine model of allergic inflammation (244).

95 75 Figure 3.1: Reduced basal and OVA induced AHR in mice lacking Itk following induction of Allergic Asthma. AHR of WT and Itk -/- mice immunized with OVA, then exposed IN to (a) PBS, *p<0.05, students t test, WT (n=8), Itk -/- (n=6); or (b) OVA, *p<0.05, **p<0.07, ***p<0.08, students t test, n=6. Filled circles, WT mice; open circles, Itk -/- mice.

96 Figure 3.2: Reduced tracheal responses to cholinergic stimulation in mice lacking ITK. (a) Tracheae from mice exposed to PBS IN stimulated with (a) ACh, WT (n=8), Itk - /- (n=5). (b) CCh, WT (n=8), Itk -/- (n=5). (c) Tracheae from mice exposed to OVA IN stimulated with (a) Ach (n=9). (d) CCh (n=9). All graphs are p<0.05 by 2 way ANOVA. Filled circles, WT mice; open circles, Itk -/- mice. These experiments were done by Ferrara.TJ. 76

97 77 Figure 3.3: Reduced tracheal responses to KCl stimulation in mice lacking ITK. (a) Tracheae from mice exposed to PBS IN, n=7 for WT mice, n=6 for Itk -/- mice. (b) Tracheae from mice exposed to OVA IN, n=10 for WT and Itk -/- mice. Both p<0.05 by 2 way ANOVA. Filled circles, WT mice; open circles, Itk -/- mice. These experiments were done by Ferrara.TJ.

98 Figure 3.4: Reduced T H 2 cytokine message in lungs of mice lacking Itk during allergic asthma induction. (a) Quantitative RT-PCR for the indicated genes from mrna isolated from lungs of WT and Itk -/- mice treated as in figure 3.1 *p<0.05 by student s t test, n=12. (b) ELISA for the indicated proteins. *p<0.05 by student s t test, n=5. 78

99 Figure 3.5: Rescue of AHR in mice lacking Itk by transfer of CD4 + T cells. (a) ( - ), Naïve Itk -/- mice (n=9); ( - ), Naïve WT recipients/itk -/- OVA splenocytes (n=8); ( - ), Naïve Itk -/- recipients/wt OVA splenocytes (n=12). Data for OVA challenged WT strain from fig. 3.1 is included for ease of comparison ( - ). (b) ( - ), Naïve Itk -/- recipients/itk - /- OVA splenocytes (n=12); ( - ), Naïve Itk -/- recipients/wt OVA CD4 - cells (n=13); ( - ), Naïve Itk -/- recipients/wt OVA CD4 + T cells (n=8). *p<0.05, 2 way ANOVA. 79

100 Figure 3.6: Rescue of airway inflammation in mice lacking Itk by transfer of CD4 + T cells. Lung sections from mice treated as in figure 3.5 stained with H&E for inflammation. (a) Naïve Itk -/- mice. (b) Naïve Itk -/- recipients/wt OVA splenocytes. (c) Naïve Itk -/- recipients/wt OVA CD4 - cells. (d) Naïve Itk -/- recipients/ WT OVA CD4 + T cells. All images were taken at 20X. 80

101 81 Chapter 4 Differential sensitivity to Itk kinase signals for T helper 2 cytokine production and chemokine mediated migration This chapter is a partial reprint of a paper originally published in the Journal of Immunology Mar 15;180(6): (2008). The authors are Sahu N, Mueller C, Fischer A and August A. Copyright permission for the use of the materials has been approved by the Journal of Immunology, Copyright 2008 The American Association of Immunologists, Inc. 4.1 Introduction Allergic asthma develops due to a complex interplay between different types of immune cells and airway resident cells. Upon exposure to allergen, airway epithelial cells and macrophages secrete chemokines, while T H 2 cells secrete cytokines such as IL-4, IL- 5 and IL-13 (233, 245). These cytokines, along with chemokine gradients induce the infiltration of inflammatory cells into the lungs (233). These events can lead to pathophysiological dysfunctions including airway hyperresponsiveness, lung inflammation and mucous production (233). Tec family tyrosine kinases, including Itk which is expressed in T cells, have been shown to be important for proper immune response against a number of microbial insults, including infections caused by T. gondii, L. major, N. brasiliensis and S. mansoni (77, 229, 230). We have also shown that Itk is important for the development of allergic

102 82 asthma (215, 216). This importance stems from the ability of Itk to modulate the development of normal T H 2 responses in vivo (229, 230, 246). Indeed, during the induction of allergic asthma, Itk null mice exhibit reduced symptoms including decreased airway hyperresponsiveness, tracheal responses, lung inflammation, eosinophil infiltration, mucous production and T H 2 cytokine production (215, 216). In addition, Itk has also been shown to control the ability of T cells to migrate to a chemokine gradient (90, 247). The kinase domain of tyrosine kinases is responsible for their catalytic activity. The importance of the kinase domain in the function of Tec kinases is illustrated in the recognition that 50% of the mutations that are responsible for X-linked agammaglobulinemia (XLA) are found in the kinase domain of Btk, a Tec kinase predominantly expressed in B cells (248). More directly, we and others have identified signaling pathways that are modulated by Btk or Itk that are kinase independent. These pathways include the rescue of B cell development by a kinase inactive Btk, which may be due to its ability to partially activate the NFκB pathway (249). Btk also has tumor suppressive activity that is independent of its kinase domain (250). Antigen receptor induced actin cytoskeletal rearrangements and activation of the transcription factor SRF have also been reported to be Itk kinase independent (212, 251, 252), raising the possibility that other functions may be kinase independent. Since mice lacking Itk are resistant to developing allergic asthma (215, 216), a number of inhibitors have recently been developed that target the kinase activity of Itk (244, ). However, the role of kinase activity of Itk in the induction of allergic

103 83 asthma is still unknown. In addition, it is unclear if the function of Itk in controlling T H 2 cytokine secretion and chemokine migration is separable during the development of allergic asthma. Using novel transgenic mice specifically carrying a mutant Itk without any kinase activity, we show here that active kinase signaling is required for the control of T H 2 responses and the development of allergic asthma. However, reduction of Itk signals allowed normal T H 2 responses, while significantly affecting chemokine mediated migration. Our findings thus suggest that signals required for T H 2 responses and migration is differentially sensitive to Itk kinase activity. 4.2 Results The kinase domain of Itk is required for development of AHR and airway inflammation To analyze the role of kinase activity of Itk in allergic asthma, WT, Itk -/-, and transgenic mice carrying a mutant Itk lacking the kinase domain on an Itk -/- background (Tg(Lckpr-Itk Kin)/Itk -/- ) were immunized and challenged with OVA to induce allergic airway responses. Mice were then analyzed for airways resistance in response to methacholine. We found that while WT mice developed significant airways resistance in comparison to Itk -/- mice as previously reported (216), the Tg (Lckpr-Itk Kin)/Itk -/- mice showed much reduced responses (Figure 4.1A). These data indicate that the kinase activity of Itk is essential for the development of airway hyperresponsiveness. We also analyzed mice carrying the mutant Itk on a WT background (i.e. in the presence of

104 84 endogenous Itk, referred to as Tg(Lckpr-Itk Kin)/WT) and found that the latter mice also showed significantly reduced airways resistance compared to WT mice, although their responses were higher than Itk -/- mice (Figure 4.1A). This decreased airway resistance in Tg(Lckpr-Itk Kin)/WT mice was an unexpected finding because these mice express endogenous Itk in the periphery at a level approximately 3 fold higher than the transgenic Itk (data not shown). Histological analysis of airway inflammation and mucous production in the lungs of these mice also showed the same pattern, with less inflammation observed in both Tg(Lckpr-Itk Kin)/Itk -/- and Tg(Lckpr-Itk Kin)/WT mice compared to the WT mice (Figure 4.1B). These data suggest that the kinase domain and thus activity of Itk is required for the induction of airway inflammation, airway constriction and mucous production. More importantly, these data suggest that the kinase domain deleted Itk has the capacity to dominantly suppress the normal function of endogenous Itk in vivo The kinase domain of Itk is required for the expression of T H 2 cytokines in the lung during allergic airway inflammation We also analyzed the expression level of T H 2 cytokines IL-4 and IL-13 in the lungs of these animals and found that the Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and surprisingly Tg(Lckpr-Itk Kin)/WT mice, all had reduced levels of message for these cytokines (Figure 4.2A). Analysis of protein levels confirmed these results, although the Tg(Lckpr- Itk Kin)/WT had higher levels of IL-4 and IL-5 than Itk -/- and Tg(Lckpr-Itk Kin)/Itk -/- mice (Figure 4.2B). These results indicate that the kinase domain of Itk is required for

105 85 the induction of T H 2 cytokines in the lungs of mice during the development of allergic asthma. In addition, the kinase domain deleted mutant dominantly affects the production of these cytokines in the lungs of Tg(Lckpr-Itk Kin)/WT Role of the kinase domain of Itk in modulating the ability of T cells to proliferate and induce T H 2 responses to OVA We further analyzed the recall response of splenocytes from OVA-exposed mice to subsequent OVA challenge in vitro. T cells from Tg(Lckpr-Itk Kin)/Itk -/- mice had low proliferative responses to OVA similar to those lacking Itk, while T cells from Tg(Lckpr-Itk Kin)/WT mice behaved similarly to WT mice and proliferated normally (Figure 4.3A). T cells from Itk -/- and Tg(Lckpr-Itk Kin)/Itk -/- mice had low levels of T H 2 cytokines, IL-4, -5 and IL-13; however those from WT and surprisingly the Tg(Lckpr- Itk Kin)/WT mice had similar high levels of T H 2 cytokines (Figure 4.3B). By contrast, cells from all mice secreted similar levels of the T H 1 cytokine IFNγ, suggesting that the Itk -/- and Tg(Lckpr-Itk Kin)/Itk -/- were able to generate a normal T H 1 response. In agreement with these findings, we also found that Tg(Lckpr-Itk Kin)/WT mice were able to generate a normal antigen-specific IgE response following OVA exposure (Figure 4.3 C). Thus the kinase domain of Itk is required for T cell proliferation and T H 2 cytokine secretion in vitro in response to antigen restimulation. However, reducing kinase signaling via Itk does not affect antigen specific proliferation or T H 2 cytokine secretion in vitro, or the development of IgE responses in vivo.

106 4.2.4 The kinase domain of Itk is required for T cell recruitment into the lung and chemokine induced migration in vitro 86 One potential explanation for the observation of reduced T H 2 cytokines in the lungs of Tg(Lckpr-Itk Kin)/WT mice is a reduction in the recruitment of T cells into the lungs as we have previously reported in mice lacking Itk (215, 216). Indeed, Itk is involved in chemokine-mediated migration, which requires alterations in the actin cytoskeleton (90, 247), and Itk can modulate chemokine mediated actin cytoskeleton rearrangements, which is suggested to be kinase dependent (247). We, therefore, determined if CD4 + T cell recruitment into the BALF of mice was affected by the presence of the transgene. We found that while WT mice had significant levels of CD4 + T cells in the BAL fluid (Figure 4.4A) and lung (data not shown), Itk -/- or Tg(Lckpr- Itk Kin)/Itk -/- mice had significantly reduced recruitment of these cells into the lungs. More importantly, Tg(Lckpr-Itk Kin)/WT also had significantly reduced recruitment of CD4 + T cells in the lung. This was accompanied by an overall reduction in cellular infiltration in BAL (Figure 4.4B). Analysis of the ability of in vitro differentiated T H 2 cells from WT and Tg(Lckpr-Itk Kin)/WT to migrate in response to the T H 2 chemokine CCL11/eotaxin-1 in vitro confirmed that Tg(Lckpr-Itk Kin)/WT T H 2 cells have defects in chemokine mediated migration (note that these cells differentiated to T H 2 cells and expressed similar levels of CCR3, the receptor for CCL11, data not shown) (Figure 4.4 C). This reduction in migratory response reflected reduced activation and increase in actin polymerization in these cells, which is required for effective migration (Figure 4.4 D). By contrast, we did not observe any differences in either actin polymerization (data

107 87 not shown) or nuclear localization of NFAT-c1, events previously shown to be affected by the absence of Itk upon TCR stimulation in T cells from these mice (Figure 4.4E) (229, 230). These data thus indicate that chemokine, but not TCR signal is highly dependent on the signaling threshold regulated by the kinase activity of Itk. Together, these data suggest that there is a threshold of Itk signaling dependent on the kinase domain that controls the development of symptoms of allergic asthma in vivo, and that this signaling threshold does not affect the development of a T H 2 response in the Tg(Lckpr-Itk Kin)/WT mice, but affects the migratory capacity of the T cells in these mice. 4.3 Discussion Itk regulates the differentiation and/or cytokine production capacity of T H 2 producing cells, as well as the development of allergic asthma (215, 216, 229, 230, 246). We and others have also shown that certain functions of Itk are kinase independent, including activation of the transcription factor SRF, modulating the localization of Vav GEF and induction of actin cytoskeleton rearrangements downstream of the TCR (212, 252). However, our data indicate that Itk functions controlling the development of T H 2 producing cells in vivo are kinase domain dependent. In addition, our findings show that reducing signaling through Itk can leave the development of T H 2 producing cells intact, while selectively affecting recruitment of T cells into the lungs. This indicates that the activity of Itk can be manipulated to separate functions involved in T H 2 cytokine production vs. chemokine induced migration. Our data further suggest that T cell

108 88 activation, differentiation and cytokine secretion is dependent on different levels of kinase activity and subsequent events downstream of Itk, in comparison to signals required for migration. Itk can regulate signaling pathways induced by the TCR as well as chemokine receptors. Downstream of the TCR, Itk functions to increase intracellular calcium and activate the NFAT transcription factor (81). In addition, Itk is also involved in TCR activation of Ras/MAPK signaling pathways leading to the activation of transcription factors such as AP-1 (257). These pathways eventually influence the secretion of cytokines such as IL-2 and IL-4, both of which have been shown to be dependent on the kinase activity of Itk (246, 258). Similarly, in cell culture models, signals regulated by Itk downstream of chemokine receptors have been shown to be dependent on the kinase activity (247). However, Itk also has been shown to function as a scaffold for the assembly of signaling proteins, such that downstream of antigen receptors, the kinase activity of Itk is not required for the induction of actin polymerization or the activation of the transcription factor SRF (212, 251, 252). Using its SH2 and SH3 domains, Itk can interact with the multiprotein complex containing LAT, Slp-76 and Vav, which may explain its ability to regulate SRF activation in a kinase independent fashion since this factor is downstream of actin rearrangements (252, 259). However, our data here indicate that kinase activity of Itk is required for the development of allergic asthma. It is possible that the PH, SH2 and/or SH3 domains of Itk interact with the LAT/Slp-76/Vav multiprotein complex to affect the actin cytoskeleton and migration in response to chemokine. This reduction in chemokine induced migration reduces T cell recruitment

109 89 into the lung, reducing T H 2 cytokines, thus leading to the observed reduction in AHR in the Tg(Lckpr-Itk Kin)/WT. However, the interaction with these effectors may have different functional outcomes downstream of the TCR signaling pathway (252, 259), and thus T H 2 cytokine secretion from spleen and lymph nodes is unaffected. The absence of Itk has also been shown to affect the development of CD4 + as well as CD8 + T cells (89, ). Analysis of our transgenic mice carrying the kinase deleted Itk indicates that further reduction of Tec kinase signal does not significantly affect this process, similar to what has been observed in mice lacking both Itk and Txk/Rlk (data not shown, (260)). Indeed, when expressed on the WT background, T cell development (data not shown), or the development of T H 2 cytokine secreting T cells is not affected, suggesting that the reduced signaling that occurs in the presence of the kinase deleted Itk mutant is not sufficient to affect T cell development or T cell differentiation in response to specific antigen. The expression level of Itk has been reported to change during the process of differentiation to T H 2 cells (59). However analysis of the expression of the transgene relative to endogenous Itk in naïve as well as differentiated T H 2 cells revealed that the transgene is expressed at approximately 30% of endogenous Itk in naïve CD4 + T cells, while in T H 2 cells, it is expressed at approximately 60% of endogenous Itk (data not shown). Thus, reduced expression of the transgene relative to endogenous Itk in T H 2 cells is unlikely to explain the relative sensitivity of chemokine vs. T H 2 differentiation pathways for Itk signals. It is possible that level of transgene expression in our mice may not be sufficient to observe potential roles for the other domains of Itk when expressed on the Itk null background. We do note that Itk

110 90 heterozygous mice have a WT phenotype, and a WT Itk transgene can rescue all aspects of T cell function when expressed on the Itk null background at similar levels to this transgene (Hu and August, unpublished). However, it is possible that if expressed at higher levels, potential functional rescue may be observed with the Itk Kin mutant. In contrast to the T H 2 pathway, signals regulated by Itk that control chemokine receptor mediated migration is affected by reducing signals, as observed in the Tg(Lckpr- Itk Kin)/WT mice. Thus there appears to be a hierarchy of signals that are regulated by this kinase with different processes differentially dependent on these signals. Signals that control development of T cells, and T H 2 cytokine production may have a lower threshold, which is reached in our transgenic animals. By contrast, those signals that control migration have a higher threshold and so are preferentially affected with the reduction in signal as in the Tg(Lckpr-Itk Kin)/WT mice. A role for migration and recruitment of T cells into the lung during the development of allergic asthma is strongly supported by reports in the literature (233). Chemokine and chemokine receptor modulation of T cell migration has been shown to play an important role in the recruitment of T cells into the lung, leading to disease. Indeed, the compound FTY720 prevents the development of allergic asthma in part by affecting T cell migration out of lymph nodes and into the lung (264). Thus the ability of Itk to control both T cell migration and T cell differentiation implicates Itk as an important player in the development of this disease. In addition, the ability to reduce Itk signaling to preferentially affect migration and not T H 2 differentiation suggest that it may be possible to do this to affect the course of disease. Our data suggest that by altering

111 91 signals coming from this kinase one can manipulate these T cell responses. A number of inhibitors have recently been developed that target the kinase activity of Itk which completely inhibit T cell responses (244, ), but can potentially lead to immunosuppression. Our work here suggest that manipulation of the dose of these inhibitors to selectively alter T cell migration while maintaining normal systemic T cell responses, may provide with a better and more effective strategy to treat allergic asthma.

112 Figure 4.1: The kinase domain of Itk is required for the induction of AHR and allergic inflammation. (a) WT, Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and Tg(Lckpr- Itk Kin)/WT mice were immunized and challenged intranasally with OVA, followed by analysis of AHR. -, WT; -, Itk -/- ; -, Tg(Lckpr-Itk Kin)/WT, -, Tg(Lckpr- Itk Kin)/Itk -/-. **Differences were statistically significant between WT and Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and Tg(Lckpr-Itk Kin)/WT; *Differences were statistically significant between Tg(Lckpr-Itk Kin)/WT mice and Itk -/- and Tg(Lckpr-Itk Kin)/Itk -/-, as well as between WT and Tg(Lckpr-Itk Kin)/WT mice (p<0.05 in both cases, n=12). (b) WT, Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and Tg(Lckpr-Itk Kin)/WT mice were immunized and challenged intranasally with OVA, followed by analysis of lung sections by H&E (top panels) or PAS staining (bottom panels). 92

113 Figure 4.2: The kinase domain of Itk is required for the production of T H 2 cytokines in the lung during the development of allergic asthma. (a) WT, Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and Tg(Lckpr-Itk Kin)/WT mice were treated as in figure 1, lungs isolated and mrna for the IL-4, -13 and IFN-γ quantified by Q-RT-PCR. (b) BAL fluid from WT, Itk -/-, Tg(Lckpr-Itk Kin /Itk -/-, and Tg(Lckpr-Itk Kin)/WT mice treated as in Figure 4.1 were isolated and the amounts of IL-4, -5 and -13 quantified. 93

114 Figure 4.3: Role of the kinase domain of Itk in T cell responses and T H 2 development in response to OVA immunization. (a) WT, Itk -/-, Tg(Lckpr-Itk Kin)/Itk - /-, and Tg(Lckpr-Itk Kin)/WT mice were treated as in figure 1, and spleens and lymph nodes collected, stimulated with the indicated concentration of OVA, and proliferation determined. (b) Supernatants from splenocytes and lymph node cells stimulated with 100 µg/ml OVA in vitro for 96 hours, were collected and assayed for IL-4, -5, -13 and IFNγ. (c) WT, Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and Tg(Lckpr-Itk Kin)/WT mice were treated as in figure 4.1, and serum collected and analyzed for OVA specific IgE as described in the materials and methods section. ND, none detected. 94

115 Figure 4.4: Reduced Itk signals specifically affect chemokine mediated migration in Itk Kin Tg /WT T cells. (a) WT, Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and Tg(Lckpr-Itk Kin)/WT mice were treated as in figure 1, and BALF collected and analyzed for the number of CD4 + T cells. (b) WT, Itk -/-, Tg(Lckpr-Itk Kin)/Itk -/-, and Tg(Lckpr-Itk Kin)/WT mice were treated as in figure 1, and BALF collected and analyzed for the number of lymphocytes. (c) Migration of WT and Tg(Lckpr-Itk Kin)/WT T H 2 cells in response to 100 ng/ml CCL11 in vitro was determined. (d) Actin polymerization in response to PBS (C) or CCL11 signals (100 ng/ml) were analyzed in WT and Tg(Lckpr-Itk Kin)/WT T H 2 cells. (e) Nuclear translocation of NFAT-c1 in response to PBS (C) or TCR signals (anti- CD3) in WT and Tg(Lckpr-Itk Kin)/WT CD4 + T cells. *p<0.05 vs. WT mice; NS, not significant. 95

116 96 Chapter 5 Selective expression rather than specific function of Txk and Itk regulate T H 1 and T H 2 responses 5.1 Introduction Txk/Rlk (hereafter referred to as Txk) and Itk are distantly related members of the Tec family of tyrosine kinases that are involved in signaling downstream from the TCR. While Txk has a palmitoylation site instead of a PH domain that allows it to be constitutively associated with lipid raft membrane fractions, Itk requires the activation of PI3 kinase for recruitment to the membrane via its PH domain (265, 266). Mutations affecting Itk in mice lead to altered T cell development and mature T cell function with reduced TCR-induced proliferation and impaired IL-2 production in vitro ((85, 260, 261, 267) see (257) for review)). The most dramatic phenotype of Itk -/- mice is their defect in T H 2 responses in vivo. Itk -/- mice are incapable of developing allergic asthma and have decreased responses to challenge with a number of T H 2-inducing parasites including the eggs of Schistosome mansoni or the worm Nippostrongyloides brasilienses (215, 216, 229, 230). Indeed, in some cases, Itk-deficient mice have been found to mount T H 1 responses to T H 2-inducing pathogens. In contrast, overexpression of Txk has been associated with increased expression of IFN-γ, a T H 1 cytokine (214, ). Txk has been found to bind directly to a sequence in the IFN-γ promoter, suggesting a direct role for Txk in driving IFN-γ transcription and T H 1 responses (271). Together, these data

117 97 suggested that Itk and Txk have distinct roles in T H 2 and T H 1 differentiation or function respectively. Nonetheless, the exact mechanism by which the Tec kinases influence T helper cell differentiation remains controversial. While some data suggest that Itk induces T H 2 differentiation by suppressing the expression of T-bet (59), other reports propose that Itk but not Txk directly interacts with and tyrosine phosphorylates T-bet, promoting its interaction with GATA3, which suppresses the latter s activity (134). It has also been proposed that Itk may modulate T H 2 differentiation by virtue of its expression: Itk is expressed at higher levels than Txk in naïve T cells and while both Itk and Txk are expressed in T H 1 cells, Txk is downregulated in T H 2 cells, leaving Itk as the major Tec kinase (59). Consistent with this idea, recent data argue that Itk is not required for T H 2 differentiation per se, but rather is required for effector function of differentiated T H 2 cells (246). These data suggest it may not be the intrinsic function of these kinases, but rather their patterns of expression that determines their roles in T helper cell differentiation and function. To evaluate these questions, we examined whether forced over-expression of Txk in T cells could rescue T H 2 responses in Itk -/- mice. If these kinases have distinct T H 1 and T H 2-inducing properties, one would predict that overexpression of Txk would preferentially drive T H 1 responses and T H 2 defects in Itk -/- mice may be exacerbated. However, if defects in T H 2 responses in Itk -/- mice are secondary to the low levels of expression of Tec kinases in T H 2 cells in the absence of Itk, expression of the Txk transgene may rescue these responses. Utilizing the murine model of allergic asthma, we

118 98 demonstrate that Itk -/- mice expressing a Txk transgene can rescue T H 2 responses, with no evidence of overexpression of T H 1 cytokines. Our results thus strongly suggest that the effects of Itk and Txk on T helper cell function may result from the differential patterns of expression of these kinases. 5.2 Results The CD2 promoter driven Txk transgene is expressed in T H 2 cells To evaluate whether Txk can compensate for Itk in T H 2 responses, we utilized a transgenic mouse model in which Txk was overexpressed using the CD2 promoter (Tg(CD2-Txk)). Previous work has demonstrated that this transgene can partially rescue certain T cell defects in Itk -/- mice including Ca 2+ mobilization following TCR crosslinking (214). To further evaluate expression of the Txk transgene, CD4 + cells from naïve Tg(CD2-Txk) mice were differentiated under T H 2-inducing conditions in the presence of IL-4 and anti-il-12 for 7 days and Txk-specific message was evaluated by QRT-PCR. Similar to previous observations in resting naïve T cells, the Txk transgene was also highly expressed in T H 2 cells (data not shown). We therefore utilized this transgene on the Itk -/- background to evaluate the ability of Txk to complement Itk function in T H 2 cells.

119 Rescue of allergic airway inflammation and airway hyperresponsiveness (AHR) in mice lacking Itk by expression of Txk transgene To determine the effect of expression of Txk in T H 2 cells, we analyzed the development of allergic asthma, a disease dependent on T H 2 cells and cytokines. OVA immunized and challenged WT and Itk -/- mice, as well as mice expressing the Txk transgene on an Itk -/- background (Tg(CD2-Txk)Itk -/- ) were evaluated for airway resistance in response to methacholine challenge as a measure of AHR. In these experiments, WT mice developed significant airway resistance in comparison to Itk -/- mice as previously reported (Figure 5.1A) (216). However, Tg(CD2-Txk)Itk -/- transgenic mice also exhibited significant levels of airway resistance, similar to the WT mice (Figure 5.1A). We next analyzed airway inflammation and mucous production, factors that can contribute to the development of allergic asthma in this model. Histological evaluation of lung sections revealed that Tg(CD2-Txk)Itk -/- mice exhibit massive leukocyte infiltration in the lung, which was similar or higher than that observed in WT mice (Figure 5.1B). In contrast, Itk -/- mice showed reduced leukocyte infiltration, as previously reported (215, 216). Increased thickening of the epithelial cell lining of the bronchioles and mucous production by airway goblet cells was also observed in Tg(CD2-Txk)Itk -/- mice, similar to that seen in WT mice (Figure 5.1B).

120 Expression of Txk transgene in Itk-null mice enhances production of T H 2 cytokines in response to allergic inflammation T H 2 specific cytokines such as IL-4, IL-5 and IL-13 are involved in inducing allergic airway inflammation (187, 233). To examine whether expression of Txk transgene rescued production of T H 2 cytokines, we first analyzed cytokine production from splenic T cells of mice immunized and challenged with OVA. Stimulation with OVA in vitro induced proliferation of T cells from WT, Tg(CD2-Txk)Itk -/- and Itk -/- OVA-challenged mice, although those from Itk -/- mice exhibited reduced proliferation in comparison to WT mice (Figure 5.2A) (215). Splenocytes from Tg(CD2-Txk)Itk -/- mice, however, had proliferative responses equivalent to WT mice, and the Txk transgene rescued IL-4, IL-5 and IL-13 secretion from these cells in vitro (Figure 5.2B). Since Txk is suggested to regulate the expression of T H 1 cytokines such as IFN-γ, we also examined the expression of IFN-γ. Strikingly, Tg(CD2-Txk)Itk -/- mice did not secrete elevated levels of IFN-γ as would be expected if it specifically regulated IFNγ (Figure 5.2B). To further examine the expression level of T H 2 cytokines in the lungs upon induction of allergic asthma, we measured the expression level of IL-4 and IL-13 mrna in the lung of OVA immunized and challenged WT, Itk -/- and Tg(CD2-Txk)Itk -/- mice using real-time quantitative RT-PCR. There was enhanced expression of IL-4 and IL-13 mrna in the Tg(CD2-Txk)Itk -/- mice in comparison to WT and Itk -/- mice. By contrast, the mrna levels of IFN-γ were similar in all three strains of mice (Figure 5.2C). These findings were further confirmed by the analysis of BAL fluid for the level of T H 2

121 101 cytokines in these mice, indicating that expression of Txk rescued the T H 2 mediated inflammation in the lungs of OVA challenged mice (Figure 5.2D). Therefore the Txk transgene does not appear to lead to enhanced T H 1 differentiation or IFNγ production, nor does it block T H 2 differentiation as previously suggested (214, ). Higher levels of IL-13 mrna but not protein was observed in the Tg(CD2-Txk)Itk -/- mice in comparison to WT. This could be due to IL-13 mrna expression in other inflammatory cells such as eosinophils and mast cells, recruited to the lungs in response to the disease. However, since we did not observe higher levels of the IL-13 protein, this message may not be translated in these cells. Altogether, these data indicate that in the absence of Itk, Txk expression can lead to the generation of a T H 2 response both systemically and in the lungs Rescue of CD4 + T cell recruitment in Itk-null mice expressing Txk transgene Analysis of CD4 + T cell numbers in the lungs revealed that while WT mice could recruit these cells into the lung during airway inflammation, as previously reported, Itknull mice could not (90, 215). However, Tg(CD2-Txk)Itk -/- mice had similar numbers of CD4 + T cells in the lungs compared to WT mice, indicating that expression of Txk was able to rescue migration and recruitment of T cells into the lung (Figure 5.3A). These results were not secondary to increased numbers of CD4 + T cells in Tg(CD2-Txk)Itk -/- mice as these mice have similar numbers of mature CD4 + T cells as Itk -/- mice (data not shown). We further found significantly elevated levels of Txk expression in the lungs of the Tg(CD2-Txk)Itk -/- mice (Figure 5.3B). These data thus confirm that in vivo, Txk can

122 rescue specific functions of Itk that lead to the recruitment of leukocytes into the lungs during the development of allergic asthma Discussion The Tec kinases Itk and Txk are both expressed in T cells and regulate their development, activation and function (257). A role for Itk has been demonstrated for the production of T H 2 cytokines, while roles for Txk are less clear (59, 215, 216, 229, 230, 246). Prior work suggests that Txk regulates IFN-γ production and thus T H 1 development; however, a specific role of Txk in the regulation of T H 1 specific cytokine production is still unclear (214, ). Our data argue that Txk does not specifically regulate this T H 1 cytokine. In our models, Txk was incapable of enhancing T H 1 differentiation or induce elevated levels of IFN-γ in order to prevent the development of T H 2 cell mediated disease. Instead, we observed elevated levels of T H 2 type cytokines including IL-4, -5 and 13 in the lungs of Tg(CD2-Txk)Itk -/- mice in response to T H 2- inducing agents. However, it should be noted that these observations do not rule out the possibility that Txk could indirectly regulate IFN-γ production via interaction with other factors which are specifically expressed in T H 1 cells. Txk is the most distantly related family member of the Tec family of tyrosine kinases. This protein has a N-terminal palmitoylation site instead of a PH domain found in Itk and other Tec kinases, allowing it to be anchored constitutively in the plasma membrane. Thus Txk, unlike Itk, does not require the activation of PI3 kinase in order to

123 103 be recruited to the plasma membrane (265, 266). This would suggest specific and unique functions for Txk. However, we show that when overexpressed, Txk can functionally replace Itk for the induction of predominant T H 2 responses in vivo by enhancing the expression of T H 2 specific cytokines. Our data provide strong evidence that Txk can function to rescue T H 2 responses in the absence of Itk, including AHR, airway inflammation, granuloma formation, T cell recruitment and cytokine production in the lungs in vivo as well as T H 2 cytokine production from T cells restimulated in vitro. Our data thus support that these kinases have overlapping functions. Although Txk is not normally expressed in T H 2 cells, our observations with these transgenic mice nonetheless help provide a better understanding of the specific functions of Txk and Itk which would have been otherwise difficult to address. In addition, these observations provided greater insight about the relevance of selective expression of Txk and Itk in T H 1 and T H 2 cells respectively. Previous studies by Berg and colleagues have shown that when differentiated under T H 1 and T H 2 conditions, Txk expression is specifically downregulated in T H 2 cells (59). In contrast, they observed similar levels of Itk expression in T H 1 as well as T H 2 cells. Thus negative regulation of Txk expression may occur in a T H 2 specific manner, perhaps via negatively regulation by T H 2 specific transcription factors. Alternatively, Itk s expression may be maintained by T H 2 specific transcription factors. This expression pattern however, results in a critical dependence of T H 2 cells on the function of Itk. Our work here lends support to this model since expression of Txk using the T cell specific CD2 promoter would allow for continuous expression of Txk, even in differentiating T H 2 cells, and provide crucial Tec kinase

124 104 signals needed for their functional response in the absence of Itk. Hence, our observations suggest that the selective expression of Txk and Itk in T H 1 and T H 2 cells respectively may provide the delicate balance of signals required for inducing or maintaining different types of T cell responses. In previous studies, we have found that while the absence of Itk leads to defective T H 2 responses, absence of both Txk and Itk surprisingly leads to normal T H 2 responses in Rlk -/- Itk -/- mice (230). Although these results may appear to contradict our current findings, it is possible that these findings result from the fact that CD4 T cells lacking both Txk and Itk maintain high levels of GATA3 following stimulation compared to WT and Itk null mice, suggesting that a defect in GATA3 downregulation may lead to a propensity to develop into T H 2 cells in T cells from the Txk/Itk double knockout mice, perhaps because this is the only type of response that can occur. Our data presented here indicates that overexpression of Txk can also lead to normalized T H 2 responses in Itkdeficient mice, and that potential redundancy in function between Txk and Itk may explain the T H 2 specific response observed in Tg(CD2-Txk)Itk -/- mice. Our data are also in support of recent data from Fowell and colleagues, who suggested that Itk deficient T cells can differentiate into T H 2 cells, however, they cannot elaborate and secrete T H 2 cytokines upon restimulation (246). The enforced expression of Txk in these T H 2 cells allows for functional rescue of this event, most likely via calcium signaling, as discussed below. Data reported by Sommers and colleagues showed that expression of this Txk transgene can rescue thymic selection defects and

125 105 Ca 2+ mobilization in Itk-null thymocytes (214). These observations suggest that Txk may be able to rescue T H 2 responses by restoration of intracellular Ca 2+ increases that are defective in Itk -/- T cells, perhaps by participating in the Slp76/GADS/LAT complex, which regulate PLCγ1 activation. Note that rescue of the Ca 2+ pathway could induce normal T H 2 cytokine secretion in Itk -/- T cells (229, 246). Indeed expression of the Txk transgene has been shown to be able to rescue PLCγ1 tyrosine phosphorylation and Ca 2+ increase in Itk -/- double positive thymocytes (214). Other cell types such as mast cells and eosinophils have also been suggested to play an important role in the development of T H 2 specific responses ( ). These cells are capable of producing T H 2 cytokines and chemokines that can induce initial immune responses and mediate further activation and migration of T H 2 cells to the lungs thereby exacerbating the immune responses (275). However, since the CD2 promoter cassette that drives Txk expression in these transgenic mice is primarily active in T cells, including NKT cells, these findings suggest that the defect in Itk-null mice in developing effective T H 2 responses in our models is unlikely to be due to the lack of expression of Itk in other cell types such as mast cells (276). In recent years there has been a growing interest in the Tec kinases Itk and Txk as potential therapeutic targets for T H 2 and T H 1-mediated diseases, respectively. Our data here strongly support the model that Tec kinases, Txk and Itk regulate T helper cell mediated responses via their differential expression in T H 1 and T H 2 cells respectively and not due to intrinsic functional differences as previously suggested (214, ). Our

126 106 data also suggest that the defective T H 2 response in the absence of Itk is due to reduced Tec kinase signals, and that either Itk or Txk can fulfill this role. Overall, these findings provide novel insight into the role of Tec kinases Txk and Itk in the regulation of T helper cell differentiation/function and disease.

127 107 Figure 5.1: Rescue of AHR in Itk-null mice by expression of the related kinase Txk. (a) WT, Itk-null or Tg(CD2-Txk)Itk -/- mice were immunized and challenged intranasally with OVA, followed by analysis of AHR by mechanical ventilation. Filled circles, WT; open circles Itk -/- ; Filled triangles, Tg(CD2-Txk)Itk -/- mice. O/O indicates mice that have been immunized with OVA, then challenged intranasally with OVA. Differences are statistically significant between WT and Itk -/- or Tg(CD2-Txk)Itk -/- and Itk -/- (p<0.05) but not significant between WT and Tg(CD2-Txk)Itk -/-. (n=5-7 mice per group, representative of three experiments). (b) Mice were immunized and challenged intranasally with OVA, followed by analysis of lung sections by H&E (top panels) or PAS staining (bottom panels), representative of three experiments.

128 108 Figure 5.2: Txk-mediated rescue of cytokine production in vitro and in vivo. (a) Splenocytes from WT, Itk-null or Tg(CD2-Txk)Itk -/- mice treated as in figure 5.1 were incubated in vitro with the indicated concentration of OVA, and analyzed for proliferative responses after 96 h of culture. Data are the mean ± SEM of triplicate cultures and are representative of three experiments, *p<0.05. (b) Splenocytes were treated as in (a) with 100 µg/ml OVA and supernatants analyzed for IL-4, -5 and -13 and IFN-γ after 72 h of culture. Data are the mean ± SEM of triplicate cultures and are representative of three experiments, *p<0.05, NS, Not significant. (c) Lungs from WT, Itk -/- or Tg(CD2-Txk)Itk -/- mice treated as in figure 5.1 were analyzed by QRT-PCR for IL-4, IL-13 and IFN-γ. n = 6, *p<0.05, NS, not statistically significant. (d) BAL from lungs of mice treated as in figure 5.1 was analyzed for IL-4, -5, -13 and IFNγ. n = 6, *p<0.05, NS, not statistically significant.

129 109 Figure 5.3: Rescue of CD4 + T cell recruitment into the lungs of Itk-null mice by expression of the related kinase Txk. (a) BAL fluid from lungs from WT, Itk -/- or Tg(CD2-Txk)Itk -/- mice treated as in figure 5.1 were analyzed for the number of CD4 + T cells by flow cytometry. (b) Lungs from mice treated as in figure 5.1 were analyzed by QRT-PCR for Txk. *p<0.05, n = 6, NS, not statistically significant.

130 110 Chapter 6 Absence of ITK prevents the development of allergic airway disease irrespective of the level of allergen exposure 6.1 Introduction Using mouse models, we have previously shown that Itk plays an important role in the induction of T H 2 cell mediated allergic asthma (160, 277). The absence of Itk abrogates the development of various symptoms associated with allergic asthma such as airway hyperresponsiveness, mucous production, lymphocyte infiltration and inflammatory cytokine production. We used a commonly used approach to induce allergic asthma in our models, using ovalbumin (OVA) as our surrogate antigen, which requires the initial sensitization with the antigen via intraperitoneal administration of the antigen OVA in conjunction with chemical adjuvant aluminum hydroxide. The mice are then challenged with antigen OVA by respiratory exposure to induce the development of the disease. However, this model has numerous limitations. Firstly, it may not mimic the actual mode of exposure to the allergens in humans. Humans most likely develop allergic asthma through direct inhalation of the allergens without prior systemic sensitization. Secondly, prolonged and continual respiratory exposure to OVA leads to inhalation tolerance and inhibits local airway responses to the antigen ( ). Thus OVA cannot

131 111 be used to exactly mimic aeroallergen induced allergic asthma and prevents the better understanding of the mechanism underlying this disease. Previous studies have suggested that antigen concentration and strength of the signal can influence the differentiation of T H 1 and T H 2 cells and thus can influence the development of allergic asthma (282, 283). While both weak and strong signals have been suggested to induce T H 2 differentiation, moderate signals have been suggested to induce T H 1 differentiation ( ). Furthermore, Itk has been suggested to act as an amplifier of the signals generated by Src kinases downstream of TCR signaling and has been suggested to enable the cells to reach the required signaling thresholds required for their activation (289). In the absence of Itk, PLCγ1 is reduced which is required for mediating calcium influx in the cells and inducing NFAT activation that is required for inducing IL-2 and IL-4. As previously discussed, these two cytokines are involved in the initiation of T H 2 differentiation (157, 229). However there is still some residual calcium signaling and NFAT activation in the Itk -/- T cells, most likely due to another Tec kinase, Rlk/Txk (61). In addition, another related Tec kinase, Tec is also expressed in T cells, albeit at a much lower level, and may have redundant functions with Itk (39). Thus, it is probable that by manipulating the antigen concentration, one may be able to trigger sufficient TCR s to generate signals sufficient for the cells to differentiate into T H 2 cells in Itk -/- mice. We therefore set out to use a new mouse model of allergic asthma to study the role of Itk, using allergens that cause the disease in humans. We used whole body extracts of house dust mite (HDM) species Dermatophagoides pteronyssinus as our allergen because these are perennial indoor allergens that are responsible for inducing

132 112 allergic asthma in at least 10 percent of the general population, and for exacerbating the effects in 90 percent of people with allergic asthma ( ). We exposed WT and Itk -/- mice intranasally to various concentrations of the whole body extracts of HDM for 10 days and analyzed the inflammatory responses developed against the allergen. We observed that WT mice responded to various concentrations of HDM in a dose specific manner. However, as previously observed in the OVA model, the absence of Itk prevented the development of allergic asthma against HDM. There was decreased airway hyperresponsivess against methacholine, decreased airway inflammation and mucous production, decreased leukocyte infiltration and inflammatory cytokine production compared to WT mice. These data thus suggest that absence of Itk can prevent the development of allergic asthma against HDM irrespective of the concentration of the allergen and that targeting Itk may be a promising strategy for the development of therapeutic drugs against allergic asthma. 6.2 Results Exposure to varying doses of HDM extract does not lead to the development AHR in mice lacking Itk To investigate the development of allergic asthma, we measured the development of airway hyperresponsiveness, one of the key symptoms of allergic asthma. Using the flow interrupter technique, we measured the total respiratory resistance in WT and Itk null mice exposed to various doses of HDM. We observed that WT mice were

133 113 hyperresponsive to increasing doses of methacholine. In contrast, Itk null mice did not develop AHR irrespective of the concentration of HDM they were exposed to (Figure 6.1). In our previous studies, we had analyzed the development of AHR in Itk null mice in response to single dose of ovalbumin, a simple antigen and shown that absence of Itk can prevent the development of AHR in response to ovalbumin. Using whole body extract of house dust mite which is a complex allergen, we now show here that absence of Itk can also prevent the development of AHR in response to more complex allergens WT but not Itk null mice develop lung pathology in response to HDM extract We also analyzed the pathology of the lungs of WT and Itk null mice after exposure to various concentrations of HDM extract. While H&E staining was used for analyzing lymphocyte infiltration and airway inflammation (Figure 6.2), PAS staining was used for mucous production (Figure 6.3). We observed increase in airway inflammation, mucous production and lymphocyte infiltration in the lungs of WT mice to HDM extract. However, Itk null mice did not have any significant levels of airway inflammation, mucous production or lymphocyte infiltration in response to the various doses of exposure to HDM extract. These data therefore explain the lack of AHR response observed in the Itk null mice, since the AHR response is highly influenced by mucous production and airway inflammation in the lungs.

134 6.2.3 Dose dependent effects of HDM extract exposure on T H 2 cytokine and 114 chemokine production in WT but not Itk null mice T H 2 cell mediated cytokines such as IL-4 and IL-13 and chemokines such as CCL7 and CCL11 play key roles in the development of mucous production and airway inflammation. We therefore measured the levels of message for these proteins produced in the lungs of WT and Itk null mice in response to various concentrations of HDM extract using QRT-PCR. WT mice produced a robust response and generated high levels of IL-4, IL-13, CCL-7 and CCL-11 message which was dose dependent (Figure 6.4 and 6.5). We observed increasing amounts of message for these cytokines and chemokines with increasing doses of HDM extract. Although, the levels of CCL-11 message produced was drastically reduced in WT mice when exposed to 200µg/day of HDM. This could possibly be due to suppressive effects of increased amounts of effector cytokines present in the lungs. However, Itk null mice produced extremely low levels of these effector cytokines and chemokines. In addition, the levels of cytokine and chemokine message produced were not dose dependent in these mice. These data indicate that Itk is important in T H 2 cells to induce the production of effector cytokines IL-4 and IL-13 in response to HDM extract exposure. These data also imply that the absence of Itk influences the production of chemokines CCL-7 and CCL-11 either directly or indirectly via regulation of cytokine production. We also determined the message levels for IFN-γ, a T H 1 specific cytokine. We observed no difference between the levels of IFN-γ produced in WT and Itk -/- mice suggesting that Itk -/- mice generate normal T H 1 response to HDM

135 (Figure 6.4C). Furthermore, these results suggest that inhibiting the function of Itk may be efficient in preventing allergic asthma No difference in dendritic cell mediated allergen uptake between WT and Itk null mice In order to confirm that this difference in response to HDM between WT and Itk null mice is not due to a difference in the uptake of antigen by the dendritic cells, which eventually present the antigen to the T cells, we analyzed the antigen uptake by dendritic cells in WT and Itk null mice. We conjugated HDM to the fluorophore Alexa-750 and exposed to WT and Itk null mice intranasally to 25 µg of the conjugated allergen. Dendritic cells were then extracted from the lungs after 40 hours and the level of antigen uptake was analyzed using flow cytometry. We observed that antigen uptake between dendritic cells from WT and Itk null mice were similar (Figure 6.6A). In particular, there was no difference in the antigen uptake by the mdc s (CD11b +, CD11c + and Gr1 - ), which are immunoregulatory and mediate an immune response by activating T cells (Figure 6.6B). These data thus confirm that the difference in immune response seen between WT and Itk null mice in response to HDM allergen is not due to difference in antigen uptake by dendritic cells in these mice.

136 6.3 Discussion 116 The importance of Itk in the T H 2 cell differentiation and function has been suggested using various disease models such as infection with N. brasiliensis, S. manasoni and L. major (78, 163, 229). The absence of Itk prevents the generation of effective T H 2 responses but since these organisms can manipulate the immune response and can alter the type of immune response, it is not clear if Itk would be important in the development of T H 2 mediated diseases due to exposure to allergens. We had further investigated the effect of Itk on a T H 2 cell mediated disease, allergic asthma, using the model allergen, OVA (160, 277). We showed previously that Itk -/- mice failed to develop the disease in this model and lack of Itk contributed to attenuation of airway inflammation, mucous production and T H 2 cytokines, IL-4, IL-5 and IL-13 production. However, there is a lot of skepticism in the field of allergic asthma about using OVA as the model antigen for studying the disease as the exposure to the model allergen most likely does not mimic the complex real-life antigens. OVA requires the presence of adjuvant in order to sensitize for an immune response. In addition, it is difficult to study the effect of antigen concentration on the development of the disease as OVA can induce inhalation tolerance after continual exposure ( ). It was thus necessary to analyze the effect of Itk on allergic asthma against real life allergens. In addition, T helper cell development and function have been shown to be sensitive to antigen dose and strength of the signals delivered by the TCR (282, 283). Low and high concentrations of antigen has been suggested to induce T H 2 differentiation while moderate concentrations lead to T H 1 cell differentiation ( ). In this light, Itk

137 117 has been suggested to play a crucial role as it is believed to be an amplifier of the signals generated downstream of the TCR, inducing PLCγ mediated calcium mobilization via second messenger, IP 3, that leads to NFAT activation and IL-4 production. Cells lacking Itk have been shown to have decreased levels of intracellular calcium and reduced, although not absent NFAT activation (157, 229, 289). This suggests that the pathways downstream of Itk are partially functional in the absence of Itk through mechanisms which are still not clear. One possible explanation is the presence of other Tec kinases, Rlk/Txk and Tec in the T cells which may be able to compensate for Itk due to potential redundancy in their functions (39, 61) (see chapter 5 on Txktg mice). The other possible explanation is that in the cells lacking Itk, there may still be low levels of intracellular calcium increase, mainly from intracellular calcium stores, which may still be able to mediate residual levels of NFAT activation. Thus if the levels of IP3 can be elevated via Src and Syk kinases to the levels required for extracellular calcium flux it may be possible to induce normal levels of NFAT activation and IL-4 production required for T H 2 differentiation. Hence, it is tempting to speculate that the amount of signal generated can be manipulated by the amount of antigen exposure. We hypothesized that if the main function of Itk is to amplify the signal generated by the TCR and to induce calcium mobilization that leads to T H 2 cell differentiation and activation, then activation of large number of TCR s with higher concentrations of antigen in the Itk -/- T cells may be able to generate sufficient increases in calcium to induce high levels of NFAT activation and T H 2 cell differentiation. We therefore decided to use the in vivo mouse model of allergic asthma using varying concentrations of allergen to test this hypothesis. However, in vivo, many TCRs exist with varying affinities to antigens found in HDM, leading to the

138 activation of a heterogenous population of T cells that may respond differently to the 118 same amount of allergen, making interpretation of results complicated. Nonetheless, even if different cells carrying TCRs of varying affinities generate different amounts of IL-4, as long as the signals are elevated in all the cells, one might still observe increased levels of IL-4 when compared to exposure to low levels of allergen. This should therefore be able to induce increased T H 2 response and lead to the development of allergic asthma in the Itk null mice. However, if Itk mediates T H 2 differentiation through a very specific mechanism then we would not see any alteration in the amount of IL-4 produced by T cells in the Itk -/- mice regardless of antigen concentration exposure. In this investigation, we used an authentic complex allergen, house dust mite (HDM), to induce allergic asthma through respiratory exposure in WT and Itk -/- mice and demonstrated that the absence of Itk can indeed prevent the development of allergic asthma. We used increasing concentrations of HDM extract in order to test the impact of varied allergen exposure in the development of the disease. Itk -/- mice did not develop airway hyperresponsiveness in response to increasing concentrations of methacholine when exposed to any of these allergen concentrations when compared to WT mice. Lung histopathology also indicated that WT mice developed airway constriction and inflammation with high levels of mucous production and lymphocyte infiltration. However, Itk -/- mice did not develop any lung pathology to any of the doses of the allergen. The airways appeared clear with no inflammation and lymphocyte infiltration. They also did not show any mucous production. We also investigated the systemic T H 2 immune activity in these mice after HDM exposure. Our data demonstrate that WT mice produce elevated levels of T H 2 cytokines IL-4 and IL-13. In addition, the T H 2 response

139 119 observed was dose dependent with the level of cytokine message generated increasing with increasing allergen dose. However, Itk -/- mice did not produce any T H 2 cytokines above baseline in response to any of the allergen doses. This suggests that the absence of Itk can prevent the development of the disease against large doses of allergen. This also suggests that Itk mediates T H 2 differentiation and function via specific signaling mechanisms which can not be overcome by increasing the amount of antigenic exposure. We also analyzed the amount of T H 1 cytokine IFN-γ produced during the course of the disease and did not see any difference in the amount of message generated between WT and Itk -/- mice. This indicates that the absence of allergic asthma disease in Itk -/- mice is not due to T H 1 mediated suppression of T H 2 responses. Because chemokines such as CCL-7 and CCL-11 act as mediators to induce lymphocyte migration in to the airways and for induction of T H 2 cytokines, we also analyzed the levels of these chemokines that are generated during the development of the disease (201, 210). We observed increased production of CCL-7 and CCL-11 mrna levels in WT mice but not in Itk -/- mice. Antigen presenting cells, mainly dendritic cells in the airways, are specialized for the uptake and processing of the inhaled allergen for effective presentation of allergen to the T cells (294, 295). This initial process is crucial for activation and differentiation of T cells. Thus, differences in allergen uptake by the dendritic cells can contribute to differences in immune response between the WT and Itk -/- mice. Hence, we analyzed the level of HDM uptake by the dendritic cells in the lungs between WT and Itk -/- mice. We did not observe any difference in the amount of allergen uptake between these mice, suggesting that there is no difference in antigen uptake by Itk null dendritic cells. However, we have not looked specifically at plasmacytoid and myeloid dendritic cells

140 120 which have been suggested to have immunosuppressive and immunostimulatory roles respectively in allergic immune responses (296). Hence it is possible that type of dendritic cells that take up the antigen in Itk -/- mice could be immunosuppressive and thus further investigations are required in this direction. Our data thus suggest that Itk indeed plays a very crucial role in the development of a T H 2 cell mediated allergic asthma in response to real life allergen, house dust mite. More importantly, our data indicate that inhibition of Itk should efficiently prevent the development of the disease in response to large concentrations of allergens. Hence, development of drug candidates against Itk can definitely offer new possibilities for treatment of allergic asthma.

141 Figure 6.1: Reduced AHR to varying doses of HDM in mice lacking Itk following induction of Allergic Asthma. AHR of WT and Itk -/- mice exposed IN for 10 days to (A) 5µg/day HDM, or (B) 25µg/day HDM, or (C) 100µg/day HDM, or (D) 200µg/day HDM, *p<0.05, students t test, n=6. Filled squares, WT mice; filled triangles, Itk -/- mice. 121

142 122 Figure 6.2: Reduced airway inflammation in mice lacking Itk after exposure to different doses of HDM. Lungs sections from mice treated as in figure 6.1 were stained with H&E staining. All images were taken at 20X.

143 123 Figure 6.3: Reduced mucous production in mice lacking Itk after exposure to different doses of HDM. Lungs sections from mice treated as in figure 6.1 were stained with PAS staining. All images were taken at 20X.

144 Figure 6.4: Dose dependent effects of HDM extract exposure on T H 2 cytokine production in WT but not Itk null mice. WT and Itk -/- mice were treated as in figure 6.1, lungs isolated and mrna for A) IL-4, B) IL-13 and C) IFN-γ were quantified by Q- RT-PCR. *p<0.05 vs.wt PBS, **p<0.05 vs.wt 5µg, ***p<0.05 vs.wt 25µg, ****p<0.05 vs.wt 100µg. 124

145 Figure 6.5: Dose dependent effects of HDM extract exposure on chemokine production in WT but not Itk null mice. WT and Itk -/- mice were treated as in figure 6.1, lungs isolated and mrna for A) CCL-7 and B) CCL-11 was quantified by Q-RT- PCR. *p<0.05 vs.wt PBS, ***p<0.05 vs.wt 25µg, ****p<0.05 vs.wt 100µg, NS Not significant.. 125

146 Figure 6.6: No difference in dendritic cell mediated allergen uptake between WT and Itk null mice. Lungs from WT and Itk -/- mice exposed IN to 25µg of HDM- Alexa750 were stained with anti-cd11b, CD11c and Gr1 to detect A) total dendritic cells and B) myeloid dendritic cells using flow cytometry. NS; Not significant. 126

147 127 Chapter 7 Eotaxin/CCL-11 rescues airway hyperresponsiveness and airway inflammation in mice lacking Tec kinase, Itk 7.1 Introduction It is widely accepted that the characteristic features associated with allergic asthma, namely, airway hyperresponsiveness, airway inflammation, infiltration of leukocytes, mucous hypersecretion, airway remodeling and high serum levels of IgE are mediated by a repertoire of cytokines secreted by T H 2 cells (171). The repertoire of cytokines observed in this disease includes IL-4, IL-5 and IL-13, with each of them having specific functions. IL-4 is mainly responsible for modulating differentiation of T lymphocytes into the T H 2 phenotype (176, 178). IL-4 in association with IL-13 induces the secretion of chemokines such as eotaxin/ccl-11 from airway epithelial cells and smooth muscle cells and elicit increased leukocyte trafficking and infiltration (210, 211, ). IL-4 also induces expression of adhesive molecule VCAM1 on endothelial cells and helps in the retention of leukocytes in the lungs during the course of the disease (180, 181). IL-5 regulates the development of eosinophils, and along with CCL-11, regulates the migration of eosinophils (189, 190, ). IL-13 is mainly responsible

148 for the events that lead to the development of mucous production and airway 128 hyperresponsiveness ( ). The Tec kinase, Itk has been shown to play an important role in inducing T H 2 specific responses against parasitic infections caused by N. brasiliensis, S. manasoni and L. major and also for the development of allergic asthma (61, 78, 160, 229, 277). The absence of Itk leads to defective secretion of cytokines IL-4, IL-5 and IL-13 in T cells due to impaired TCR mediated signaling, and thereby prevents the development of effector T H 2 responses. We thus hypothesized that exogenous addition of these cytokines should be able to rescue the defective T H 2 differentiation as well as effects in the lung as a result of the lack of Itk during the development of allergic asthma. Since chemokines have also been suggested to regulate T cell signaling via costimulation through the chemokine receptors, we also wanted to investigate the effects of administration of chemokine CCL-11 in mice lacking Itk (303) (Figure 7.1). We therefore analyzed the effects of local (lung) administration of cytokines IL-4 or IL-13 and chemokine CCL-11 in mice lacking Itk on the development of allergic asthma using the standard OVA model. 7.2 Results Effect of IL-4 and IL-13 cytokine addition on Itk null mice in response to OVA We have previously reported that Itk -/- mice are incapable of producing normal amounts of effector cytokines IL-4, IL-5 and IL-13 in response to antigen and do not

149 129 develop allergic asthma (160, 277). Since, IL-4, -5 and -13 have been shown to play a crucial role in mediating AHR, airway inflammation and mucous production; we speculated that the decreased levels of these effector cytokines in the lungs of Itk -/- mice are responsible for the absence of allergic airway disease in these mice. Hence, we sought to investigate whether exogenous delivery of these cytokines directly into the lungs can rescue the defects seen in Itk -/- mice and be able to induce all the symptoms associated with allergic asthma, including AHR, airway inflammation and mucous production. We therefore delivered 8 µg of IL-4 or IL-13 individually along with OVA into OVA sensitized Itk -/- mice during the last four days of OVA-challenge. We first measured the development of AHR in these mice using the flow interrupter technique in response to increasing concentrations of methacholine. We observed that both IL-4 and IL-13 could rescue the development of AHR in the Itk -/- mice to normal levels as seen in WT mice (Figure 7.2A and B). Histopathology of the lungs showed increased leukocyte infiltration and airway inflammation and constriction in response to IL-4 as well as IL-13 (Figure 7.3). IL-4 addition significantly increased the number of CD4 + T cells and eosinophils recruited into the lungs of Itk -/- mice (Figure 7.6) and also resulted in significant increases in the levels of message for cytokines IL-4, IL-13 and chemokines CCL-7 and CCL-11 in the lungs (Figure 7.4). However, addition of IL-13 alone only induced increased production of mrna for chemokines CCL-7 and CCL-11, with less of an effect on T H 2 cytokines (Figure 7.5). Although the levels of these cytokines and chemokines produced were lower than WT mice in some cases, they were still significantly higher than Itk -/- mice challenged with OVA alone suggesting that even a

150 partial rescue of these cytokines and chemokines can mediate the development of 130 symptoms associated with allergic asthma Introduction of exogenous Eotaxin/CCL-11 into the lungs of Itk -/- mice induces AHR and leukocyte recruitment The chemokine, Eotaxin/CCL-11 has been shown to play an important role in mediating allergic airway disease by inducing migration of eosinophils and T H 2 lymphocytes via CCR3, the receptor for CCL-11 ( ). Effector cytokines IL-4 and IL-13, mostly generated by T H 2 cells, further induce the production of CCL-11 by the airway epithelial cells and smooth muscle cells (298, 299). However, since the levels of these cytokines are reduced in Itk -/- mice, the levels of CCL-11 produced in Itk -/- mice during allergic asthma are also significantly low in comparison to WT mice. Hence, we investigated the effect of exogenous addition of CCL-11 on Itk -/- mice during the development of allergic asthma. Instillation of 3 µg of CCL-11 along with OVA into the lungs of Itk -/- mice surprisingly induced AHR in these mice at similar levels to WT mice (Figure 7.7A). We also observed a full rescue in the development of AHR when CCL-11 was administered in association with IL-13 (Figure 7.7B). Histopathology analysis of lungs further revealed leukocyte infiltration and airway inflammation in the lungs which was significantly higher than Itk -/- mice but not as high as WT mice (Figure 7.8). We also isolated leukocytes from the lungs exposed to CCL-11 alone and analyzed the number of CD4 + T cells and eosinophils using flow cytometry (Figure 7.7C). Although the numbers of CD4 + T cells were lower than WT mice, they were still significantly higher than ITK -/- mice. However, the percentage of eosinophils recovered was similar to WT mice and

151 significantly higher than ITK -/- mice. Thus, the increased recruitment of leukocytes 131 directly correlated with the induction of AHR Eotaxin/CCL-11 partially rescues effector cytokines IL-4, IL-5 and IL-13 and chemokine CCL-7 production in the lungs of Itk -/- mice Our next objective was to further investigate the effect of exogenous CCL-11 on the levels of effector cytokines, IL-4, IL-5, IL-13 and chemokines CCL-7 and CCl-11 in the lungs of Itk null mice after OVA challenge. We observed significant increases in the cytokine message of IL-4 and IL-13 as well as chemokine CCL-7 in the lungs of Itk -/- mice that were exposed to CCL-11, although they were lower than WT levels (Figure 7.9A, B and C). However, we did not see any increase in the CCL-11 message levels in these lungs (Figure 7.9D). We observed an even higher production of IL-4 and CCL-7 message when CCL-11 was administered with IL-13 (Figure 7.9A and C). Administration of CCL-11 along with IL-13 also induced normal expression of CCL-11 message in the lungs of Itk -/- mice (Figure 7.9D). Further analysis of BAL fluid for protein levels of IL-4, IL-5 and IL-13 also showed a significant increase of these cytokines in lungs exposed to CCL-11 only in Itk -/- mice and confirmed the pattern observed with cytokine message (Figure 7.10). These data indicate that CCL-11 alone is capable of rescuing the defects of Itk -/- mice in developing symptoms of allergic asthma, by producing increased levels of cytokines and chemokines that mediate this disease.

152 7.2.4 Eotaxin/CCL-11 triggers eosinophil mediated IL-4 production in lungs of OVA-challenged Itk -/- mice 132 Since, T cells lacking Itk have been shown to be defective in producing T H 2 cytokines we investigated the source of secretion of these cytokines in the lungs of these mice. Since the results in (Figure 7.9A) shows that among all the cytokines, CCL-11 was able to induce IL-4 most efficiently, we analyzed various subsets of leukocytes ranging from CD4 + T cells, CD8 + T cells, NKT cells, γδ T cells, B cells and eosinophils for the production of IL-4. We found that only eosinophils were capable of producing significantly amounts of IL-4 in the lungs of Itk -/- mice exposed to CCL-11, compared to control mice (Figure 7.13A). Since, exposure to IL-4 alone could induce AHR and airway inflammation of the lungs as seen in Figure 7.2A and Figure 7.3 respectively, these data indicate that CCL-11 mediated increased secretion of IL-4 by eosinophils and is possibly responsible for the rescue of the symptoms associated with allergic asthma in Itk -/- mice In vivo exposure to Eotaxin/CCL-11 rescues in vitro proliferation and cytokine production of Itk -/- T lymphocytes in response to OVA Analysis of recall response to OVA revealed that splenocytes from Itk -/- mice exposed to CCL-11 were able to proliferate to normal levels as compared to Itk -/- mice that had not been exposed to CCL-11 (Figure 7.11). Measurement of secreted cytokines further revealed that these splenocytes were also capable of secreting significant amounts of IL-4, IL-5 and IL-13 cytokines upon rechallenge with OVA (Figure 7.12). Again, these levels observed were still less than that observed with WT mice, however these data

153 suggest that CCL-11 is able to partially increase the levels of cytokines secreted by Itk -/- lymphocytes and is capable of rescuing defects caused due to lack of Itk Effect of CCL-11 on expression of Tec in T H 2 cells from WT and Itk -/- mice Since another Tec kinase, Tec has also been shown to be expressed in T H 2 cells and shares similar functions with Itk, we measured the level of Tec that is generated after in vitro T H 2 differentiation in the presence or absence of CCL-11. We observed no difference in the expression of Tec in the WT T H 2 cells. However, we observed a significant increase in the levels of Tec in ITK -/- T H 2 cells that were differentiated in the presence of CCL-11 (Figure 7.13B). These data suggest that CCL-11 may be able to mediate some effects in T cells via increased signaling through Tec. However, since we did not observe the same increase in expression of Tec in WT T H 2 cells, it is difficult to conclude any specific role of CCL-11 in the induction of this Tec kinase CCL-11 mediates increased IL-4 production in in vitro differentiated WT T H 2 cells but not Itk -/- T H 2 cells Since we observed an increase in expression of Tec after in vitro differentiation of T H 2 cells in the presence of CCL-11, we further looked at the effect of CCL-11 on IL-4 production in these cells. We differentiated T H 2 cells from WT and Itk -/- mice in the presence or absence of CCL-11. We observed that in the presence of CCL-11, WT T H 2 cells produced significantly increased levels of IL-4 message in comparison to T H 2 cells differentiated without CCL-11. However, CCL-11 did not have any effect on the IL-4

154 134 production in the Itk -/- T H 2 cells (Figure 7.13C). These data indicate that Tec is incapable of inducing IL-4 production. These data further imply that Itk is required for the induction of IL-4 production in response to CCL Discussion Itk plays a crucial role in TCR mediated signaling. Itk is responsible for phosphorylating PLCγ which modulates calcium signaling downstream and activates transcription factor NFAT, which induces activation of IL-2 and IL-4 genes (61, 78, 229). Hence the absence of Itk is associated with decreases in calcium signaling, decreased nuclear translocation and activation of NFATc1 and decreased activation of IL-2 and IL- 4 cytokines. Consequently, lack of Itk also leads to defective T H 2 differentiation and activation, preventing the development of T H 2 cell dependent immune responses. However, T H 2 cytokine production is not completely abrogated in the absence of Itk but the levels of cytokine secretion is significantly reduced. T H 2 cells and eosinophils have been recognized as the critical components in signaling events that lead to the development of allergic asthma (171). The expression of IL-4, IL-5 and IL-13 by the T H 2 cells have in particular been lionized for induction of various symptoms associated with allergic asthma. In addition to inducing T H 2 differentiation, IL-4 also modulates VCAM-1 expression on endothelial cells that allows infiltrating leukocytes to adhere to these cells and retain them in the airways (176, 178, 180, 181). IL-5 mobilizes eosinophils from the bone marrow into the lungs upon allergen exposure and induces their activation and mediates eosinophilic airway inflammation and

155 135 perhaps mucous production indirectly via eosinophils (189, 190, ). However, IL- 13 has been shown to play the most crucial role in the development of AHR. Sustained inhibition of IL-13 only during the period of allergen challenge has been shown to completely inhibit AHR and also decreases airway inflammation and mucous production ( ). In fact, AHR, airway inflammation and mucous production have been shown to be induced in naïve mice with just repeated administration of IL-13 into the airways. In addition, administration of IL4 and IL-13 has been shown to be sufficient to induce AHR by directly acting on the resident airway cells. Chemokines have also been shown to play an important role in orchestrating the movement of T H 2 cells and eosinophils and promote their migration into the lungs during allergic asthma. CCL-11/Eotaxin in particular has been shown to coordinate with IL-5 in regulating the activation and function of eosinophils (306). Eotaxin/CCL-11 has also been shown to increase calcium flux in T H 2 cells and induces chemotaxis of T H 2 cells as well as eosinophils and basophils through the CCR3 receptor that is preferentially expressed in these cells (304, 305, 307, 308). CCL-11 has also been shown to directly act on smooth muscle cells and induce smooth muscle cell contraction, that contributes to AHR (298). Recent reports have also suggested that chemokine receptors can act as costimulators during T cell activation (303). In this investigation we have attempted to study the ability of exogenous exposure to IL-4 or IL-13 or CCL-11 to rescue allergic asthma in Itk -/- mice. We found that both IL-4 and IL-13 could rescue AHR in the Itk -/- mice in response to OVA challenge. Since Itk -/- mice already produce low levels of IL-4, IL-5 and IL-13, exogenous addition of more IL-4 or IL-13 probably helped in bringing the cytokines to levels sufficient to

156 136 induce AHR. They also mediated leukocyte infiltration and airway inflammation and mucous production in the lungs. IL-4 addition alone was able to partially rescue the production of IL-4, IL-13 and chemokines CCL-7 and CCL-11 to significant levels in comparison to Itk -/- mice that were only exposed to OVA. Addition of IL-13 however, was only capable of rescuing the production of chemokines CCL-7 and CCL-11 without having any effect on the expression of IL-4 or IL-13. Previous studies have also shown that IL-4 and IL-13 induce CCL-11 secretion from smooth muscle cells and airway epithelial cells. Thus our data suggest that both IL-4 and IL-13 are capable of inducing AHR in Itk -/- mice and inducing chemokine production in the lungs. However, only IL-4 is capable of further inducing the expression of IL-4 and IL-13. Exposure of Itk -/- mice with both IL-13 and CCL-11 led not only to the rescue of AHR but also fully rescued the expression of IL-4, CCL-7 and CCL-11. They also displayed increased airway inflammation and mucous secretion. These data thus support the view that chemokines in conjunction with cytokines are sufficient for complete induction of all the symptoms associated with allergic asthma. In order to further analyze the specific function of CCL-11 in the induction of allergic asthma, we administered CCL-11 alone into the lungs of Itk -/- mice during the induction of allergic asthma. Surprisingly we observed that CCL-11 alone could fully rescue AHR in these mice. CCL-11 addition could also rescue the recruitment of CD4 + T cells and eosinophils into the lungs. This could be due to induction of chemotaxis via CCL-11 for eosinophils and CD4 + T cells either directly or indirectly. Further analysis of cytokine and chemokine production in the lungs showed that there was significant increase in the levels of IL-4, IL-5 and IL-13 cytokines, especially IL-4 and chemokine

157 137 CCL-7 in response to CCL-11 exposure. However, we did not observe any significant increase in the production of CCL-11. These data indicate that CCL-11 is able to modulate the production of these effector cytokines through a pathway independent of Itk. We next determined the source of IL-4 production in the lungs after CCL-11 exposure and OVA challenge. Intracellular staining of leukocytes obtained indicated that eosinophils were the major producers of IL-4 in these lungs. In addition, since IL-4 alone is capable of inducing AHR in Itk -/- mice and IL-4 is also capable of inducing the expression of IL-5 and IL-13, our data here suggest that CCL-11 is inducing increased production of IL-4 in eosinophils which is contributing to the production of other cytokines as well as AHR in Itk -/- mice. In addition, CCL-11 has been shown to induce calcium flux mediated smooth muscle cell contraction which can also contribute to AHR we observe in Itk -/- mice. We also induced a recall response with the splenocytes obtained from Itk -/- mice exposed with OVA and CCL-11 using ovalbumin. These cells proliferated normally and produced significantly higher levels of IL-4, IL-5 and IL-13 compared to Itk -/- splenocytes that were not exposed to CCL-11 during ovalbumin challenge. Since, CCL- 11 has also been shown to induce calcium signaling in T H 2 cells which is defective in T H 2 cells from Itk -/- mice, it is also possible that CCL-11 is able to induce normal calcium signaling in Itk -/- T cells thereby rescuing IL-4 production. In order to investigate this, we differentiated WT and Itk -/- T cells under T H 2 specific conditions in the presence or absence of CCL-11. We observed that while CCL-11 was able to induce increased production of IL-4 from WT T H 2 cells, we did not observe any difference in the IL-4 production from the Itk -/- T H 2 cells. Since, Tec and Itk have redundant functions during T

158 cell signaling, we also analyzed the expression of Tec in the T H 2 cells under these 138 conditions and observed that Itk -/- T H 2 cells had a significant increase in expression of Tec. However, we did not observe the same increase in WT T H 2 cells. These data suggest that CCL-11 is not capable of rescuing IL-4 production via rescue of calcium signaling in Itk -/- T H 2 cells. These data also indicate that increased expression of Tec is not capable of rescuing the defects caused by Itk in T H 2 cells. More importantly these data indicate that Itk is also required for CCL-11 mediated signaling in T cells. However, the rescue in proliferation and effector cytokine production by the splenocytes from CCL-11 exposed Itk -/- mice during recall response could be due to other cells such as eosinophils which respond to CCL-11. Overall our data here suggests that while IL-4 and IL-13 can rescue the defects caused by the lack of Itk and induce allergic asthma in Itk -/- mice, chemokine CCL-11 is also capable of rescuing allergic asthma in Itk -/- mice. CCL-11 may mediate these effects by influencing various factors that are required for induction of the disease. CCL-11 may be able to induce AHR by directly acting on smooth muscle cells independent of any cytokines such as IL-4 or IL-13, or it could work in association with these cytokines that are significantly increased after CCL-11 addition. CCL-11 may also rescue the infiltration of CD4 + T cells and eosinophils in the lungs due to chemotaxis. In addition, we have shown that T H 2 cells from mice lacking kinase domain of Itk had defects in migration and actin polymerization in response to CCL-11 suggesting that Itk is required for normal chemotaxis of T H 2 cells when tested in vitro. Thus, our data here suggest that CCL-11 may be able to rescue the migration of CD4 + T cells into the lungs with the help of other factors, that may be absent in vitro and in the lungs of Itk -/- mice. However,

159 specific studies are required to confirm this. Furthermore, our data here suggests that 139 CCL-11 is able to induce increased production of IL-4 in the lungs via eosinophils suggesting that CCL-11 is probably modulating its affects independent of T H 2 cells for the induction of allergic asthma in Itk -/- mice. However, basophils have also been suggested to secrete IL-4 in response to CCL-11 and thus we also need to determine their contribution to the increased levels of IL-4 in the lungs of Itk -/- mice after CCL-11 exposure. Nevertheless, our data suggest that chemokine CCL-11 can rescue the defects in Itk -/- mice and induce allergic asthma, although the underlying mechanism still needs to be further investigated.

160 140 Figure 7.1: CCR3 chemokine mediated signaling pathway. G-protein coupled chemokine receptor CCR3 is activated by chemokine, CCL-11 in T H 2 cells and leads to the induction of Ras family of GTPases that eventually activates the MAPK signaling pathway. CCR3 also induces calcium flux through a yet unknown mechanism. The MAPK and calcium mediated signaling pathways induce the activation of T H 2 genes.

161 141 Figure 7.2: Rescue of AHR by addition of IL-4 and IL-13 cytokine in Itk null mice in response to OVA. (A) WT and Itk -/- mice were immunized and challenged intranasally with OVA or OVA and IL-4, followed by analysis of AHR. -, WT; -, Itk -/- (OVA); -, Itk -/- (OVA+ IL-4). (B) WT and Itk -/- mice were immunized and challenged intranasally with OVA or OVA and IL-13, followed by analysis of AHR. -, WT; -, Itk -/- (OVA); -, Itk -/- (OVA+ IL-13). *p<0.05 vs. Itk -/- mice

162 142 Figure 7.3: Rescue of airway inflammation and mucous production by addition of IL-4 and IL-13 cytokine in Itk null mice in response to OVA. WT and Itk -/- mice were immunized and challenged intranasally with OVA or OVA+IL-4 or OVA+IL-13 followed by analysis of lung sections by H&E (left panel) or PAS staining (right panel).

163 Figure 7.4: Rescue of T H 2 cytokines and chemokines in the lung of Itk -/- mice during the development of allergic asthma by addition of IL-4. WT and Itk -/- mice were treated as in figure 7.2, lungs isolated and mrna for (A) IL-4, (B) IL-13, (C) CCL-7 and (D) CCL-11 quantified by Q-RT-PCR. *p<0.05 vs. Itk -/- mice. 143

164 Figure 7.5: Rescue of chemokines in the lung of Itk -/- mice during the development of allergic asthma by addition of IL-13. WT and Itk -/- mice were treated as in figure 7.2, lungs isolated and mrna for (A) IL-4, (B) IL-13, (C) CCL-7 and (D) CCL-11 quantified by Q-RT-PCR. *p<0.05 vs. Itk -/- mice 144

165 145 Figure 7.6: Rescue of CD4 + T cell and eosinophil recruitment into the lungs of Itknull mice by addition of IL-4. Lymphocytes from lungs from WT and Itk -/- mice treated as in figure 7.2A were analyzed for the number of (A) CD4 + T cells and (B) eosinophils by flow cytometry. *p<0.05 vs. Itk -/- mice

166 Figure 7.7: Rescue of AHR and leukocyte recruitment in the lungs by addition of CCL-11 with or without IL-13 cytokine on Itk null mice in response to OVA. WT and Itk -/- mice were immunized and challenged intranasally with (A) OVA or OVA and CCL-11 (B) OVA or OVA and CCL-11 and IL-13, followed by analysis of AHR. (C) lymphocytes and eosinophils from lungs of these mice were analyzed by flow cytometry. *p<0.05 vs. Itk -/- mice 146

167 147 Figure 7.8: Rescue of airway inflammation and mucous production by addition of CCL-11 with or without IL-13 cytokine on Itk null mice in response to OVA. WT and Itk -/- mice were immunized and challenged intranasally with OVA or OVA+CCL-11 or OVA+CCL-11+IL-13 followed by analysis of lung sections by H&E (left panel) or PAS staining (right panel).

168 Figure 7.9: Rescue of T H 2 cytokines and chemokines in the lung of Itk -/- mice during the development of allergic asthma by addition of CCL-11 or CCL-11 and IL-13. WT and Itk -/- mice were treated as in figure 7.7, lungs isolated and mrna for (A) IL-4, (B) IL-13, (C) CCL-7 and (D) CCL-11 quantified by Q-RT-PCR. *p<0.05 Itk -/- (OVA+CCL-11) vs. Itk -/- mice, **p<0.05 Itk -/- (OVA+CCL-11+IL-13) vs. Itk -/- mice. 148

169 149 Figure 7.10: CCL-11 mediated rescue of cytokine production in BAL fluid. BAL fluid from WT and Itk -/- mice treated as in figure 7.7A was analyzed for (A) IL-4, (B) IL- 5 and (C) IL-13. Data are the mean ± SEM of triplicate cultures and are representative of three experiments, *p<0.05 vs. Itk -/- mice.

170 150 Figure 7.11: CCL-11 mediated rescue of proliferation in vitro. Splenocytes from WT and Itk -/- mice treated as in figure 7.7A were incubated in vitro with 100µg/ml of OVA for 72 hrs and H 3 dt was added in the last 16hrs and amount of proliferation analyzed.

171 151 Figure 7.12: CCL-11 mediated rescue of cytokine production in vitro. Splenocytes from WT and Itk -/- mice treated as in figure 7.7A were incubated in vitro with 100µg/ml of OVA for 96hrs and analyzed for (A) IL-4, (B) IL-5 and (C) IL -13. Data are the mean ± SEM of triplicate cultures and are representative of three experiments, *p<0.05 vs. Itk -/- mice.

172 Figure 7.13: CCL-11 rescues IL-4 production. (A) Eosinophils isolated from lungs as in figure 7.7A were analyzed by intracellular staining for IL-4 production. (B) WT and Itk -/- CD4 + T cells were differentiated under T H 2 conditions in the presence or absence of CCL-11, restimulated for 48hrs with CD3+28 and the amount of message generated was measured by QRT-PCR for Tec and (C) IL-4. *p<

173 153 Chapter 8 Conclusions and Discussion Studies of TCR and costimulatory pathways have provided valuable information about the role of Tec kinases in T cell signaling as modulators and amplifiers of signals generated by Src and Syk kinases, by regulating the duration of calcium flux that affects the TCR contacts with APC s and signaling. Mutations affecting these kinases have also been shown to alter T cell differentiation and/or function as well as development of diseases such as allergic asthma. In addition, selective expression of Txk/Rlk and Itk in T helper cells suggests distinct roles for these kinases in T helper cells. However, their specific roles in T helper cells are still unclear. We have therefore utilized various transgenic and knockout mouse model systems to further elucidate the role of Itk and Txk in T helper cell differentiation and/or function, in the development of allergic asthma and to investigate whether active signaling is involved in their function. Mice lacking Itk have been shown to induce weak T H 2 responses to pathogens Schistosoma manasoni, Nippostrongylus brasiliensis, Leishmania major (99, 159). Our laboratory has demonstrated that absence of Itk prevents the development of airway inflammation, mucous production and T H 2 cytokine secretion by splenocytes during recall response in response to ovalbumin. We have further discovered that Itk is also required for the development of airway hyperresponsiveness and tracheal responses in the lungs. The importance of Itk in allergic asthma was further confirmed using a real life

174 154 allergen, house dust mite. Mice lacking Itk did not develop allergic asthma in response to HDM. Variation in the antigen concentration has been shown to affect the strength of the signal that can contribute to the outcome of T helper cell differentiation (282, 283). Use of low and high affinity TCRs has been shown to induce T H 2 differentiation while intermediate affinity TCR engagement has been suggested to induce T H 1 differentiation ( ). The number of TCR s that get activated has also been suggested to affect the outcome of the amount of signal generated (309). Itk has been suggested to regulate the strength of the signal generated in response to TCR stimulation via amplification of the signal generated by proximal molecules (289). Lack of Itk has been suggested to bring the signal strength generated to intermediate levels causing the cells to undergo T H 1 differentiation under T H 2 conditions due to suboptimal TCR stimulation. Under low signal strength, which usually leads to T H 2 differentiation by default, absence of Itk is suggested to prevent the cells from going into this default pathway. Thus, the manipulation of the antigen concentrations could lead to the activation of just the desired number of TCR s when the optimal antigen concentration is reached that could possibly generate signals sufficient to induce T H 2 differentiation. We observed that manipulation of signal strength using varying concentrations of HDM did not alter the pathogenesis of the disease in mice lacking Itk. WT mice generated a dose dependent response to the varying allergen concentrations and developed the disease. But Itk -/- mice were not responsive to any of the concentrations of the allergen and did not develop the disease. These data suggest that the concentrations of HDM used in these experiments were in the range of signals that could generate a T H 2 response. The failure of Itk -/- mice to develop a T H 2 response suggests that in addition to acting as an amplifier, Itk may perform other

175 155 specific functions during T H 2 differentiation. Previous studies have suggested that Itk regulates T H 2 differentiation at the initial stages by negatively regulating T-bet expression, without having any effect on GATA-3 expression. (59). In the absence of Itk, expression of T-bet is not suppressed, which causes the cells to undergo T H 1 differentiation. But the cells that do get committed to T H 2 phenotype in the absence of Itk via GATA-3 and IL-4R signaling pathway are still defective in producing effector cytokines suggesting that signaling pathways regulated specifically by Itk are incapable of inducing effector cytokine production in its absence. Our studies indicate that even in response to varying concentrations of allergen, the same phenomenon possibly occurs. Cells lacking Itk initially have defects in T H 2 differentiation due to ineffective suppression of T-bet and those that manage to get differentiated have a defect in cytokine secretion due to decreased NFATc1 nuclear translocation and other factors. Normal levels of IFN-γ production seen in our Itk -/- mice could be attributed to the fact that lack of Itk does not affect the T H 1 differentiation or function after the T cells are committed to the T H 1 phenotype. This suggests that absence of the disease in Itk -/- mice is not due to the induction of a protective T H 1 response. Although the use of complex allergens which could induce TCR s with varying affinities was a crude approach to mimic the concentration variation done in previous studies, it enabled us to mimic the responses that occur in real life against real allergens. These data also give the valuable information that lack of Itk can prevent the development of allergic asthma in response to high doses of allergens which could be promising for therapeutic strategies that are targeting Itk for treatment of allergic asthma.

176 Itk activates transcription factors NFATc1, AP-1 and NF-қB, all of which 156 contribute to the activation of T H 2 cytokine gene locus (81). The gene encoding Itk also has binding sites for transcription factor GATA-3, the master regulator of T H 2 cell differentiation (53). Itk also activates MAP kinase p38 through Ras pathway which has been shown to induce nuclear translocation of GATA-3 by directly phosphorylating GATA-3 (310, 311). At later stages of T H 2 differentiation, activation of GATA-3 and IL- 4 has been shown to be independent of Itk because IL-4R mediated JAK/STAT signaling pathway has been shown to function normally in the absence of Itk (53). However, Itk could indirectly influence this process by regulating the secretion of the effector cytokines that is crucial for the stimulation of IL-4R. However, contribution of these cytokines by other cell types could possibly overcome this limitation. We observed that lack of Itk influenced the production of effector T H 2 cytokines IL-4, IL-5 and IL-13. Lack of Itk prevented the development of these T H 2 cytokines in the lungs in response to allergic asthma. Rescue of disease development in these mice by addition of WT CD4 + T cells further supports the idea that T H 1 specific responses are not responsible for the suppression of the disease in mice lacking Itk. In addition, exogenous exposure of IL-4 or IL-13 rescued the development of allergic asthma in mice lacking Itk. These observations suggest that the key role of Itk during the development of allergic asthma is the activation of T H 2 cytokine locus and production of IL-4, IL-5 and IL-13 most likely via transcription factors NFATc1 and GATA-3 which could upregulate T H 2 genes and AP-1 which could possibly downregulate T-bet. Certain functions of Tec kinases are dependent on their kinase activity while some are not. Activation of PLC-γ1, LAT, Vav, SLP-76, Dok1 and 2, CD28, calcium

177 157 mobilization and chemokine SDF-1α mediated actin polarization and activation of Rac and Cdc42 have been shown to be dependent on the catalytic activity of Tec kinases (90, 247). Activation of the NFκB pathway (249), suppression of tumor development (250) and induction of antigen receptor mediated actin cytoskeletal rearrangements and activation of the transcription factor SRF (212, 251, 252) are shown to be independent of the kinase activity of Tec kinases. We observed that kinase activity of Itk is required for the development of T H 2 responses during allergic asthma. Mice expressing only the kinase domain deleted form of Itk did not develop airway hyperresponsiveness, mucous production or T H 2 effector cytokines. Chemokine mediated responses also play an important role during infections. Chemotactic gradients induce migration of T cells to the site of infection which then exert their effects at the site after activation. T H 2 cells mainly express chemokine receptor CCR3 that helps their migration to lungs during allergic asthma in response to CCL-11 secreted in the lungs. Itk has been shown to be involved in signaling pathways regulating Rho GTPases, cell polarization and migration, downstream of chemokine receptors (90, 91). Our analysis suggests that signaling pathways involved in T H 2 cytokine production and chemokine migration are differentially sensitive to kinase activity of Itk. Mice co-expressing kinase domain deleted Itk and WT Itk display defective CCL-11 mediated actin cytoskeletal reorganization and migration, but normal T H 2 cytokine production. The kinase deleted Itk also imparts dominant negative effects on the WT Itk. Our data therefore propose a new model for the requirement of catalytic activity of Itk in TCR and CCR3 regulated signaling pathways. Low level of catalytic activity of Itk is required for T H 2 cytokine production. However, higher level of catalytic activity of Itk is required for chemokine mediated migration (Figure 8.2). Hence,

178 158 manipulation of the catalytic activity of Itk can provide a novel therapeutic strategy for the treatment of allergic asthma by specifically preventing the T cell migration while leaving the T H 2 responses intact thereby preventing specific immunosuppression. In addition to demonstrating the role of Itk in chemokine mediated migration, our analysis also shows that Itk is required for signaling pathways downstream of chemokine CCR3 for the induction of IL-4 production. We have observed positive costimulation by CCL-11 in inducing IL-4 production in WT T H 2 cells but not Itk -/- T H 2 cells. CCL-11 is shown to induce calcium flux in T H 2 cells and importance of Itk in regulating intracellular calcium signals suggest that Itk probably regulates signaling pathways downstream of chemokine receptors by controlling the calcium flux (304, 305, 307, 308). Rescue of allergic asthma in Itk -/- mice via IL-4 secretion by eosinophils upon exposure to CCL-11 suggests that CCL-11 is capable of increasing the concentration of effector T H 2 cytokines to the threshold level that is sufficient for inducing allergic asthma. This phenomenon is not observed in Itk -/- mice in the absence of exogenous addition of CCL- 11 because induction of CCL-11 secretion is dependent on activation of smooth muscle cells and airway epithelial cells by T H 2 cytokines which are expressed at very low levels in these mice. Hence, low level of CCL-11 is secreted in the Itk -/- mice, and is insufficient to induce migration and activation of eosinophils. Tec kinases Txk/Rlk and Itk have been suggested to have distinct functions in T helper cells although they have partially redundant functions in calcium signaling pathways. Txk has been proposed to specifically regulate T H 1 differentiation due to its selective expression in T H 1 cells. Itk has been proposed to play a specific role in T H 2

179 159 cells due to the reduced T H 2 responses observed in the absence of Itk in various disease models. Txk has been suggested to directly bind to a sequence in the IFN-γ promoter and driving IFN-γ transcription and T H 1 differentiation and function. Overexpression of Txk has been shown to increase the expression of IFN-γ (214, ). Our studies show that overexpression of Txk does not induce increased production of IFN-γ and hence Txk is not a direct inducer of IFN-γ (Figure 8.1). Our observations also indicate that Txk is not a specific regulator for T H 1 cell differentiation or function. We observed full rescue of T H 2 mediated allergic asthma in Itk -/- mice overexpressing Txk in T H 2 cells. T H 2 cell differentiation occurred normally with normal levels of T H 2 cytokine production in the presence of Txk suggesting that signals generated by Txk induce the same pathways as generated by Itk that is required for the activation of T H 2 gene locus. Some studies have suggested that Itk negatively regulates T H 2 differentiation by suppressing GATA3 activity via direct interaction and phosphorylation of T-bet, which promotes interaction of T-bet with GATA3 (134). Our studies strongly indicate that Itk is not a negative regulator of GATA-3. Lack of Itk prevented the development of T H 2 responses during allergic asthma which is dependent on activation by GATA-3 instead of upregulating its activation and function. Itk has also been suggested to be not required for T H 2 differentiation per se, but is rather required for effector function of differentiated T H 2 cells (246). We have also observed similar responses when Itk -/- cells are differentiated under T H 2 conditions in vitro. These cells undergo T H 2 differentiation normally with upregulation of GATA-3 and downregulation of T-bet. However, they still remain defective in the production of IL-4. Hence, these data suggest that during allergic asthma,

180 160 although the Itk -/- T cells maybe able to undergo normal T H 2 differentiation, lack of Itk eventually prevents the production and secretion of T H 2 cytokines, thereby causing the attenuation of responses dependent on T H 2 cytokines. Based on this report we propose a model in which Txk and Itk regulate T H 1 and T H 2 functions by selective upregulation of expression in T H 1 and T H 2 cells respectively (Figure 8.3). In T H 1 cells, upregulation of Txk leads to activation of IFN-γ indirectly through association with other T H 1 specific proteins such as RIBP that can induce IFN-γ production. In T H 2 cells, upregulation of Itk leads to activation of signaling pathways downstream of both T cell receptor and chemokine receptor CCR3. Downstream of TCR, kinase activity of Itk induces mobilization of calcium and NFATc1 activation that leads to upregulation of T H 2 cytokines IL-4. Itk also upregulates activation of GATA-3 by inhibiting the expression of T-bet and further induces the expression of IL-4, IL-5 and IL- 13. Downstream of CCR3, kinase activity of Itk induces actin polymerization and migration in response to chemokines and also regulates chemokine mediated IL-4 production. During induction of allergic asthma, upon encounter with allergens presented by APC s, T H 2 cells in the draining mediasteinal lymph nodes get activated and migrate into the lungs where they secrete IL-4, IL-5 and IL-13 in an Itk dependent manner. These cytokines activate the smooth muscle cells, airway epithelial cells and eosinophils and induce the secretion of CCL-11 and other chemokines. The chemokines induce actin polymerization and migration of more T H 2 lymphocytes from the periphery to the lungs in an Itk dependent manner. This leads to increased secretion of effector T H 2 cytokines in the lungs which further induce more secretion of chemokines and recruits other leukocytes, such as eosinophils and basophils into the lungs (Figure 8.4). The increased

181 161 secretion of cytokines and chemokines eventually leads to smooth muscle contraction, airway inflammation and mucous production leading to the development of allergic asthma. Future Work Although the importance of Itk in T H 2 differentiation and allergic asthma is now clear based on these investigations, there are still a lot of observations that remain unexplained regarding the function of Tec kinases. The mechanism by which kinase activity of Itk regulates the signaling pathways downstream of CCR3 is still unknown and needs to be investigated. In addition, factors that facilitate the development of allergic asthma in response to CCL-11 alone in the absence of Itk should also be unraveled. We show here that Txk and Itk are required for T H 1 and T H 2 responses respectively. Although, they have redundant functions, by virtue of their differential expression, Txk and Itk bring about different outcomes in T H 1 and T H 2 cells. One would thus predict that in the absence of Txk and Itk the T helper cells would be defective in both T H 1 and T H 2 responses. However, mutation of both Txk and Itk kinases in mice has been shown to be able to generate normal T H 2 responses in response to infection with Schistosome manasoni. This suggests that there still are unknown factors and signaling mechanisms that exist that can bypass the requirement of Itk to induce T H 2 responses. Although, defective downregulation of GATA3 has been suggested to be the reason for this phenomenon, the exact mechanism is still unclear. It is proposed that in the absence of both Txk and Itk, the strength of the signal generated is low that directs the T helper cells to differentiate into T H 2 cells by default. In the absence of only Itk however, the

182 162 strength of the signal generated is moderate and leads to T H 1 differentiation and under strong signals, the cells differentiate into T H 2 cells. Preliminary studies with these double knockout mice show that these mice are also capable of inducing allergic asthma. We have analyzed the expression of GATA-3 suppressors, ROG and FOG in the double knockouts but did not observe any difference in expression in comparison to WT mice. Hence the mechanism by which GATA3 is upregulated in Txk -/- Itk -/- mice still needs to be investigated. In addition the mechanism by which these cells secrete the T H 2 cytokines also needs to be investigated.

183 Figure 8.1: Functions of Itk and Txk in T helper cell differentiation. Itk does not inhibit GATA-3 activation by phosphorylating T-bet and inducing direct interaction between GATA-3 and T-bet. Instead Itk probably inhibits T-bet expression, leading to activation of GATA-3 and induction of IL-4 production. In addition, Txk does not specifically induce T H 1 differentiation and does not directly induce IFN-γ production. 163

184 164 Figure 8.2: Differential sensitivity to signal strength for T H 2 cytokine production and chemokine mediated migration. TCR and chemokine receptors such as CCR3 are differentially sensitive to signal strength generated by Tec kinase, Itk via its kinase activity for T H 2 production and T cell migration. The required threshold level for TCR mediated T H 2 cytokine production (in red box) is lower and is reached by partial catalytic activity of Itk that generates low signals. However, the threshold for chemokine receptor mediated migration of T cells (in green box) is higher and is reached only upon full catalytic activity of Itk that generates high signals.

185 Figure 8.3: Proposed model for role of Tec kinases Txk and Itk in T helper cell signaling. In naïve cells, Itk is the most highly expressed Tec kinase, with Txk expressed at lower levels and Tec expressed at the lowest level. Upon T helper cell differentiation, the expression levels change. T H 1 cells selectively express Txk at highest level, with relatively similar levels of expression of Itk, and lower levels of Tec. T H 2 cells highly express Itk along with low levels of Tec. Txk and Itk have redundant functions downstream of TCR signaling pathway in T H 1 and T H 2 cells respectively. Both activate the calcium signaling pathway leading to the activation of NFAT. In addition, in T H 1 cells, Txk may regulate T-bet indirectly and induce IFN-γ production. In T H 2 cells, Itk induces activation of GATA-3 while probably inhibiting T-bet and leads to the production of IL

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