ROLE OF TEC FAMILY KINASE ITK IN REGULATING THE DEVELOPMENT OF T CELL SUBSETS

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1 The Pennsylvania State University The Graduate School The Huck Institute for Life Sciences ROLE OF TEC FAMILY KINASE ITK IN REGULATING THE DEVELOPMENT OF T CELL SUBSETS A Dissertation in Immunology and Infectious Diseases by Jianfang Hu 2008 Jianfang Hu Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy August 2008

2 The dissertation of Jianfang Hu was reviewed and approved* by the following: Avery August Associate Professor of Immunology Dissertation Advisor Chair of Committee Biao He Associate Professor of Veterinary Sciences Margherita Cantorna Associate Professor of Immunology Co-chair of Immunology and Infectious Diseases Program Robert Paulson Associate Professor of Veterinary Sciences Director of Pathobiology Program Na Xiong Assistant Professor of Veterinary Sciences Wendy Hanna-Rose Assistant Professor of Biochemistry and Molecular Biology *Signatures are on file in the Graduate School

3 ABSTRACT iii T cells with a memory like phenotype and possessing innate immune function have been previously identified. These cells rapidly secrete IFNγ upon stimulation with IL-12/IL- 18 and are involved in innate responses to infection with Listeria monocytogenes. The signals regulating these cells are unclear. The Tec kinase Itk regulates T cell activation, and we report here that a majority of the CD8 + T cells in Itk null mice have a phenotype of CD44 hi similar to memory like innate T cells. These cells are observed in mice carrying an Itk mutant lacking the kinase domain, indicating that active Tec kinase signaling suppresses their presence. These cells carry preformed message for IFNγ and are able to rapidly produce IFNγ upon stimulation in vitro with IL-12/IL-18, and endow Itk null mice the ability to effectively respond to infection with L. monocytogenes or exposure to LPS by secretion of IFNγ. Transfer of these cells rescues the ability of IFNγ null mice to reduce bacterial burden following L. monocytogenes infection indicating that these cells are functional CD8 + CD44 hi T cells previously detected in vivo. These results indicate that active signals from Tec kinases regulate the development of memory like CD8 + T cells with innate function. In addition to regulating the development of conventional and innate CD8 + T cells, the Tec family kinase Itk also regulates the development of CD4 + T cell lineages. We show here that Itk null mice have increased percentage of CD62L Lo CD44 Hi memory phenotype CD4 + T cells compared to WT mice. These cells can arise directly in the thymus, but do not require this organ for their development. Instead MP CD4 + T cell development is bone marrow derived MHC Class II dependent, and independent of MHC Class II

4 iv expression on the thymic epithelium. These cells express high levels of transcripts for the T-bet and IFNγ and are able to produce IFNγ directly ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4 + T cells with CD62L Hi CD44 Lo naïve phenotype, but has no effect on the number of memory phenotype CD4 + T cells, indicating that the development of memory phenotype CD4 + T cells is Itk independent. We further show that the development of the naïve phenotype CD4 + T cells is dependent on active Itk kinase signals and can be rescued by expression of Itk specifically in T cells, but not the Txk overexpression. Itk is required for functional TCR signaling in these cells, but not for the innate function in response to IL-12/IL-18 or L. monocytogenes stimulation. These results indicate that CD62L Hi CD44 Lo naïve and CD62L Lo CD44 Hi innate memory phenotype CD4 + T cells may be independent populations that differ in their requirement for Itk s signals for development. Our data also suggest that CD4 + CD62L Lo CD44 Hi memory phenotype T cells have innate immune function. We have also examined whether CD4/CD8 lineage choice is affected by a combination of TCR affinity and Itk by analyzing mice lacking Itk and carrying the low affinity TCR transgene OT-II, specific for a peptide in Ovalbumin. Our results show that OT-II/Itk -/- thymocytes receive reduced TCR signals, with a reduction in CD4 + T cell development. Surprisingly, these mice develop significant numbers of MHC class II restricted TCR transgenic CD8 + T cells that resemble non-conventional or innate memory phenotype CD8 T cells. We also show that the development of these class II-restricted innate memory phenotype CD8 + T cells is bone marrow intrinsic, and may be the result of reduced TCR signaling in the absence of Itk resulting in reduced expression of the

5 v transcription factor, Th-POK, a master regulator of CD4 commitment. All together, these data suggest that Itk plays an important role in CD4/CD8 commitment by regulating signal thresholds for the lineage commitment. Our data also suggest that the lower level of TCR signaling that occurs with a low affinity TCR in the absence of Itk can redirect MHC class II restricted CD4 + T cell to develop into class II-restricted CD8 + innate memory phenotype T cells. Overall our results suggest that loss of Itk differentially affects the development of conventional vs. non-conventional MP T cells and also plays important roles in CD4/CD8 lineage commitment.

6 TABLE OF CONTENTS vi LIST OF FIGURES... ix ACKNOWLEDGEMENTS... xii Chapter 1 Introduction T cells in immune system T cell development T cell activation T cell signaling T cell differentiation Non-conventional T cell lineages develop in the thymus TEC family kinases The structure of Tec family kinase The activation of Tec family kinases Effect of Tec family kinases in The T cell development and T cell function Effect of Tec family kinases in pre-tcr signaling Effect of Tec kinases on positive selection Effect of Tec kinases on negative selection Effect of Tec kinases on CD4/CD8 commitment Effect of Tec kinases on peripheral T cell differentiation and Cytokine Production The purpose of the study Chapter 2 Materials and Methods Animals Bacterial Infection Antibodies and Flow Cytometry Real-Time PCR analysis In vitro analysis of cytokine secretion and production In vitro analysis of T cell proliferation BrdU Incorporation Fetal Thymic Organ Culture (FTOC) Bone marrow chimera Statistical analysis Chapter 3 Memory phenotype CD8 + T cells with innate function selectively develop in the absence of active Itk... 40

7 3.1 Introduction Results Increased presence of CD8 + T cells with memory like phenotype in Itk null mice Tec kinase activity suppresses the presence of CD8 memory like T cells Txk partially suppresses the presence of memory phenotype CD8 + T cells Itk -/- memory like CD8 + T cells carry preformed message for IFNγ Itk -/- mice exhibit enhanced clearance of Listeria dependent on CD8 + T cells Itk -/- memory T cells reduce bacterial burden in IFNγ null mice Discussion Chapter 4 Naïve and Innate Memory phenotype CD4 + T-cells have different requirements for active Itk for their development Introduction Results Increased percentages of CD4 + CD62L Lo CD44 Hi T cells in Itk null mice Homeostasis of naïve and MP CD4 + T cells does not contribute to the increased percentage of MP cells in Itk null mice MP CD4 + T cells carry preformed message for IFNγ and rapidly secrete this cytokine upon stimulation with P/I Phenotypic characterization of NP and MP CD4 + T cells in Itk -/- mice MP CD4 + T cells can develop in the thymus Altered CD4 + lineage development in the absence of Itk is intrinsic to bone marrow-derived cells Tec kinase activity is required for the presence of NP CD4 + T cells, but not for the MP CD4 + T cells Txk cannot replace Itk in the development of NP CD4 + T cells MP CD4 + T cells develop in MHC class II deficient recipients The development of MP CD4 + T cells is dependent on bone marrow expression of MHC class II The development of MP CD4 + T cells is thymus independent Itk is required for TCR induced but not innate signal induced elaboration of MP CD4 + effector function Discussion Chapter 5 Itk derived signals regulate the expression of Th-POK and controls CD4/CD8 lineage decisions Introduction vii

8 5.2 Results The absence of Itk results in reduced development of OT-II transgenic CD4 + T cells and enhanced development of OT-II transgenic CD8 + T cells Reduced TCR signaling in the absence of Itk during T cell development Normal survival of CD4SP and CD8SP TCR transgenic thymocytes in the absence of Itk Transgenic TCR hi CD8 + T cells respond to antigen specific stimulation to make IFN-γ The development of transgenic TCR hi thymocytes into CD8SP cells is bone marrow cell intrinsic Transgenic TCR hi CD8 + T cells that develop in OT-II/Itk -/- mice have a memory phenotype and innate function Down-regulation of the CD4 lineage commitment factor, Th-POK, in Itk null DP thymocytes Discussion Chapter 6 Conclusions and future directions Conclusions and discussion Itk plays different roles in CD4 + and CD8 + MP T cell development Txk plays different roles in CD4 + and CD8 + MP T cell development TCR signaling strength and MP T cell development The requirements for MP T cell development Difference between real memory and MP cells Future directions What other proteins regulate MP T cell development? What are the requirements for MP T cell development? What are the mechanisms regulating MP T cell development? What is the function of MP T cells? The significance of the study Bibliography viii

9 LIST OF FIGURES ix Figure 1-1: Hematopoiesis Figure 1-2: Overall Scheme of T cell development in the thymus Figure 1-3: Strengh of signal model for the CD4/CD8 commitment Figure 1-4: T cell signaling Figure 3-1: Increased percentage of CD8 + CD44 hi memory phenotype T cells in mice lacking Itk Figure 3-2: Increased absolute number of CD8 + CD44 hi memory phenotype T cells in mice lacking Itk Figure 3-3: Active Tec kinase signaling regulates the presence of CD8 + CD44 hi memory phenotype T cells Figure 3-4: Txk can partially suppress the presence of CD8 + CD44 hi memory phenotype T cells Figure 3-5: CD8 + CD44 hi T cells rapidly secrete IFNγ upon activation with IL- 12/IL Figure 3-6: CD8 + CD44 hi T cells show proliferative response to IL-2 stimulation Figure 3-7: Memory Phenotype CD8 + T cells carry preformed message for IFNγ and T-bet Figure 3-8: Itk -/- mice exhibit enhanced clearance of L. monocytogenes in vivo Figure 3-9: Itk -/- splenic macrophages produce increased TNFα upon HKLM stimulation in vitro Figure 3-10: Memory Phenotype CD8 + T cells actively secrete IFNγ in vivo after infection with L. monocytogenes Figure 3-11: CD8 + CD44 hi T cells reduce bacterial burden in IFNγ null mice infected with L. monocytogenes Figure 4-1: Increased percentage of CD4 + CD62L Lo CD44 Hi MP T cells in mice lacking Itk Figure 4-2: Homeostasis of naïve and memory phenotype CD4 + T cells

10 x Figure 4-3: MP CD4 + rapidly secrete IFNγ upon stimulation and carry high levels of preformed message for IFNγ and T-bet Figure 4-4: Surface phenotype of CD4 + CD62L Lo CD44 Hi and CD62L Hi CD44 Lo T cells from WT and Itk -/- mice Figure 4-5: Memory phenotype of CD4SP thymocytes Figure 4-6: MP CD4 + T cells can develop in the thymus Figure 4-7: Altered CD4 + T cell development in the absence of Itk is intrinsic to bone marrow-derived cells Figure 4-8: The development of memory phenotype CD4 + T cells is Itk independent Figure 4-9: The development of MP CD4 + but not NP CD4 + T cells is independent of active Itk signaling Figure 4-10: Txk cannot replace Itk in the development of NP CD4 + T cells Figure 4-11: The development of MP CD4 + T cells is independent of MHC Class II expression on the thymic epithelium Figure 4-12: The development of MP CD4 + T cells is dependent on expression of MHC Class II on bone marrow derived cells Figure 4-13: The development of MP CD4 + T cells is thymus independent Figure 4-14: TCR activation of MP CD4 + TCR is Itk dependent Figure 4-15: Innate activation of MP CD4 + TCR is Itk independent Figure 5-1: Development of MHC class II restricted TCR hi CD8SP cells in a CD4/MHC-II restricted environment in the absence of Itk Figure 5-2: The percentage and absolute number of MHC class II restricted TCR hi SP cells Figure 5-3: The strength of the TCR signal is reduced in the absence of Itk Figure 5-4: Normal survival of TCR transgenic CD4 and CD8 SP thymocytes in the absence of Itk Figure 5-5: Transgenic TCR hi CD8 + T cells produce IFN-γ upon MHC class IIrestricted OVA-peptide stimulation

11 Figure 5-6: Development of MHC class II-TCR restricted thymocytes to CD8SP lineage in the absence of Itk is cell intrinsic Figure 5-7: The TCR transgene positive CD8 + T cells that develop in OT-II/Itk -/- mice have a memory phenotype Figure 5-8: The TCR transgene positive CD8 + T cells that develop in OT-II/Itk -/- mice have innate function Figure 5-9: Itk mediated signals regulate the expression of the CD4 lineage commitment factor Th-POK xi

12 ACKNOWLEDGEMENTS xii I would first like to give my special thanks to my parents, my parents-in-law, my husband Minghao and my brother Jianping, for their great love and supports both in my life and work. Without you, I would not have made it where I am today. I am particular grateful to my thesis advisor, Dr. Avery August. He gave me extraordinary guidance and valuable help at every stage of my graduate studies at Penn State University. I greatly appreciate the excellent environment and freedom that he provided me. His intelligence and humor made my Ph.D. journey full of challenge and fun. His enthusiasm and passion for understanding the immune system has been an inspiration. Thanks to other thesis committee members: Dr. Biao He, Dr. Margherita Cantorna, Dr. Robert Paulson, Dr. Na Xiong and Dr. Wendy Hanna-Rose, for their input of time, direction and thoughtful suggestions throughout my thesis work. Special thanks to all the members of August lab, past and present, whom I had the opportunity to work with. Shengli hao, Melanie Ragin, Cynthia Mueller, Archana Iyer, Meg Potter, Margaret Kensinger, Man Kit Law, Qian Qi, Nisibeta Sahu, Elizabeth Walsh, Sonia Mohinta, J. Luis Morales and Chavez Carter. You all provided a very stimulating, interactive, educational and entertaining environment throughout the years. Working with you is really fun. Thanks to the members of laboratories in the ASI building and Henning

13 building, especially, Henderson s lab, Hankey s lab, Paulson s lab, He s lab, Xiong s lab and Harvill s lab, for your technical assistance and support. xiii Finally, I would like to give my sincere thanks to all of the people that directly and indirectly contributed to my accomplishments during my studies. I wish these people health, peace, and contentment in their future.

14 1 Chapter 1 Introduction

15 2 1.1 T cells in immune system The mammalian immune system consists of many types of organs, tissues, cells and proteins, which interact in a dynamic network to form a defense system that can protect animals from foreign organisms. A large variety of cells of the immune system are capable of recognizing and eliminating limitless foreign invaders, as well as regulating the development of autoimmunity, allergic responses, transplant rejection etc. The cells within the immune systems are various white blood cells, which arise from hematopoietic stem cells (HSC) and populate the lymphoid organs through the blood vessels and lymph. HSCs are a self-renewing cell population, which have the potential to differentiate into lymphoid and myeloid progenitors (Figure 1-1). All lymphoid cells, such as NK cells, T cell and B cells arise from a common lymphoid progenitor and all myeloid cells such as macrophages, neutrophils, eosinophils and basophils arise from a common myeloid progenitor. Among these cells, only lymphocytes contribute attributes of specificity, diversity, and memory and self/nonself recognition to the immune system, all hallmarks of an adaptive immune response. All the other cells play accessory roles in adaptive immunity, which serve to activate lymphocytes by secreting various effector factors and increase pathogen clearance by direct phagocytosis. T lymphocytes are one of the most vital cells of immune system and often critically influence the outcome of an immune response by secretion of cytokines and the expression of specific soluble and membranebound molecules (1). My thesis will focus on the role of T cells in the immune system, clarifying the development and function of T cells, especially, the role of the Tec-family kinase, Itk, in T cell development and function.

16 Figure 1-1: Hematopoiesis. HSCs give rise to many different types of cells. B-cells, T- cells, NK cells and dendritic cells arise from lymphoid progenitors while myeloid cells such as erythrocytes, megakaryocytes, granulocytes and macrophages arise from myeloid progenitors. (Adapted from M. William Lensch, Stem cells in the blood 3

17 T cell development T cells play a central role in the adaptive immune system and carry a special receptor on their cell surface called the T cell receptor (TCR). Conventional T cells arise from the HSCs in the bone marrow and migrate to the thymus to mature (2, 3). The thymus is the organ that supports the differentiation and selection of T cells, and T cell development requires signals from non-hematopoietic stromal cells including various thymic epithelial cells and mesenchymal fibroblasts (4-6). Upon entry into the thymus, precursors lack expression of coreceptors CD4 and CD8 and these earliest thymocytes are called doublenegative (CD4 - CD8 - ) cells (Figure 1-2). DN cells progress through DN1 to DN4 with changes in the expression of CD25 and CD44: DN1, CD44 + CD25 - ; DN2, CD44 + CD25 + ; DN3, CD44 - CD25 + ; and DN4, CD44 - CD25 - (7). Small percentages of DN2/DN3 thymocytes that make successful γ and δ TCR rearrangements become γδ TCR expressing T cells. However the majority of DN thymocytes express pre-tcr-α during DN3 stage and pair with a rearranged TCR-β chain, and progress down the αβ T cell development pathway (8-10). During late DN3 and DN4 stages, the pre-tcr-α is lost and a low level of mature αβ TCR assembled with the CD3 chains along with the CD3/ζ chain is displayed on the cell surface. These thymocytes also begin to express the coreceptors CD4 and CD8 to develop into double-positive (DP; CD4 + CD8 + ) thymocytes (11). During this process DP thymocytes must express TCR that can recognize self-major histocompatibility (MHC) - self peptide complex to escape a default fate of programmed cell death. Briefly, thymic development of T cells consists of several important processes: death by neglect, positive selection, negative selection and CD4/CD8 commitment (11-13). 90% of DP thymocytes express TCRs with poor or strong affinity

18 5 to self-peptide-mhc ligands and therefore die by neglect or by negative selection, respectively. Few DP thymocytes with TCRs bind adequately to self-ligand to be selected to mature. If the cells expressing TCRs with strong affinity to self-ligands fail to be deleted by negative selection and are permitted to leave the thymus, these lymphocytes could cause autoimmune disease. Negative selection eliminates these T cells that react too strongly with self-mhc. This process is therefore important in generating a primary T cell repertoire that is self-tolerant. Positive selection permits the survival of only those T cells with TCRs that are capable of recognizing self-mhc molecules with weak affinity. This process is responsible for the creation of a self-mhc-restricted repertoire of T cells. It is well documented that TCR signals mediate positive and negative selection in developing T cells (6). This process ultimately results in lineage commitment into either CD4 + or CD8 + mature T cells.

19 Figure1-2: Overall Scheme of T cell development in the thymus. Lymphoid progenitors arise in the bone marrow and migrate to the thymus. The coreceptors are not expressed during the DN early stage, and these DN cells progress through DN1 to DN4 with the changes in the expression of CD25 and CD44: DN1, CD44 + CD25 - ; DN2, CD44 + CD25 + ; DN3, CD44 - CD25 + ; and DN4, CD44 - CD25 -. Successful expression of pre-tcr leads the cells to progress from DN to DP stage. DP thymocytes must express TCR that can recognize self-major histocompatibility (MHC) - self peptides complex to escape a default fate of programmed cell death. Thymic development of T cells consists of several important processes: death by neglect, positive selection, negative selection and CD4/CD8 commitment. (Adapted from Germain, RN; Nature Reviews, 2002) 6

20 7 CD4/CD8 commitment involves a change from DP to a CD4 or CD8 SP state, which is accompanied by the silencing of transcription of one co-receptor locus (14-17). In CD4 + CD8 + DP thymocytes, TCR signals generated by interaction with Class II MHC- TCR-peptide complexes are required for the CD4 + T cell fate, while TCR signals generated by interaction with Class I MHC-TCR-peptide complexes are required for the CD8 + T cell fate. There are two original models of lineage commitment that describes the role of TCR signaling in this process (18). The instructive model proposes that different signals are generated upon coengagement of the TCR and CD4 or CD8, and these differences direct lineage choice (19, 20). Whereas the stochastic/selection model suggests that the lineage is randomly decided and followed by elimination of mismatched cells and differentiation of correct cells. As more evidence appears, it is apparent that these basic models are no longer tenable. Several lines of evidence suggests that the strength of TCR intracellular signaling determines CD4 and CD8 lineages (21-25). These data suggest that attenuating TCR signaling could redirect thymocytes with Class II specific TCRs from CD4 to the CD8 lineage, while the enhanced signaling could redirect thymocytes with Class I specific TCRs from CD8 to CD4 lineage. On the basis of these data, Itano first proposed the strength of signal model. This model suggests that the intensity and duration of signaling from the T cell receptor determines the CD4 versus CD8 lineage. Short-duration signals can lead to CD8 development, while strong and prolonged signals lead to CD4 development. This model is also supported by work of a number of other investigators (26-32)(Figure 1-3).

21 Figure 1-3: Strength of signal model for the CD4/CD8 commitment. This model suggests that the intensity and duration of signaling from the T cell receptor determines the development of the CD4 versus CD8 lineages. Short-duration signals can lead to CD8 development, while strong and prolonged signals lead to CD4 development. (Adapted from Germain, RN; Nature Reviews, 2002) 8

22 9 With lots of evidence showing that the quantity of TCR signaling determines the CD4/CD8 T cell lineage, how signals from the TCR directs changes in gene expression that specify CD4/CD8 T cell lineage commitment and how the transcriptional factors control the lineage-specific coreceptor expression have become major goals of research in this area. With the considerable effort, several transcription factors essential for CD4/CD8 T cell lineage commitment have been identified, such as Runx, TOX, GATA3 and Th-POK. The Runx family of transcription factors has transcriptional repressor activities and contains three members, Runx1, Runx2 and Runx3. Runx1 is required for CD4 silencing at the DN stage and also contributes to CD8 expression at the DP stage (33-35). Runx3 is expressed in CD8SP thymocytes but not in CD4SP thymocytes, binds to CD8 enhancers and contributes to CD8 expression in CD8SP and mature CD8 lineage T cells, but not DN thymocytes (36). The different repressive functions in immature and mature thymocytes of Runx proteins indicate their unique requirement in these cell subsets. The fact that the full derepression of CD4 is observed upon mutation of both Runx binding sites, but not in Runx3 -/- mice, suggest that these Runx proteins may be redundant in some functions (35). Multiple Runx protein deficient mice will need to be analyzed to determine whether Runx proteins play a role in CD4/CD8 lineage commitment. TOX, a high mobility group (HMG) box family member, was identified as a transcription factor that is able to promote CD8 + T cell development. In TOX-transgenic mice, the development of CD8SP thymocytes is increased, while the development of CD4SP thymocytes is impaired, which indicates that TOX plays a role in CD8 + T cell lineage commitment (37, 38). Tox -/- mice show a severe block at completion of positive selection

23 10 of the CD4 lineage, leading to severely reduced numbers of CD4 + T cells, which indicates that TOX is required for the development of the CD4 T cell lineage (39). However, class II MHC-restricted T cells are unable to be misdirected to the CD8 T cells in the absence of TOX, which cast doubt on the role of TOX as a master lineage regulator of CD4/CD8 T cell lineage commitment (39). GATA3 is a Zinc-finger transcription factor that has also been reported to be involved in CD4/CD8 T cell lineage commitment. GATA3 is unregulated in late DP thymocytes and maintained at higher levels in CD4SP thymocytes than in CD8SP thymocytes (40, 41). Overexpression of GATA3 increases the development of CD4SP thymocytes and inhibits the development of CD8SP thymocytes, while inhibition of GATA3 enhances the generation of CD8 SP and inhibits the CD4SP thymocyte development, indicating that GATA3 could be essential for CD4/CD8 lineage commitment (40, 41). Thymocytes from GATA3 conditional deficient mice that are committed to the CD4 lineage either cannot be generated or fail to survive, indicating that GATA3 is essential for CD4 T cell commitment (42). To further confirm the role of GATA3 in the CD4/CD8 commitment, it will be useful to test this issue with a transgenic MHC class II restricted TCR model to see if the GATA3 deficiency is able to misdirect these T cells. Although transcription factors Runx, TOX and GATA3 are all involved in regulating CD4/CD8 lineage commitment, no factors appeared to be sufficient to direct CD4/CD8 T lineage choice. Recently, Th-POK was identified as a transcription factor that can act as a master regulator of CD4/CD8 commitment, which is an important step in understanding the transcriptional control of lineage commitment. Th-POK (T help inducing POK factor), a Zn finger transcription factor, was identified based on the analysis of a

24 11 spontaneous mutant mouse line, HD mice. In HD mice, the development of CD4 + T cells are abolished owing to a fundamental disruption of CD4 + T cell development because all positively selected thymocytes including class II-restricted cells develop to the CD8 lineage (43, 44). Through the genetic mapping of the HD defect, a point mutant within the Th-POK gene was recognized as responsible for the defect (45). Th-POK is specifically expressed in the CD4 lineage and enforced constitutive expression of Th- POK not only restores normal development of Class II-restricted T cells to the CD4 lineage in HD -/- mice but also causes redirection of Class I-restricted cells to the CD4 lineage (45, 46). Since Th-POK is necessary and sufficient to induce the CD4 + T cell commitment and repress the CD8 + T cell commitment, it is recognized as the master regulator of CD4/CD8 T cell lineage commitment. Although the discovery of Th-POK was a significant advance in understanding CD4/CD8 T cell commitment, how Th-POK is linked to TCR signaling and how Th-POK is regulated are still unknown. Following development, self-mhc -restricted non-self- reactive mature CD4 + and CD8 + T cells are released from thymus to peripheral tissues. There they are referred to as naïve T cells and are ready to protect the body from invading pathogens T cell activation Naïve T cells are T cells that have not yet encountered antigen, and are characterized by the surface expression of CD62L and the absence of activation markers, such as CD25, CD44 and CD69 (47). Naïve T cells also have condensed chromatin, very little cytoplasm and little transcriptional activity. These T cells continually recirculate between the blood and lymph systems (48, 49). During recirculation, these cells reside in secondary

25 12 lymphoid tissues such as lymph nodes and spleens. Upon encounter with invading organisms, antigen-presenting cells (APCs) resident in the host pick up these organisms and/or their proteins, and process them into short peptides, which are loaded onto MHC class I and II proteins (50). APCs from infected tissues migrate to the draining lymph nodes that allow circulating naïve T cells to survey the resident APCs for MHC-peptide complexes (51). If a naïve T cell recognizes an antigen-mhc complex on an appropriate APC, it will be activated and initiate a primary response. T cells can recognize antigen only when it is presented by a self-mhc molecule and activation depends on a signal induced by engagement of the TCR complex and co-stimulatory signals T cell signaling T cell activation is initiated by the interaction of the TCR with peptide-mhc complex on the surface of an antigen presenting cell. The TCR transduces extracellular signals by initiating a wide array of intracellular signaling pathways that ultimately determine cell fate through regulating cytokine production, cell survival, proliferation and differentiation (52). Upon activation, one of the first biochemical events is the phosphorylation of the immunoreceptor tyrosine-base activation motif (ITAMs) on the intracellular side of the TCR-CD3 complex by Src family tyrosine kinase Lck. ζ-chain associated protein kinase (ZAP70) is recruited to the TCR/CD3 complex and becomes activated, which promotes recruitment and phosphorylation of downstream adaptor or scaffold proteins. Phosphorylation of SLP-76 by ZAP70 promotes recruitment of Vav (a guanine nucleotide exchange factor) and, the adaptor proteins Nck and GADS and inducible T cell kinase, ITK. ITK then phosphorylates phospholipase Cγ1 (PLCγ1) which

26 13 leads to its activation. Activation of PLCγ results in hydrolysis of phosphatidylinositol4, 5-bisphosphate (PIP2), and generation second messages Diacylglycerol (DAG) and inositol trisphosphate (IP3). DAG activates protein kinase C (PKC), which in turn phosphorylates Ras, a GTPase that activates Raf leading to recruitment of the MAP kinase cascade. IP3 releases calcium from intracellular stores in the endoplasmic reticulum(er). The released Ca 2+ binds to calmodulin which in turn, activates calcineurin, a Ca 2+ /calmodulin dependent protein phosphatase. NFAT, a transcriptional regulator of interleukin-2 (IL-2) gene expression, is a direct target of calcineurin. Calcineurin dephosphorylates the cytosolic component of NFAT, NFATc, which migrates to the nucleus and induces transcription of the IL-2 gene. Feedback regulation at several points with these pathways allows for different outcomes, depending on the cell type and environment (Figure 1-4).

27 Figure 1-4. T cell signaling. Upon activation, one of the first biochemical events is the phosphorylation of the ITAMs on the intracellular side of the TCR-CD3 complex by Src family tyrosine kinase Lck. ZAP70 is recruited to the TCR/CD3 complex and becomes activated, which promotes recruitment and phosphorylation of downstream adaptor or scaffold proteins. Phosphorylation of SLP-76 by ZAP70 promotes recruitment of Vav (a guanine nucleotide exchange factor), and the adaptor proteins Nck and GADS and ITK. ITK then phosphorylates PLCγ1 which leads to its activation. Activation of PLCγ results in hydrolysis of PIP2 and generation of second messages DAG and IP3. DAG activates PKC, which in turn phosphorylates Ras, a GTPase that activates Raf leading to recruitment of the MAP kinase cascade. IP3 releases calcium from intracellular stores in the ER and activates the Ca 2+ pathway to further activate the transcription factors. (Adapted from Berg, L J. Annu. Rev Immunol : ) 14

28 T cell differentiation When naïve T cells are activated after recognizing an antigen-mhc complex on an antigen presenting cell, a series of signaling cascades are initiated. The signals induce transcription of the gene for T cell growth factor IL-2 as well as the α chain of the highaffinity IL-2 receptor among other genes. Secretion of IL-2 and its binding to the highaffinity IL-2 receptor induces the activated naïve T cell to proliferation and differentiate into effector and memory T cell populations. Effector T cells are short-lived cells and carry out specialized function such as cytokine secretion (CD4 + T cells) and cytotoxic killing activity (CD8 + CTL). The effector and naïve T cell populations express different cell surface markers, such as CD44 and CD62L, which contribute to different recirculation patterns. In 1986, Mossman and Coffman identified two CD4 + effector subpopulations distinguished by the different types of cytokines they make. One CD4 + subpopulation was found to secrete IL-2, IFN-γ and TNF-α and was termed a Th1 cell, which is responsible for classic cell-mediated functions such as delayed-type hypersensitivity and the activation of CD8 + effector T cells. Another subset described was the Th2 subset, which secretes IL-4, IL-5 and IL-10 and were able to help B cell activation (53). The ability of the naïve CD4 + T cells to differentiate into Th1 or Th2 subsets is influenced by the cytokine environment during the initial activation, and two subset specific transcription factors are reported to be critical for directing Th1 and Th2 cell differentiation (54). The formation of Th1 cells requires IL-12 production by APCs (55), and the evidence shows that Th2 cell development requires IL-4 (56). Since recently activated dendritic cells produce high levels of IL-12, IFN-γ production by T effector

29 16 cells may be imprinted early in the immune response and continue until the response begins to wane. By contrast it is thought that the Th2 pathway may be a default pathway that develops in the absence of exposure to IL-12, along with exposure to IL-4 (55). IFN-γ and IL-4 affect Th1 and Th2 differentiation by controlling the expression of transcription factors T-bet and GATA3, respectively (57, 58). IL-4 activates the transcription factor STAT6, which then rapidly induces the GATA-3 expression (59). Expression of GATA-3 forms a positive feedback for Th2 differentiation and also regulates the expression of IL-4 (60). Th1 development is dependent on IL-12 and IFN-γ signaling, which activate STAT4 and STAT1, respectively, both of which induce the expression of T-bet (61). T-bet activates the IFN-γ gene by chromatin remodeling, leading to transcription and secretion of IFN-γ (62). T-bet can also inhibit GATA-3, although the mechanism is still unclear, but this provides positive feedback for Th1 development, and negative feedback for Th2 development (57). Thus the fate of T cells depends on both the GATA-3 and T-bet expression, as well as the cytokines produced by these cells. Furthermore, the balance between of these two subsets is essential for proper immune response to pathogens. The specific interaction of peptide-mhc with TCR on CD8 + T cells leads to signaling events that in turn result in effector functions. Unlike naïve CD8 + T cells, effector CD8 + T cells are called CTL and are characterized by certain membrane bound and/or soluble effector molecules (63). The membrane-bound molecules belong to the tumor necrosis factor (TNF) family of membrane proteins and include Fas ligand (FasL) expressed on CD8 + CTLs (64). CTLs also secrete the cytotoxins perforin and granzyme as well as cytokines, IFN-γ and TNF-β. Each of these membrane-bound and secreted molecules

30 17 plays an important role in various CD8 + T cell effector functions. Fas ligand, perforins and granzymes are involved in target-cell destruction. IFN-γ and TNF-β play roles in macrophage activation. The most remarkable characteristic of the immune system is its ability to set aside memory cells that maintain a library of all previous immunologic exposures. This library ensures that when an animal encounters a specific pathogen for a second time, the system will respond rapidly and robustly. The generation memory T cells have been a subject of intense study and debate. Currently there are two general models of memory generation (65): 1) effector model: memory T cells are derived from effector T cells that have regressed to a rested stage through an unknown means; 2) No effector model: memory T cells are developed directly from naïve T cells. These two models have been extensively evaluated for T cell memory development. Although there is evidence that effector differentiation is not a prerequisite for CD8 + T cell memory generation (66), much recent data indicates that generation of CD4 + and CD8 + effector T cells is necessary for production of effective and long-term T cell memory (67, 68). Research also shows that after adoptive transfer, a large fraction of in vitro-derived effector CD4 + T cells have the potential to become memory T cells without further division (69). This data strongly supports the effector model and suggests that most properties of memory cells are predetermined during effector generation. According to the effector model memory T cell generation can be divided into three phases of the immune response: initiation, clonal expansion, and contraction (70, 71). The initiation phase of the T cell response requires efficient presentation of peptide antigen by specialized, activated APCs, which display co-stimulatory ligands that are

31 18 necessary for the activation and initial proliferation of the naïve T cells. This in turn triggers the clonal expansion phase of the response. The effectors generated in this phase are highly activated cells that begin cytokine and chemokine synthesis immediately upon stimulation. Subsequently, apoptosis occurs in most activated effector T cells. If effectors succeed in becoming resting cells without dying, they can become memory cells (71). In the past five years, more and more studies indicate that memory T lymphocytes contain distinct populations of central memory (TCM) and effector memory (TEM) cells characterized by distinct homing capacity and effector function (72). Central memory T cells constitutively express CC-chemokine receptor 7 (CCR7) and L-selectin (CD62L), two receptors required for cell extravasation through high endothelial venules (HEV) and migration to T cell areas of secondary lymphoid organs (73). Effector memory T cells lose the constitutive expression of CCR7 and CD62L, and display characteristic sets of chemokine receptors and adhesion molecules that are required for homing to inflamed tissues. As a result, TCM and TEM would be expected to have distinct re-circulatory properties in vivo. Several studies showed that TCM migrate efficiently to peripheral lymph nodes, whereas TEM can be found in other sites, such as the liver and lung (74). When the functional properties of TCM and TEM are compared, some interesting properties are observed. TCM have little or no effector function, but they proliferate and differentiate to effector cells and produce IL-2 but little IFN-γ, IL-4 or IL-5 in response to antigenic stimulation in vitro, while TEM display immediate effector function and can produce IFN-γ, IL-4 and IL-5 but less IL-2 within hours following antigenic stimulation (75) Non-conventional T cell lineages develop in the thymus

32 19 Cytokines are important in regulating the immune response, and conventional T cells are essential sources of cytokines during infection. Typically, it takes several days after initial exposure to the pathogen for T cells to respond. In addition to conventional CD4 + and CD8 + T cells, the immune system also includes non-conventional T cell lineages such as NKT cells, CD8αα + intraepithelial T cells found in the gut, T-CD4 T cells and CD4 + CD25 + T cells that also develop from DP thymocytes. These cells are thought to provide rapid immune responses, similar to the innate response, prior to the induction of antigen-specific cytokine production. The development of these cells does not fit the conventional rules of thymic selection, and these cells all have the characteristic of being able to rapidly secrete cytokines within minutes to hours of infection. NKT cells express characteristics of both natural killer (NK) and conventional T cells, and were originally identified by their ability to rapidly produce IL-4 and IFN-γ, and other cytokines such as IL-2, TNF-α, IL-5 and IL-13 (76). NKT cells express the NK1.1 antigen and a predominant population of NKT cells expresses a unique invariant α-chain (Vα14) TCR. These latter cells also termed inkt, and are selected by the MHC-class-Ilike protein CD1d. inkt cells can therefore be specifically identified by using CD1d tetramers loaded with the glycolipid α-galactosyl ceramide (α-galcer) (77, 78). A thymic precursor to NKT cell lineage has been identified using specific CD1d/glycolipid tetramers in normal mice. This precursor undergoes expansion and expresses specific activation markers and gain regulatory properties over time (79, 80). inkt cells have innate immunity-like behavior by producing a large amount of cytokines including IL-4, IL-13, and IFN-γ within minutes to hours after stimulation (81). The rapid production of these cytokines by inkt cells amplifies and regulates adaptive immune responses, which

33 20 enhance the function of dendritic cells, NK cells, and B cells, as well as conventional CD4 + and CD8 + T cells, thus linking innate and adaptive immunity (82). NKT cells have been implicated in regulating several different autoimmune diseases in mice and human, including type I diabetes, experimental autoimmune encephalomyelitis (a mouse model for multiple sclerosis), allergic asthma and atherosclerosis (76, 83). CD8αα + T cells, which develop in the thymus and predominate among the intraepithelial lymphocytes (IEL) of the small intestine, are another specialized T cell subset which displays an activated phenotype and are thought to have innate-like function (84-87). Although CD8αβ is constitutively expressed on MHC class I-restricted T cells and serves as a TCR coreceptor to enhance the T cell activation, CD8αα can be transiently or permanently expressed on T cells, regardless of the MHC restriction of the TCR or the presence of conventional coreceptor, since CD8αα can be expressed together with CD4 coreceptor, CD8αβ or activated mature TCR αβ T cells (88, 89). Enforced transgenic expression of CD8αα on DN thymocytes greatly decreases intracellular calcium response and blocks the tyrosine phosphorylation of intracellular proteins, suggesting that CD8αα + T cells function as a TCR corepressor to negatively regulate T cell activation, which is distinct from the conventional MHC class I restricted CD8 + αβ T cells (86, 90, 91). IEL activated by the interaction of CD8αα-TL (thymus leukemia antigen) show enhanced cytokine production, but they also show reduced proliferation and cytotoxicity, which allows for protection without destruction of the epithelial cells (92). CD8αα TCRαβ IEL have also been implicated in exerting a regulatory role in a transfer model of colitis induction (93).

34 21 T-CD4 T cells can develop from human cord blood cells in immunocompromised mice (94, 95). T-CD4 T cells also develop in transgenic mice expressing human MHC class II on mouse DP thymocytes and respond to APC expressing human MHC class II, but not mouse MHC (96). T-CD4 T cells produce both IFN-γ and IL-4 cytokines immediately after in vivo and in vitro T cell receptor stimulation, which is similar to NKT cells, and require the same signaling components as NKT cells for their development (97). Mice with T-CD4 T cells are protected from the development of allergen-induced allergic airway inflammation and they are also protected from experimental autoimmune encephalomyelitis (EAE) (98). All these data suggest that T-CD4 T cells have important immunoregulatory roles. CD4 + CD25 + T cells, a specialized subset of T cells, are regulatory T cells (also known as suppressor T cells) that act to suppress activation of the immune system to maintain immune system homeostasis and tolerance to self (99). They are characterized by constitutive expression of CD25, Foxp3 and glucocorticoid-induced TNF receptor-related protein (GITR) (100). The development of CD4 + CD25 + T cells also occurrs in the thymus and these cells exhibit an activated phenotype (101). CD4 + CD25 + T cells produce antiinflammatory cytokines such as IL-10 and TGF-β, and are powerful inhibitors of T-cell activation both in vivo and in vitro (102). The enhancement of CD4 + CD25 + suppressive function might be useful for the treatment of immune-mediated diseases, whereas the down-regulation of the function of these cells might be beneficial for the enhancement of the immunogenicity of vaccines (102). In addition to these well-studied examples, there are other lineages of non-conventional T cell that develop in the thymus, including T cells restricted by the non-conventional MHC

35 22 class I H2-M3 and MR1 proteins ( ). All these subsets have several features in common: (a) They all arise in the thymus; (b) They are selected in the thymus by interactions with haematopoietic cells instead of epithelial cells; (c) They also share features with conventional memory T cells, such as high levels of expression of surface markers including CD44, CD122 and NK1.1 (107, 108); (d) They display activated phenotypes and acquire effector functions before antigenic encounter (109), a feature shared with NK cells (110). NK cells are the first lymphocytes that were shown to have effector function as a result of their maturation process rather than a consequence of the activation (110). Based on this similarity, T cells sharing this feature have been termed innate T cells (109). 1.2 TEC family kinases The founding member of the Tec family of non-receptor tyrosine kinases, Tec, was originally identified in 1990 as being highly expressed in the liver (111), Several related proteins were subsequently found and recognized as members of this family. In 1993, the importance of Tec family of kinases was highlighted by the discovery that the mutations in Btk, a Tec family kinase predominantly expressed in B cells, were associated with both the human genetic disorder X-linked agammaglobulinemia (XLA) and the murine mutant X-linked immunodeficiency (xid) (112, 113). The Tec family kinases are the second largest family of non-receptor tyrosine kinases next to the Src family kinases. Tec-family of kinases consists of five members in mammals: Tec (Tyrosine kinase expressed in hepatocellular carcinoma), Btk (Bruton s tyrosine kinase), Itk (Interleukin-2 inducible T cell kinase, also known as Tsk and Emt), Rlk (Resting lymphocyte kinase, also known as

36 23 Txk), and Bmx (Bone-marrow tyrosine kinase gene on chromosome X, also known as Etk) (114). Three of the family members are expressed in T cells, Itk, Txk and Tec, and they are expressed at different levels. All three of these kinases are activated downstream of TCR (115). Itk is the predominant Tec kinase in T cells, and is also expressed in NK cells, NKT cells and mast cells (116) The structure of Tec family kinases The Tec family kinases contain five protein domains. An N-terminal Pleckstrin homology (PH) domain followed by a Tec homology (TH), Src homology 3 (SH3) and SH2 domains, and a C-terminal kinase domain. The individual functions of each of these domains are well established. Four of the Tec family kinases are unique among the tyrosine kinases in possessing a PH domain, which is involved in recruiting Tec family members to the plasma membrane by binding the PI3K product PI(3,4,5)P 3 (117). By contrast, Txk lacks the PH domain and instead contains a string of cysteines that can be palmitoylated, which allows Txk to be constitutively associated with membrane (118, 119). The TH domain includes a Btk motif and one or two proline-rich regions (PRR), and are thought to be important for intra- and intermolecular interactions (120). The SH3 and SH2 domains are involved in protein-protein interactions that regulate the binding partners of the Tec kinases and thus determine their proximity to signaling complexes and potential substrates (115).The kinase domains have catalytic activity and are responsible for transferring phosphates onto tyrosine residues of substrate proteins. The kinase domains of Tec family kinase share conserved structural features of all

37 24 catalytically active kinase, and the X-ray crystallographic data confirms that the kinase domains of Itk and Btk are similar to those of Src family kinases (121, 122) The activation of Tec family kinases Tec family kinases can be activated by a number of cell surface molecules, including the antigen receptors of B and T cells, Fc receptors, chemokine and G-protein-coupled receptors, cytokine receptors, Toll-like receptors, as well as CD28 and CD2 (115, 123). Most studies have focused on activation of Tec kinases downstream of antigen receptors. Activation of Tec family kinases involves several interrelated steps: localization to the plasma membrane through interactions between their PH domains and products of PI3K, and phosphorylation by Src family kinases, the latter of which is necessary for activation. Activation also requires interactions between Tec family kinases and other proteins that bring the Tec-family kinases into antigen-receptor signaling complex (115, 124). Immediately after TCR stimulation, Src family kinases such as Lck and Syk family kinases are activated, leading to the phosphorylation of two adaptor proteins LAT (linker for activation of T cells), which is constitutively associated with lipid rafts, and SLP76 (SH2-domain-containing leukocyte protein of 76kDa), which is a cytoplasmic adaptor protein. Stimulation also leads to activation of PI3K, which converts PIP2 into PIP3 within the cell membrane. The enrichment of PIP3 at the site of the activated receptor recruits Tec-family kinases to the membrane in a PH domain-dependent manner ( ). Following membrane localization, a multi-molecular complex containing Itk, SLP76, Gads, and Grb2 is formed around LAT within lipid rafts (128, 129). Tec-family

38 25 kinases are then phosphorylated by Src kinases within the activating loop of the kinase domain (130), resulting in activation of the Tec-family kinase. While Src and Syk family kinases play the role of initiating TCR signaling, Tec-family kinases are essential in amplifying and propagation of the signal by regulating downstream signaling cascades (131, 132). Once the Tec family members have been activated, they are capable of phosphorylating phospholipase C-γ1 (PLC-γ1) (133). This phosphorylation leads to the activation of PLC-γ1, which then hydrolyzes PIP2 into second messengers IP3, which is required for Ca 2+ mobilization, and DAG, which activates PKC and RASGRP, thereby leading to activation of MAPK pathways, as well as the downstream transcription factors such as NF-κB and AP-1 (activator protein-1) complex, c-fos and c-jun. The latter factors regulate genes involved in cytokine signaling, survival and differentiation ( ). Subsequent to Ca 2+ mobilization, the transcription factors NFATs (nuclear factor for activated T cells) are activated and induce the activation of a number of genes essential for lymphocyte activation, such as IL-2. Thus, Itk can affect multiple processes important for T cell development, activation, effector function and homeostasis in part through activation of PLC-γ1 (6, 137). Impairments in Itk activation can affect several downstream events in T lymphocytes. Signaling events downstream of PLC-γ1 in Itk-deficient cells are all impaired following TCR activation, such as calcium mobilization, the activation of NFAT family members, MAP kinases, as well as AP-1 components(135, 138, 139) Effect of Tec family kinases on T cell development and function

39 26 A role for Tec-family kinases in the T cell development and T cell function is supported by studies of single or double knockouts of the genes encoding Itk, Txk and Tec in mice. The development of mature T cells takes place in the thymus and is critically dependent on both the environment and signals through the TCR. Thus the proteins that are important for TCR signaling such as Lck and ZAP70 play essential roles in T cell development, and disruption of these genes results in severe defects in T cell development (140, 141). By contrast T cell development is not completely abolished by the disruption of the Itk gene or genes encoding both Itk and Txk, indicating that the loss of Itk and/or Txk only partially affects T cell development. Among these single knockouts, the deficiency of Itk has the most overt effect on T cell development, while the deficiency of Txk or Tec has no reported effect (136, 138, ). These phenotypes correlate with the mrna levels for these genes. In T cells, Itk mrna is present at the highest level, Txk mrna levels are two to three fold lower and Tec mrna is present at levels close to 100-fold lower than that of Itk (146). Combined disruption of Itk and Txk leads to more severe effect on T cell development than does the Itk disruption alone, indicating the possibility of some levels of functional redundancy between these two Tec kinases (138). Itk-deficient mice were first generated by Liao and colleagues in 1995 and they showed that such animals had decreased numbers of mature thymocytes and mature T cells. Itkdeficient mice had reduced proliferative responses to allogeneic MHC stimulation and to anti-tcr cross-linking, but responded normally to stimulation with phorbol ester plus ionomycin or with IL-2. These results provide genetic evidence that Itk is involved in T cell development and also suggest that Itk has an important role in proximal events in

40 27 TCR-mediated signaling pathways (136). More and more research has been done since then, and it appears that the loss of Itk affects most stages of thymopoiesis Effect of Tec family kinases on pre-tcr signaling Tec kinases are important for pre-tcr signaling. Early studies by northern blot analysis showed that both Itk and Txk mrna can be detected by day 14 of fetal development, a time at which the thymus consists solely of DN1 and DN2 precursor cells (116, 147). Tec kinases are expressed in early thymic progenitor cells suggesting that Tec kinases might play a role in pre-tcr signaling. Recent reports from Lucas and colleagues demonstrated that DN thymocytes lacking Itk, or Itk and Txk, are impaired in their ability to generate normal numbers of DP thymocytes, the impairments are more significant when placed in direct competition with WT DN thymocytes. They also showed that Itk is required for maximal pre-tcr signaling in DN thymocytes. All of their data demonstrate that the Tec kinases Itk and Txk are involved in, but are not essential for, pre-tcr signaling in the thymus (146) Effect of Tec kinases on positive selection The strength of the signals received by DP thymocytes through their TCR can determine selection of the repertoire, which ensures that mature T cells can respond to non-self, but not self. For T cells developing in the thymus, this process includes positive selection and negative selection. Several Src and Syk family kinase proteins proximal to TCR signaling are critical for positive selection (148). ZAP-70-deficient mice revealed the importance of ZAP-70 in positive selection, whose T cells are strongly impaired for positive selection

41 28 (140). The importance of signals generated by a Ca 2+ flux in the positive selection have been appreciated for some time, because positive selection can be blocked by cyclosporin A or FK506, inhibitors of the Ca 2+ dependent calcineurin pathway (149, 150). The significance of ERK activation in positive selection was appreciated when dominant interfering forms of Ras and MEK were found to block positive selection (151, 152) and this was confirmed upon analysis of ERK1-deficient mice (153). The Tec family kinases are recruited to TCR signaling complexes upon activation and are important downstream tyrosine kinases of Src and Syk family in TCR signaling. Tec family kinases are also critical in generating a Ca 2+ flux and full activation of ERK pathway. Not surprisingly, Tec family kinases are required for optimal positive selection (154, 155) Effect of Tec kinases on negative selection Many of the proteins involved in TCR proximal signal transduction are required for both positive and negative selection, such as Lck and ZAP-70 (6, 156). In addition, Grb2, an adaptor protein associated with TCR signaling, was recently shown to be important in negative but not positive selection (157). The role of Itk in negative selection is less clear. As discussed above, while the ERK MAP kinase pathway is important for positive selection, two other MAP kinase pathways, JNK and p38, have been implicated in negative selection (158). The absence of Tec family kinases only affects the activity of ERK1 and ERK2, not the activity of p38 (155). While Schwartzberg and colleagues have suggested that Itk may play an important role in negative selection, Berg and colleagues have questioned this conclusion, although this may be dependent on the TCR transgenic

42 29 system used (124). Different systems and more research need to be done to resolve this discrepancy Effect of Tec kinases on CD4/CD8 commitment CD4 and CD8 lineage commitment is the third developmental step after positive and negative selection. The signals that determine lineage commitment are not completely clarified, but a role for TCR signaling in the CD4/CD8 lineage commitment is supported by many lines of experimental evidence (22). High activity of Lck and MAP kinases ERK1/2 leads to CD4SP development, while the low activity of Lck, ZAP-70 and ERK1/2 leads to CD8SP development ( ). Considering the role of Tec-family kinases in TCR signaling, it seems likely that the Tec-family kinases may also influence CD4/CD8 commitment. However, Berg and colleagues have demonstrated that Itk does not affect this process (154). However, they did not rule out the possibility that the strength of the TCR signal in the models that they used were above a threshold where Itk deficiency could not influence lineage commitment Effect of Tec kinases on peripheral T cell differentiation and cytokine production As a result of biochemical defects and impaired thymic development, peripheral T cells from Itk knockout or Itk and Txk double knockout mice show multiple functional defects: decreased proliferation of mature T cells; reduced tyrosine phosphorylation and activation of phospholipase-cγ1 (PLCγ-1), Ca 2+ mobilization, activation of transcription factors such as NFAT NF-κB and Elk, which regulate cytokine genes as well as genes

43 30 involved in cytokine signaling, survival and differentiation ( ). Mice deficient in Itk exhibit defects in T cell development and function. Naïve Itk -/- CD4 + T cells have decreased production of IL-4 and ITK -/- mice cannot mount effective Th2 cell response to infection with many pathogens, such as Leishmania major and Nippostrongylus brasiliensis, which are used to evaluate Th2 cell differentiation (134, 162). Itk deficient mice also do not develop allergic asthma, which is induced by an increased number of Th2 cells, increased Th2 cytokine production and increased mucus production in the lungs (163, 164). While Txk is involved in the Th1 cell differentiation, overexpression of human Txk in Jurkat T cells result in increased production of IFN-γ, whereas IL-2 and IL-4 levels remained the same (165). However, Txk knockout mice show only minor defects in response to T. gondii, which is a pathogen that induces strong Th1 cell response, among other responses (138, 162). The effects of Txk on Th1 cell differentiation require further evaluation. Mice lacking both Itk and Txk show more severe defects in TCR-signaling and cellular defects than those observed in Itk single knockout mice (138), which suggests that there is redundancy between these two kinases. Surprisingly, Itk and Txk double knockout mice have been shown to have effective Th2 response to infection and near normal levels of Th2 cytokine production (162), suggesting complex mechanisms regulating T cell differentiation by Tec family kinases.

44 The purpose of the study In addition to the conventional CD4 + SP and CD8 + SP T cells, DP thymocytes also give rise to lineages such as T reg and NKT cells. Using knockout and transgenic mice, a number of proteins have been identified that are required for the development and function of T reg and NKT cells. Although T cell development has been studied for more than twenty years, the subsets under the main T cell lineages and the proteins that are crucial for their development are still not completely studied. The analysis of the T cell lineages in Itk -/- mice has revealed a consistent increase in the percentage of memory phenotype CD4 + and CD8 + T cells comparing to WT mice. The reasons that cause this increase in the absence of Itk are not clear and the development and the function of these memory phenotype cells are unknown. Using Itk -/- mouse models, we have examined the role of Itk in the T cell lineage development, particularly the role of the Itk in the MP T cell development and function. We hypothesized that Itk regulted the T cell lineage development. Although the role of the Itk in the conventional CD4 + and CD8 + T cells is well studied, the role of Itk in the CD4/CD8 commitment is controversial. There are significantly more CD8 + SP T cells in the thymus of Itk -/- compared to WT mice, and lower CD4/CD8 ratios in the periphery, indicating that Itk may play important role in CD4/CD8 commitment. The Berg laboratory has previously examined this issue by analyzing Itk -/- mice crossed with five different MHC Class II specific TCR transgenic lines, and observed no difference in the number of TCR transgenic CD8 + SP thymocytes, indicating that the Itk is not involved in the lineage commitment. However, they did not rule out the possibility that the strength of the TCR signal in the models that they used were above a threshold

45 32 where Itk deficiency could not influence lineage commitment. We have examined mice lacking Itk and carrying a low affinity TCR transgene specific for a class II restricted peptide derived from Ovalbumin (OVA, OT-II mice), to determine if Itk play a role in the CD4/CD8 commitment. We hypothesized that Itk regulated the CD4/ CD8 commitment in this low affinity TCR transgenic modle.

46 33 Chapter 2 Materials and Methods

47 Animals WT and Itk null mice (166) used were 2 days to greater than 12 months of age and were kept in specific pathogen free conditions. Itk Kin Tg /Itk -/- were generated by cloning a mutant Itk with the kinase domain replaced with EGFP (167) into a transgenic expression cassette driven by the Lck proximal promoter and CD2 enhancer (kind gift of Dr. Anuradha Ray, University of Pittsburgh). Tg(CD2-hItk)Itk -/- mice were generated by cloning a human Itk cdna into a transgenic expression cassette driven by the CD2 promoter and CD2 enhancer. These mice were backcrossed >5 generations. In both cases, the expression level of the transgene was roughly 30% of endogenous Itk as determined by quantitative RT-PCR. These mice were backcrossed to the C57BL/6 background >10 generations. IFNγ -/- mice, MHC class II -/-, nude mice and OT-II transgenic mice were from Jackson Labs (Bar Harbor, Maine) and bred in our lab. OT- II/Itk -/- mice were generated by crossing Itk -/- mice to OT-II transgenic mice. These mice were intercrossed, then backcrossed >5 generations. Itk Kin Tg /Itk -/- and Tg(CD2-Txk)WT were kindly provided byy Dr. Pamlea Schwartzberg (NHGRI/NIH) by permission of Drs. Paul Love and Connie Sommers (NICHD/NIH). Experiments were approved by the IACUC at Pennsylvania State University. 2.2 Bacterial infection Mice (age matched 6-8 weeks old) were infected with erythromycin-resistant L. monocytogenes (2X10 3 CFU, kind gift of Dr. Hao Shen, University of Pennsylvania) and sacrificed after 72 hours (similar results were observed after 24 and 48 hours). Bacterial CFU were determined by plating splenic extracts on BHI agar containing 5 μg/ml

48 35 erythromycin. To analyze T cell secretion of cytokine ex vivo following infection, mice were infected with L. monocytogenes (2X10 3 CFU) for 24 hrs, and splenocytes isolated and incubated in vitro with Brefeldin A (10 μg/ml) for a further 6 hours, followed by analysis of intracellular IFNγ as described below. Analysis of secretion of IFNγ in vivo was analyzed as previously described (168). CD8 + T cells were depleted by injection of 1 mg anti-cd8 mab (YTS168.4, kind gift of Dr. Eric Harvill, Penn State University) and 24 hours later, infected with bacteria as described above, followed by analysis 3 days later. In experiments where CD8 + or CD8 + CD44 hi T cells were transferred into IFNγ null mice, 3X10 6 sorted CD8 + or CD8 + CD44 hi T cells were transferred into these mice, then the mice were infected with 2X10 3 CFU L. monocytogenes. Mice were analyzed at 3 days post infection. 2.3 Antibodies and Flow Cytometry Cells were incubated for half an hour at 4 o C with antibodies in 100 μl PBS/2% FBS, followed by two washes in PBS/2% FBS for surface staining. The following antibodies were purchased from BD Pharmingen and used as suggested by the manufacturer (BD Pharmingen, San Diego, California): anti-cd8-fitc, Vα2-PE, Vβ5-FITC, CD44- Cychrome, CD5-PE, CD45.2-FITC, IL-4-PE, CD122-PE, IFNγ-PE-Cy7, CCR7-PE, CD127-PE, NK1.1-PE, and BrdU-FITC. α-galactosyl Ceramide/CD1d tetramers were from the NIH tetramer Facility (Atlanta, GA). Anti-CD62L-APC-Alexa-Fluo750 was purchased from ebioscience (San Diego, California). Anti-CD4-ECD was from Invitrogen (Carlsbad, California) and anti-murine-bcl-2-pe was from Santa Cruz Biotechnology (Santa Cruz, California). Intracellular staining of Bcl-2 was carried out

49 36 using a Fixation/Permeabilization kit from BD Pharmingen (San Diego, California). Apoptosis was detected using the Annexin V-PE apoptosis detection kit from BD Pharmingen. Cells were analyzed using a FC500 Cytometer from Beckman Coulter (Fullerton, California). 2.4 Real-Time PCR analysis CD8 + CD44 lo or CD8 + CD44 hi T cells and CD4 + CD62L Lo or CD4 + CD62L Hi T cells were sorted from the spleens of WT and Itk null mice using a Cytopeia influx Cell Sorter (Cytopeia Inc., Seattle, WA). Total RNA was prepared from sorted cells using RNease mini kit (Qiagen Sciences, Maryland). cdna was generated using You Prime First- Strand beads (GE healthcare, Buckinghamshire, UK), and quantitative PCR performed using primer/probe sets for IFN-γ and T-bet, with GAPDH as a housekeeping gene as described (169). Data was analyzed using the Comparative C T (threshold cycle) method and normalized to GAPDH. The relative gene expression levels were then determined by comparing to the expression found in the WT CD8 + CD44 lo populations or WT CD4 + CD62L hi populations, which were set at 1 or as indicated in the respective figure legends. In chapter 5, TCR hi DP thymocytes were sorted from the thymus of transgenic OT-II and OT-II/Itk -/- mice and quantitative PCR was performed using primer/probe sets for Th-POK, TOX, Runx3 and GATA3 (Applied BioSystems, Branchburg, New Jersey), with GAPDH as a housekeeping gene. Data was analyzed using the ΔΔ Comparative CT (threshold cycle) method and normalized to GAPDH and relative to a calibrator sample. The relative gene expression levels were then determined by comparing to the expression found in the transgenic TCR hi OT-II DP populations, which were set as 1.

50 In vitro analysis of cytokine secretion and production Splenocytes were stimulated with 50 ng/ml PMA/0.5 μm Ionomycin for 6 hours, or IL- 12 (5 ng/ml, R&D Systems, Minneapolis, MN) and IL-18 (10 ng/ml, MBL, Watertown, MA) overnight, in the presence of Brefeldin (10 μg/ml) and analyzed for intracellular IFNγ and cell surface CD8 or CD4 and CD44 by flow cytometry using specific antibodies. Splenocytes or peritoneal macrophages were incubated with heat killed L. monocytogenes for 6 hours, and analyzed for TNF-α using a specific ELISA (R&D Systems) or intracellular TNF-α in F4/80 + cells by flow cytometry. In chapter 5, spleens were removed from mice and total splenocytes from OT-II and OT-II/Itk -/- mice were cultured with 1 μg/ml OVA peptide (Research Genetics, Huntsville, AL) for 7 days. Dead cells were removed using lympholyte-m (Cedarlane Laboratories, Burlington, NC), and viable cells then stimulated with OVA (1 μg/ml) or PMA/Ionomycin (50 ng/ml/0.5 μm) for 6 hours in the presence of Brefeldin A (10 μg/ml), followed by analysis of cytokine secretion by intracellular cytokine staining. 2.6 In vitro analysis of T cell proliferation To analyze responses to IL-2, sorted CD8 + CD44 hi from WT and Itk -/- mice were stimulated at 1X10 5 /well in triplicate with recombinant murine IL-2 (250 U/ml) for 3 days and proliferation measured by H 3 -thymidine incorporation. In chapter 4, Purified CD4 + CD62L Lo and CD4 + CD62L Hi T cells from WT and Itk -/- mice were stimulated at cells/well in triplicate with 1 μg/ml anti-cd3 or 1 μg/ml anti-cd3 plus 1 μg/ml anti-cd28 for 3 days and proliferation was measured by 3 H-thymidine incorporation over the final 18 hours.

51 BrdU Incorporation Mice were treated with BrdU in drinking water (dissolved at 0.8 mg/ml) for 9 days, with mice given fresh BrdU-containing drinking water daily. Splenocytes were collected and stained for surface marker using appropriate antibodies, followed by washing in PBS and resuspending in ice-cold 0.15 M NaCl. The cells were fixed and permeabilized in icecold 95% ethanol for 30 min on ice, then washed with PBS and fixed again in 1% paraformaldehyde for 30 min at room temperature. To detect BrdU, the cells were spun down and resuspended in 1 ml DNase I solution (50 U/ml deoxyribonuclease I in 4.2 mm MgCl 2 /0.15 M NaCl, ph=5) for 10 min at room temperature. The cells were then washed and resuspended in 100 μl of 1:10 dilution of anti-brdu-fitc for 30 min at room temperature. The cells were washed and resuspended in 500 μl PBS and analyzed by flow cytometry. For turnover analysis, the mice were treated with BrdU-containing water for 9 days and then treated with normal water for indicated days, and then analyzed by the procedure described above. 2.8 Fetal Thymic Organ Culture (FTOC) Fetal thymi were harvested from E16 pregnant females. Thymic lobes were placed in transwell plates (Costar) with 2 ml DMEM medium (containing 15% Fetal calf serum) for the indicated time periods. 2.9 Bone marrow chimera Bone marrow was isolated from femurs and tibia of Thy1.1 WT and Itk -/- mice cells were injected into lethally irradiated congenic WT (Thy1.2) mice. Mice were

52 39 analyzed 8 weeks after reconstitution. To determine if Itk -/- T cells can compete with WT T cells during development, a 1:1 mixture ( cells each) of bone marrow from Thy1.2/CD45.1 congenic WT and Thy1.2/CD45.2 congenic Itk -/- mice were injected into irradiated Thy1.1/CD45.2 WT mice, followed by analysis of donor derived WT (Thy1.2/CD45.1) and Itk -/- (Thy1.2/CD45.2) T cells 6 weeks after reconstitution. In chapter 5, Bone marrow was isolated from femurs and tibia of OT-II mice (CD45.2) and OT-II/Itk -/- mice (CD45.2). Cells ( ) were injected into lethally irradiated congenic WT (CD45.1) mice. Mice were analyzed 12 weeks after reconstitution Statistical analysis Data was analyzed using Prizm, and significance determined using Students t test, with a value of p<0.05 considered statistically significant.

53 40 Chapter 3 Memory phenotype CD8 + T cells with innate function selectively develop in the absence of active Itk Chapter adapted from the manuscript entitled: Memory phenotype CD8 + T-cells with innate function selectively develop in the absence of active Itk Authors: Jianfang Hu, Nisebita Sahu, Elizabeth Walsh, Avery August Eur J Immunol Oct; 37(10):

54 INTRODUCTION Itk, a Tec family kinase, regulates signals emanating from the T cell Receptor (166, ). Mice lacking Itk have reduced numbers of CD4 + T cells, and these cells exhibit reduced proliferation and IL-2 production in vitro and in vivo (166, 172, 173). Itk also modulates the differentiation of helper T cells into Th2 cells, or alternatively, controls their production of Th2 cytokines such as IL-4 ( ). Indeed, infection of Itk deficient mice on a Balb/c background with parasites such as S. mansoni or L. donovani, results in the generation of a predominant Th1 response (172, 174, 175). In agreement with these findings, Itk null mice are resistant to developing Th2 mediated allergic asthma (169, 177). By contrast, these mice are more susceptible to infection with the intracellular parasite T. gondii (171), as well as infection with VSV and LCMV (178, 179). T cells are generally characterized as naïve cells, generally identified as CD62L hi and/or CD44 lo or memory cells, generally identified as CD62L lo and/or CD44 hi (for review see (180, 181)). Recently, a population of CD8 + T cells with innate like function expressing a similar memory like phenotype has been identified (182, 183). These cells are CD8 + CD44 hi, can rapidly secrete IFNγ upon stimulation with IL-12 and IL-18, and play important roles in the innate response against infections such as L. monocytogenes, or chronic infections with viruses such as γherpes virus ( ) (for review see (188)). The origin of these cells or the regulation of their function is poorly understood.

55 42 During analysis of mice lacking Itk, it has been noted that these mice have increased percentage of memory like CD4 + as well as CD8 + T cells ( ). Most recently, the memory like CD8 + T cells were shown to be CD44 hi /CD122 +, and to be restricted by MHC class 1b ( ). Whether these cells represent the previously described innate type memory T cells is unclear ( ). We report here that a majority of the CD8 + T cells in Itk null mice have a phenotype similar to memory like CD8 + innate T cells. These cells do not require active Tec kinase signaling for their development, carry preformed message for IFNγ, which they are able to rapidly produce upon stimulation with IL-12/IL-18. Finally, we show that Itk null mice exhibit enhanced response to infection with L. monocytogenes in vivo and that transfer of Itk null CD8 + CD44 hi reduces bacterial burden in L. monocytogenes infected IFNγ null mice. These results indicate that active signals from Tec kinases regulate the development and function of memory like T cells with innate function.

56 RESULTS Increased presence of CD8 + T cells with memory like phenotype in Itk null mice CD8 + CD44 hi T cells which resemble memory cells represent an increased percentage of total CD8 + T cells in Itk null mice. This increased population of cells are present in the youngest mice tested (age <1 month) and the increase persists out to >12 months of age (Fig. 3-1a, shown for the spleen). The percentage of these cells is increased in the lymph nodes, blood and spleen of mice lacking Itk, suggesting that this is not dependent on preferential enrichment of these cells in specific tissues (data not shown). Expression of CD122, the IL-2Rβ chain, has been reported to define this population of memory like CD8 + T cells (193), and we found that the CD8 + CD44 hi population in both WT and Itk null mice expressed CD122, although the Itk null mice carry a larger percentage of these cells (Fig.3-1b). Analysis of the number of CD8 + CD44 hi T cells in 6-8 week old WT and Itk null mice indicated that the Itk -/- mice had significantly more of these T cells in the spleen (Fig.3-2). These data suggest that signals from Itk negatively regulate the development of CD8 + T cells having a memory like phenotype. Alternatively, Itk signals are required for the development of conventional CD8 + T cells expressing low levels of CD44 and in their absence, CD8 + CD44 hi T cells predominate Tec kinase activity suppresses the presence of CD8 memory like T cells We and others have reported that the kinase domain and/or activity of Itk is dispensable for specific functions of Itk in vitro (167, 194, 195). To determine if this domain or active

57 44 signaling by Itk is required for the presence of the memory like T cells, we analyzed mice carrying a mutant Itk lacking the kinase domain, replaced with GFP, instead of WT Itk (ItkΔKin Tg /Itk -/- mice). These mice had the memory like CD8 + T cells similar to those seen in Itk null mice (Fig.3-3a). WT mice had significantly lower percentage of CD8 + CD44 hi CD122 + than those found in Itk -/- and ItkΔKin/Itk -/- mice (Fig. 3-3b). As a control, we also analyzed mice carrying wild-type Itk (Itk tg Itk -/- ). These mice had memory like CD8 + T cells similar to those seen in WT mice. These data indicate that active signaling by Tec kinases reduces the development of these cells or enhances the development of conventional CD8 + CD44 lo T cells Txk partially suppresses the presence of memory phenotype CD8 + T cells Txk is another Tec family kinse expressed in T cells (147). Txk -/- mice exhibit only a mild impairment in TCR signaling (138). However, Txk -/- /Itk -/- mice exhibit more severe impairment in TCR signaling than Txk -/- or Itk -/- mice, suggesting that these proteins may perform redundant functions (138). To study Txk function, in 1999, the Love lab generated transgenic mice that overexpress Txk throughout T cell development (196). They showed that Txk overexpression resulted in enhanced IL-2 production after TCR engagement, and partially rescued the defect in positive selection in Itk -/- mice (196). To determine if Txk can replace Itk for the presence of the memory like CD8 + T cells, we analyzed the memory like CD8 + T cells in these Txk transgenic mice on an Itk -/- background. We found that these mice had a lower percentage of memory like CD8 + T cells compared to Itk -/- mice, but the percentage was still significant higher compared

58 45 with Itk tg Itk -/- mice (Fig. 3-4). These data indicate that Txk signals can partially suppress the development of memory phenotype CD8 + T cells Itk -/- memory like CD8 + T cells carry preformed message for IFNγ To determine if these memory like CD8 + T cells exhibit similar functional behavior in vitro, we analyzed their ability to rapidly secrete IFNγ upon stimulation. Comparison of IFNγ production by PMA/Ionomycin stimulated CD8 + CD44 hi T cells in vitro indicates that the Itk null cells behave similar to those from WT mice in rapidly (within 6 hours) secreting this cytokine, with a similar percentage of CD8 + CD44 hi T cells responding from both WT and Itk -/- mice (Fig. 3-5a). By contrast, CD8 + CD44 lo T cells did not secrete any appreciable IFNγ in this time period. A role for memory like CD8 + CD44 hi T cells in the early innate response following exposure to LPS or infection with the intracellular bacteria L. monocytogenes has been previously reported (185). Infection of macrophages by L. monocytogenes results in the secretion of IL-12 and IL-18 (197), and these cytokines can stimulate CD8 + CD44 + memory like T cells to secrete IFNγ, thus activating macrophages for TNF-α production and bacterial clearance (186). We therefore determined if this population is the same population that is expanded in mice lacking Itk. Analysis of IFNγ production in cells from WT and Itk null mice stimulated in vitro with IL-12 and IL-18 revealed that WT and Itk null CD8 + CD44 hi secreted significant levels of this cytokine, and again a similar percentage of Itk null CD8 + CD44 hi T cells responded compared to WT cells (Fig. 3-5b). Similarly, incubation of purified CD8 + CD44 hi T cells from WT and Itk revealed that they had similar proliferative responses to IL-2, in agreement with their expression of CD122,

59 46 while the CD8 + CD44 lo population did not proliferate to any significant extent (Fig. 3-6). This population of cells also rapidly secretes IFNγ in response to exposure to LPS in vivo, as revealed by injection of mice with LPS, followed by analysis of intracellular cytokine in CD8 + CD44 hi T cells from WT and Itk null mice (data not shown). Since these cells were able to rapidly secrete IFNγ, we evaluated freshly isolated unstimulated CD8 + CD44 hi T cells and found that these cells carry high levels of preformed message for IFNγ, as well as high levels of the transcription factor T-bet (compared to the CD8 + CD44 lo T cell population) (Fig. 3-7). Thus these memory like CD8 + T cells with innate function are poised to rapidly secrete IFNγ Itk -/- mice exhibit enhanced clearance of Listeria dependent on CD8 + T cells To determine if this population of memory like T cells is involved in the response against L. monocytogenes, we infected WT and Itk null mice with low numbers of L. monocytogenes (2X10 3 CFU), and analyzed the spleens for bacterial numbers after three days. We recovered approximately 2 fold lower bacteria from Itk null mice than from WT mice, suggesting that Itk null mice were better able to mount an innate immune response against L. monocytogenes (Fig. 3-8). This is most likely due to the fact that Itk null spleens contained significantly higher percentage as well as higher numbers of CD8 + CD44 hi T cells compared to WT mice (see Fig. 3-2). When analyzed at 7 days post infection, we could not detect any bacteria in either WT or Itk null mice indicating that eventual clearance is similar in these mice (data not shown). Macrophages have been shown to be largely responsible for bacterial clearance, and we found that stimulation of splenic cells with heat killed or live L. monocytogenes resulted in significantly enhanced

60 47 TNF-α production by Itk -/- splenocytes (Fig. 3-9). Examination of the cells secreting TNF-α revealed that F4/80 + macrophages are the only population in splenocytes that secrete TNF-α in response to L. monocytogenes stimulation (data not shown), and that a significantly higher percentage of the Itk -/- macrophages secreted TNF-α upon stimulation (Fig. 3-9). By contrast, peritoneal macrophages from the same mice secreted similar levels of TNF-α upon stimulation with heat killed L. monocytogenes (Fig. 3-9). Note that Itk -/- and WT mice have similar numbers of macrophages in the spleen and peritoneum. These data suggest that splenic macrophages from Itk null mice are more responsive to L. monocytogenes stimulation, and that this response may contribute to the advantage that these mice have upon infection Itk -/- memory T cells reduce bacterial burden in IFNγ null mice We reasoned that an increased numbers of CD8 + CD44 hi memory like T cells with innate function in Itk null mice may be able to secrete elevated levels of IFNγ, resulting in increased macrophage activation in the spleen, but not in the peritoneum, where there are significantly less T cells. We therefore determined if the CD8 + CD44 hi population of T cells in Itk null mice could secrete IFNγ during infection with L. monocytogenes. We found that CD8 + CD44 hi T cells actively secrete IFNγ in vivo 24 hours after infection with L. monocytogenes (Fig. 3-10a). Mice lacking IFNγ cannot reduce bacterial burden when infected with L. monocytogenes due to their inability to produce IFNγ critical for this event (184). Indeed, Itk null mice had elevated levels of IFNγ in the serum during infection (Fig. 3-10b). CD8 + T cells are

61 48 critical producers of IFNγ, and we found that antibody-mediated depletion of CD8 + T cells resulted in significantly reduced ability to clear this infection in both WT and Itk null mice, indicating that CD8 + T cells play a critical role in the early innate response to the bacteria (Fig. 3-11a). The majority of CD8 + T cells in the ITK null mice are of the CD44 hi phenotype, and we had found that ITK null mice were better at clearing the infection with L. monocytogenes. We therefore wanted to determine if these T cells could secrete IFNγ and reduce bacterial burden in IFNγ null mice, and determine if the ITK null CD8 + T cells are more effective in the anti-listeria response. We did this by determining if transfer of cells from the WT and ITK null mice could rescue antibacterial responses in the mice lacking IFNγ. Sorted total CD8 + T cells from WT and Itk - /- mice were transferred into IFNγ -/- mice, which were subsequently infected with L. monocytogenes. Analysis of bacterial burden in the spleen of these mice indicated that while transfer of WT CD8 + T cells could reduce their bacterial burden, the difference was not statistically significant (p=0.092 vs. control IFNγ null mice) (Fig. 3-11b). By contrast, transfer of total CD8 + T cells from ITK null mice was able to significantly reduce the bacterial burden on the IFNγ null mice (Fig. 3-11b). We next determine if the CD8 + CD44 hi T cells in WT and ITK null mice were equivalent in their ability to secrete IFNγ and reduce bacterial burden in the IFNγ null mice. We therefore transferred sorted CD8 + CD44 hi T cells from WT and Itk null mice into mice lacking IFNγ, and then infected them with L. monocytogenes. Analysis of bacterial burden in the spleen of these mice showed that both WT and Itk CD8 + CD44 hi T cells could reduce their bacterial burden, indicating that the CD8 + CD44 hi T cells seen in Itk null mice represent the population of cells previously described ((184, 188), Fig. 3-11c). Altogether, these data

62 49 show that the CD8 + CD44 hi T cells that develop in mice lacking signals from Itk possess innate like immune function in response to IL-12/IL-18, LPS or bacterial exposure in vitro and in vivo.

63 DISCUSSION A population of T cells having a memory like CD8 + CD44 hi phenotype has been shown to develop in the absence of classical class I molecules, and these cells possess innate immune function in their ability to rapidly secrete IFNγ in response to signals from IL-12 and IL-18, and to infection with L. monocytogenes (186). More importantly, these cells can reduce bacterial burden in L. monocytogenes infected IFNγ null mice in an antigen non-specific manner early in the innate response against this bacteria (184). Our work here demonstrates that in the absence of Tec kinase signals the relative percentage of a population of CD8 + CD44 hi CD122 + T cells increases. However, these cells behave in the same manner as WT CD8 + CD44 hi CD122 + T cells in rapidly secreting IFNγ in response to stimuli from IL-12/IL-18 and infection with L. monocytogenes, suggesting that Tec kinase signals are not required for this function. We also find that these cells carry preformed message for IFNγ as well as T-bet, suggesting a mechanism for their ability to rapidly secrete this cytokine. The origin of T cells with a CD8 + CD44 hi phenotype is controversial. Activation of naïve T cells in vitro generates a population of cells that have this phenotype, and the presence of the CD44 marker on T cells has been used to identify previously activated T cells ( ). In addition different T cell memory populations have been identified based on expression of CD44 and CD62L, including effector memory and central memory T cells (201). Thus the expression of the CD44 marker on CD8 + T cells seem to represent those cells that have been previously activated and may have effector or memory function.

64 51 Similar populations of CD8 + CD44 hi T cells have been reported in mice lacking classical class I molecules and T cells with this phenotype appear to be class 1b restricted (183, 189, 202). These T cells are reported to be thymic-independent in origin and carry CD122, which may distinguish them from memory T cells, although this distinction is not always clear since older studies on memory T cells did not examine this marker (203). While it has been suggested that these cells appear due to stimulation by normal flora, they are also present in germ-free mice (204). H2-M3 restricted T cells can have a similar CD8 + CD44 hi phenotype and can rapidly respond to L. monocytogenes infection compared to antigen specific T cell responses, although this H2-M3 specific expansion of these cells peaks at around 7 days, while the secretion of IFNγ by cells with a similar phenotype in response to IL-12/IL-18 induced by LPS activated macrophages is much quicker (186, ). However, transfer of naïve T cells into lymphopenic mice can also generate T cells bearing the CD44 marker, and these cells can take on an effector function. Similarly, individuals who have had a bone marrow transplant, and elderly individuals and mice have elevated percentages of T cells with a similar phenotype ( ). In addition, viral and other infections can lead to expanded populations of T cell bearing this phenotype (187). The appearance of these cells may represent homeostatic expansion of T cells to fill niches left empty by irradiation (during bone marrow transplant), reduced T cell output (in elderly individuals), viral exhaustion of antigen specific T cells, expansion of class 1b restricted T cells, or may represent true memory cells. It is possible that the immune system uses such a system of expanding CD8 + T cells with this phenotype

65 52 (i.e. during homeostatic expansion, cells take on an innate-effector phenotype) to ensure that time of lymphopenia does not result in an immunocompromised position. It is possible that there are several distinct population of CD8 + CD44 hi T cells that contain pools of true memory T cells as well as cells that have undergone rapid homeostatic expansion such as those seen in class 1b restricted T cells, in lymphopenic mice, in cases of infections or class 1b restricted T cells. The increased relative percentage of these cells in mice lacking Tec kinase signals may be the result of entry of CD8 + T cells into a lymphopenic compartment early in the development of these mice, leading to homeostatic expansion and the acquisition of this CD44 hi CD122 + phenotype. These cells would then have innate-like effector function with preformed IFNγ message and the ability to rapidly secrete this cytokine upon stimulation with IL-12/IL-18 or infection with L. monocytogenes. Alternatively, these cells may be selected on class 1b molecules, which result in homeostatic expansion of these T cells and the acquisition of the CD8 + CD44 hi CD122 + phenotype. Recently, Dubois et al, Broussard et al and Atherly et al reported finding similar populations of CD8 + CD44 hi CD122 + T cells in mice lacking Itk (191) or Itk/Rlk (189, 190), however, these investigators did not determine if these cells were functional in vivo, or related to the population of CD8 + CD44 + T cells previously reported to rapidly respond to bacterial infection, LPS or IL-12/IL-18 in vitro and in vivo ( , 188). It is unlikely that these CD8 + CD44 hi CD122 + T cells are memory T cells as the latter are usually CD122 -/lo (203). Our data presented here suggest that the memory-like

66 53 CD8 + CD44 hi T cell populations are equivalent in WT and Itk null mice, and represent an innate like population of T cells. Our data also indicates that the increased serum IFNγ observed in L. monocytogenes infected Itk -/- mice is likely to be due to the increased numbers of these CD8 + CD44 hi T cells in these mice, since transfer of these cells into IFNγ null mice can reduce bacterial burden in these mice, indicating that T cell-derived IFNγ is sufficient for this event. This increase in IFNγ could lead to more effective macrophage activation and provide the ITK null mice with an advantage in the macrophage response to listeria. Interestingly, while the absence of Tec kinase signals results in an enhanced relative percentage of these cells, the innate-type function of these cell does not seem to be affected by the absence of Itk in that similar percentage of these cells produce IFNγ upon stimulation in vitro or in vivo compared to their WT counterparts. In addition, when equal numbers of WT and Itk -/- CD8 + CD44 hi T cells are transferred into IFNγ null mice, they behave similarly in being able to reduce bacterial burden. Given that T cell receptor mediated stimulation is reduced in the absence of Itk, these data suggest that the innate response of these cells is Itk independent. Taken together our data indicate that in the absence of signals from Tec kinases such as Itk, there is an increased population of CD8 + T cells with a memory like phenotype and innate function that can rapidly secrete cytokine in vivo in response to bacterial infection as well as LPS.

67 54 Fig.3-1 a) b) Figure 3-1: Increased percentage of CD8 + CD44 hi memory phenotype T cells in mice lacking Itk. (a) Spleens from WT and Itk -/- mice were analyzed for the percentage of CD8 + CD44 hi cells over the indicated time frame. (b) Analysis of CD44 and CD122 expression on CD8 + T cells in WT and Itk null mice.

68 55 Fig.3-2 Figure 3-2: Increased absolute number of CD8 + CD44 hi memory phenotype T cells in mice lacking Itk. Number of CD8 + CD44 hi T cells in the spleens of WT and Itk -/- mice. (*p=0.0007, n=5).

69 56 Fig. 3-3 a) b) Figure 3-3: Active Tec kinase signaling regulates the presence of CD8 + CD44 hi memory phenotype T cells. (a) Spleens from WT, Itk -/-, Itk Tg Itk -/- and ItkΔKin Tg /Itk -/- mice were analyzed for the percentage of CD8 + CD44 hi CD122 + cells. (b) Percentage of CD8 + CD44 hi T cells that carry CD122 in the indicated mice (*p<0.05; **P<0.05; NS, Not statistically significant).

70 57 Fig.3-4 a) b) Figure 3-4: Txk can partially suppress the presence of CD8 + CD44 hi memory phenotype T cells. (a) WT, Itk and Itk Tg Itk -/- mice are the same as in figure 3-3,spleens from Txk Tg /Itk -/- mice were analyzed for the percentage of CD8 + CD44 hi CD122 + cells. (b) Percentage of CD8 + CD44 hi T cells that carry CD122 in the indicated mice (*, ** and ***, p<0.05).

71 58 Fig.3-5 a) b) Figure 3-5: CD8 + CD44 hi T cells rapidly secrete IFNγ upon activation with IL-12/IL- 18. (a) Splenocytes from WT and Itk -/- mice were stimulated with PMA/Ionomycin followed by analysis for intracellular IFNγ in gated CD8 + T cells. (b) Similar experiments as described in (a) except that cells were stimulated with IL-12 and IL-18. Percentages shown in (a) and (b) indicate those CD8 + CD44 hi that respond to stimulation (upper right) and those that did not respond (lower right). C = control; = IL-12 + IL-18.

72 59 Fig.3-6 Figure 3-6: CD8 + CD44 hi T cells show proliferative response to IL-2 stimulation. Purified CD8 + CD44 hi from WT and Itk -/- mice were stimulated with IL-2 for 3 days and proliferation measured by H 3 -thymidine incorporation.

73 60 Fig.3-7 a) b) Figure 3-7: Memory Phenotype CD8 + T cells carry preformed message for IFNγ and T-bet. (a) CD8 + CD44 hi and CD8 + CD44 lo T cells from WT and Itk -/- mice were sorted by flow cytometry and message for IFNγ was analyzed by Q-RT-PCR. (b) The same cells from (a) were analyzed for preformed message of T-bet. Data are corrected for GAPDH expression and expressed as fold over the WT CD8 + CD44 lo populations, which was set at 1. (*p<0.05 ;)

74 61 Fig.3-8 Figure 3-8: Itk -/- mice exhibit enhanced clearance of L. monocytogenes in vivo. WT and Itk -/- mice were infected with 2X10 3 CFU L. monocytogenes for 3 days and the number of bacteria in the spleen determined (*p<0.05 vs. WT mice).

75 62 Fig.3-9 a) b) Figure 3-9: Itk -/- splenic macrophages produce increased TNFα upon HKLM stimulation in vitro. (a) Splenocytes (left panels) or peritoneal macrophages (right panels) from WT and Itk -/- mice were stimulated in vitro with HKLM and cells analyzed for the percentage of TNFα + F4/80 + macrophages by flow cytometry. (b) Splenocytes (left panels) or peritoneal macrophages (right panels) from WT and Itk -/- mice were stimulated in vitro with HKLM and supernatants analyzed for TNFα by ELISA. C = control; HKLM = Heat Killed Listeria Monocytogenes.

76 63 Fig a) b) Figure 3-10: Memory Phenotype CD8 + T cells actively secrete IFNγ in vivo after infection with L. monocytogenes. (a) WT and Itk -/- mice were infected with 2X10 3 CFU L. monocytogenes, and 24 hrs later, splenocytes harvested and CD8 + CD44 hi T cells analyzed for intracellular IFNγ. Percentages shown indicate those CD8 + CD44 hi that respond to stimulation (upper right) and those that did not respond (lower right). (*p<0.05 vs. WT mice) (b) WT and Itk -/- mice were infected 2X10 4 CFU L. monocytogenes for 2 days and serum IFNγ determined by ELISA (*p<0.05 vs. WT mice). C = Control. LM = Listeria Monocytogenes.

77 64 Fig.3-11 a) b) c) Figure 3-11: CD8 + CD44 hi T cells reduce bacterial burden in IFNγ null mice infected with L. monocytogenes. (a) CD8 + T cells were depleted from WT and Itk -/- mice, and the mice infected with L. monocytogenes 24 hours later and recovered bacterial were determined. (b) Sorted total CD8 + T cells or (c) Sorted CD8 + CD44 hi T cells from WT and Itk -/- mice were transferred into IFNγ -/- mice, which were subsequently infected with 2X10 3 CFU L. monocytogenes for 3 days and the number of bacteria in the spleen determined. (*p=0.092 for recipients of WT -/- T cells vs. IFNγ -/- mice; **p<0.05 for recipients of ITK -/- T cells vs. IFNγ -/- mice; **p<0.01 for recipients of WT T cells vs. ITK -/- T cells).

78 65 Chapter 4 Naïve and Innate Memory phenotype CD4 + T-cells have different requirements for active Itk for their development Chapter adapted from the manuscript entitled: Naïve and Innate Memory phenotype CD4 + T-cells have different requirements for active Itk for their development Authors: Jianfang Hu, and Avery August J Immunol, 2008, 180:

79 INTRODUCTION Mature CD4 + SP and CD8 + SP T cells arise from CD4 + CD8 + DP T cell precursors in the thymus. The development of CD4 + and CD8 + T cells can be influenced by the strength and duration of signals received through the TCR in DP thymocytes (213, 214). In addition to conventional T cell lineages, DP thymocytes also give rise to some other lineages of mature T cells, such as T reg and NKT cells ( ), which are called nonconventional T cells. Studies of many knockout mice have identified proteins required for the T reg and NKT cells, but less is known about signaling pathways leading to the specific development of CD4 + and CD8 + T cells. Itk is the predominant Tec kinase expressed in T cells and is activated downstream of the T cell receptor (TCR) (166, , ). Specifically, Itk seems to act as an amplifier of TCR signals, and is required for the full activation of PLCγ-1, Ca 2+ mobilization, and activation of transcription factors such as NFAT, NF-κB, and AP-1. These transcription factors activate a number of genes, including cytokine and other genes involved in cytokine signaling, survival and differentiation (166, 172, 174, 218, 221). Thus, Itk can affect multiple processes important for T cell development, activation, and effector function (218). These affected processes include impaired positive selection (222, 223), altered CD4/CD8 commitment, defects in TCR induced proliferation, IL-2 production and reduced activation induced cell death (AICD) in the absence of Itk (166, 172, 173, 218). We and others have recently shown that Itk is required for the development of conventional CD44 Lo naïve CD8 + T cells but not innate memory phenotype CD44 Hi

80 67 CD8 + T cells ( , 224). In the absence of Itk, CD8 + T cells resemble activated/memory cells, express memory markers, carry high levels of preformed messages for IFNγ and T-bet, and rapidly produce IFNγ ex vivo in response to stimulation (189, 190, 224). These CD8 + T cells developed as a result of interaction with MHC molecules expressed on haematopoietic cells in the thymus (189). All these properties suggest that these cells share properties with innate T cells such as H2-M3 specific CD8 + T cells, MAIT cells and CD1d specific NKT cells (202, 206, 217, 225). Although conventional CD8 + T cell development is abolished in the absence of Itk, the development of CD4 + T cell lineage seems to be less affected (189, 190), suggesting that Itk may play different roles in the development of CD4 + and CD8 + T cell lineage. Here, we report that a higher percentage of CD4 + T cells in Itk null mice have a CD62L Lo CD44 Hi memory phenotype (MP) and show effector function ex vivo in response to stimulation. Itk deficiency greatly decreases the number of CD4 + T cells with naïve phenotype (NP), but has no effect on the numbers of the MP CD4 + T cells. We also specifically show that active Itk signaling is required for these effects, and that Itk is required for function through the TCR signaling of these cells, but not for the innate function in response to IL-12/IL-18 or L. monocytogenes stimulation. These results indicate that CD62L Hi CD44 Lo naïve and CD62L Lo CD44 Hi memory CD4 + T cells may include independent populations that differ in their requirement for Itk s signals for development.

81 RESULTS Increased percentages of CD4 + CD62L Lo CD44 Hi T cells in Itk null mice We and others have recently shown that Itk is required for the development of conventional CD44 Lo naïve CD8 + T cells but not a CD44 Hi innate MP population of CD8 + T cells ( , 224). Similarly, Berg and co-workers have observed that there is a higher proportion of CD4 + T cells with a memory phenotype in mice lacking Itk (226). To determine if Itk affects the development of the CD4 + T cell lineage, we examined the CD4 + T cell lineages in the spleens of Itk -/- and WT mice by characterizing the expression of surface maturation markers, CD62L and CD44. We found that Itk -/- mice show a higher percentage of MP (for simplicity, we refer to cells carrying CD62L Lo, CD44 Hi or CD62L Lo CD44 Hi as MP cells since similar results were observed using these markers) CD4 + T cells (Figure 4-1a). We also found that CD4 + T cells with MP were present in the youngest mice (less than one month) and the increase persists out to more than 12 month of age (Figure 4-1b). These data indicated that Itk regulates the development of CD4 + T cell lineage by altering the ratio of NP and MP CD4 + T cells Homeostasis of naïve and MP CD4 + T cells does not contribute to the increased percentage of MP cells in Itk null mice Maintenance of the size of the lymphocyte pool is critical for proper immune responses. When naïve phenotype (NP, for simplicity, we refer to cells carrying CD62L Hi, CD44 Lo or CD62L Hi CD44 Lo as NP cells since similar results were observed using these markers) T cells are introduced into a lymphopenic compartment, they undergo

82 69 homeostatic expansion, converting to a phenotype that resembles MP cells (227). It is possible that Itk null mice have altered T cell homeostasis, leading to increased percentage of MP T cells. To test this hypothesis, the turnover of CD4 + T cells in Itk -/- mice and WT mice was determined by examining the CD4 + T cell incorporation of bromodeoxyuridine (BrdU). Itk -/- and WT mice were fed with BrdU-containing water for 9 days, after which their CD4 + T cells in the spleen were analyzed for incorporation of BrdU. We found that the CD4 + CD62L Hi T cells incorporated little BrdU during this process, and CD4 + CD62L Hi cells from Itk -/- mice incorporated slightly less BrdU compared to cells from WT mice (Figure 4-2a). We also found that amount of BrdU incorporated into CD4 + CD62L Lo cells from both Itk -/- and WT mice were similar (Figure 4-2a), indicating that a similar percentage of Itk -/- and WT CD4 + CD62L Lo cells were actively incorporating BrdU over this time period. These data suggest that Itk null T cells do not have increased proliferation in vivo. Indeed, transferring NP WT and Itk -/- CD4 + T cells into RAG -/- mice indicates that Itk -/- T cells actually exhibit reduced homeostatic expansion in this lymphopenic environment (data not shown). To determine if these cells have altered turnover in vivo, we also performed pulse-chase experiments using BrdU labeling since the latter is not reused. Itk -/- and WT mice were fed BrdU containing water for 9 days and then transferred to normal water to examine the rate of turnover of labeled MP CD4 + T cells. The data show that MP CD4 + T cells from both Itk -/- and WT mice showed similar kinetics of decay (Figure 4-2b). Altogether, these data suggest that the homeostasis of NP and MP CD4 + T cells does not contribute to the altered ratio of NP and MP CD4 + T cells in Itk -/- mice, although the homeostatic expansion and conversion of NP T cells to MP CD4 + T cells in a lymphopenic environment may be Itk dependent.

83 MP CD4 + T cells carry preformed message for IFNγ and rapidly secrete this cytokine upon stimulation with P/I Previously activated or memory CD4 + T cells have the ability to produce effector cytokines immediately ex vivo after stimulation. To determine if these MP CD4 + T cells exhibit effecter function ex vivo, we examined their ability to secrete IFNγ upon stimulation. We found that a large proportion of the CD4 + CD44 Hi population produce IFNγ in response to PMA and Ionomycin stimulation, while the CD4 + CD44 Lo population did not secrete any IFNγ during this period. CD4 + CD44 Hi T cells from Itk -/- mice behave similar to those from WT mice by rapid production of IFNγ, with a similar percentage of WT as well as Itk -/- CD4 + CD44 Hi T cells making this cytokine (Figure 4-3a). These results indicate that MP CD4 + T cells exhibit effector function to secrete effector cytokines ex vivo. We and others have found that MP CD8 + T cells with innate function rapidly secrete IFNγ upon stimulation since they carry large amounts of preformed message for this cytokine as well as the IFNγ regulator T-bet (189, 190, 224). The ability of CD4 + CD44 Hi but not CD4 + CD44 Lo T cells to rapidly produce IFNγ when stimulated suggests that these two subsets may differ in the expression of preformed IFNγ message. We therefore analyzed mrna from freshly isolated unstimulated CD4 + CD62L Lo and CD4 + CD62L Hi T cells from Itk -/- and WT mice for preformed mrna for IFNγ and T-bet by real-time quantitative RT-PCR. We found that CD4 + CD62L Lo T cells carry significantly higher levels of preformed message for IFNγ, as well as higher levels of the transcription factor T-bet compared to the CD4 + CD62L Hi T cells, although there was no difference between WT and Itk -/- CD4 + CD62L Lo T cells (Figure 4-3b). Analysis of mrna for

84 71 Eomesodermin, a T-bet related transcription factor also revealed both WT and Itk -/- CD4 + CD62L Lo cells expressed 3-4 fold more Eomesodermin than CD4 + CD62L Hi cells (data not shown). These data indicated that the MP CD4 + T cells have higher levels of T- bet and IFNγ transcripts, which may contribute to the ability of these cells to rapidly secrete IFNγ upon stimulation Phenotypic characterization of NP and MP CD4 + T cells in Itk -/- mice To further characterize these two T cell populations, we examined them for expression of a variety of surface markers. As shown in Figure 4-4, WT and Itk -/- CD4 + CD44 Hi CD62L Lo T cells expressed similar levels of CD122, CCR7 and CD127, suggesting that these two populations were the same in the two strains of mice. There were difference in expression of specific markers between NP and MP T cells in the expression of CD122, CCR7 and CD127. Of interest is that the MP, but not the NP subset also express low levels of NK1.1, the marker for NK and NKT cells, and Itk -/- mice have a smaller percentage of these cells than WT mice as previously suggested (228, 229)(data not shown). The small percentage of the MP CD4 + T cells that are NK1.1 or α-galcer/cd1d tetramer positive rule out the possibility that MP CD4 + T cells are NK or NKT cells, since these cells can also carry preformed message for IFNγ and rapidly secrete cytokine upon stimulation (229)(data not shown) MP CD4 + T cells can develop in the thymus Our data show that Itk -/- mice have increased percentage of MP CD4 + T cells. One potential explanation for these results is that T cells that develop in the thymus migrated

85 72 into a lymphopenia-like environment in the Itk -/- mice, during which they proliferated and up-regulated CD44 and down-regulated CD62L. This could result in the finding of higher percentages of MP CD4 + T cells in periphery of Itk -/- mice since a higher percentage of NP T cells would undergo lymphopenia-induced proliferation in these mice. However, we have already shown that Itk -/- T cells do not undergo increased homeostatic expansion. Another potential explanation is that these MP CD4 + T cells develop in the thymus and migrate out into the periphery, and that in the absence of Itk, more of these cells develop, or alternatively, less NP phenotype cells develop in the thymus resulting in the observed increased percentage of these cells in these mice. We therefore wanted to determine if these cells originated in the thymus during T cell development. To examine this issue, we first analyzed CD4 + T cells in newborn mice from birth through the first 1 week. We found that MP CD4 + T cells were present at 2 days after birth in WT and Itk -/- mice (Figure 4-5a). Furthermore, increased percentage of CD4 + CD44 hi CD62L low was detected in Itk -/- mice compared to WT controls. The percentage of MP CD4 + T cells decreased at 4 days and 7 days after birth in WT mice, while in Itk -/- mice, this percentage also decreased but remained elevated compared to WT mice. This suggests that MP CD4 + T cells develop in the thymus. We also characterized the expression of specific surface maturation markers that identify these cells. We found that CD4 + SP thymocytes in 6-8 week old Itk -/- mice exhibited a higher percentage of CD44 Hi and CD122 Hi populations than cells in WT mice, which suggests that Itk -/- mice contained higher percentage MP CD4 + SP thymocytes than WT mice (Figure 4-5b). As previously reported, almost all CD8 + SP thymocytes in Itk -/- mice exhibit a MP (CD44 Hi CD122 Hi ) ( , 224). To further confirm this, we evaluated

86 73 fetal thymic organ cultures (FTOC) from WT and Itk -/- mice where T cell development occurs in vitro, ruling out potential recirculation of already developed cells back into the thymus as would occur in the animal. Our results show that a higher percentage of CD4 SP T cells develop the CD44 Hi CD122 + phenotype in Itk null FTOC than in the WT FTOC (Figure 4-6). These data suggest that MP CD4 + and CD8 + T cells compartment differ in some aspects of their development since the percentage of the CD4 + compartment that had this memory phenotype was lower than that seen in the CD8 + compartment, but they have similar phenotypes. These data also suggest that the increased percentage of MP CD4 + T cells observed in the absence of Itk reflects either enhanced development of these cells, or reduced development of NP CD4 + T cells Altered CD4 + lineage development in the absence of Itk is intrinsic to bone marrow-derived cells To better understand whether the altered development of Itk -/- CD4 + T cells was due to defects intrinsic to the developing T cells, we generated bone marrow chimeric mice in which WT and Itk -/- bone marrow was injected into lethally irradiated WT congenic mice. After reconstitution, the percentage of CD4 + CD44 Hi CD62L Lo T cells in the spleen was clearly higher in mice reconstitution with Itk -/- bone marrow compared to those reconstituted with WT bone marrow (Figure 4-7a). However, when we compared the number of CD4 + CD44 Hi CD62L Lo T cells in the spleen, we found similar numbers regardless of whether the mice received WT or Itk -/- bone marrow (Figure 4-7b). To further determine if CD4 + CD44 Hi CD62L Lo MP T cells are indeed able to develop

87 74 independently of Itk expression, we performed competitive mixed bone marrow chimera analyses to determine if Itk null cells cam effectively compete with WT cells in the same host for development to these two cell populations. Our results confirm that while development of Itk null CD4 + CD44 Lo CD62L Hi NP T cells were reduced compared to their WT counterparts, development of CD4 + CD44 Hi CD62L Lo MP T cells was not affected and equal numbers of WT and Itk -/- cells developed (Figure 4-8a,b). This indicates that Itk is not required for the development of CD4 + CD44 Hi CD62L Lo MP T cells, but is required for the development of CD4 + CD44 Lo CD62L Hi NP T cells, and that the increased percentage observed in the absence of Itk is due to reduced development of the latter population. These data also suggest that these two populations of T cells are distinct and have distinct requirements for their development Tec kinase activity is required for the presence of NP CD4 + T cells, but not for the MP CD4 + T cells Our data shows that Itk regulates the development of CD4 + T cells by changing the ratio of NP and MP CD4 + T cells, and suggest that these represent unique and separate populations of CD4 + T cells. Our data also shows that their development is intrinsic to bone marrow-derived cells. To further determine if this process was intrinsic to the T cells and if the Tec kinase signaling was involved, we examined the percentage and absolute numbers of these two CD4 + T cell lineages in transgenic mice carrying Itk expressed in a T cell specific manner (driven by the CD2 promoter, Tg(CD2-hItk)Itk -/- mice), as well as transgenic mice carrying a mutant Itk lacking its kinase also expressed in a T cell specific manner (Tg(Lck-ItkΔKin)Itk -/- mice, (224)). Tg(CD2-hItk)Itk -/- mice

88 75 expressed low levels of Itk under the CD2 promoter (approximately 25-30%), however this was sufficient to significantly rescue the development of NP phenotype CD4 + T cells, but did not have any effect on the numbers of MP CD4 + T cells (Figure 4-9a,b). By contrast, analysis of the NP and MP CD4 + T cells in Tg(Lck-ItkΔKin)Itk -/- mice carrying the mutant Itk lacking its kinase domain instead of WT Itk revealed that these mice had NP and MP CD4 + T cells populations similar to those seen in Itk null mice (Figure 4-9a,b). These data indicate that active signaling by Itk enhances the development of NP CD4 + T cells, but has little effect on the development of MP CD4 + T cells Txk cannot replace Itk in the development of NP CD4 + T cells In chapter 3, we showed that Txk could partially suppress the increased percentage of memory phenotype CD8 + T cells. To determine if Txk can replace Itk for the presence of the memory phenotype CD4 + T cells, we analyzed the memory phenotype CD4 + T cells in Txk overexpressed Itk -/- mice. The data revealed that these mice had NP and MP CD4 + T cells populations similar to those seen in Itk null mice (Figure 4-10). These data indicate that Txk can t replace Itk in the development of CD4 + T cell subsets MP CD4 + T cells develop in MHC class II deficient recipients To better understand the requirements for selection and development of the altered CD4 T cells, we created bone marrow chimeras with WT and MHC Class II deficient hosts. MHC class II deficient mice fail to develop mature CD4 naïve phenotype T cells due to the lack of MHC class II expression on the selecting thymic epithelium. Transfer of WT and Itk -/- bone marrow into MHC class II -/- mice gave rise to very few

89 76 CD4 + CD44 Lo CD62L Hi NP T cells, while majority of CD4 + T cells that developed in MHC class II -/- hosts were of the CD44 Hi CD62L Lo MP phenotype (Figure 4-11a). These data indicate that the development of CD4 NP T cells requires the expression of MHC class II molecules on the thymic epithelium, while the development of CD4 + MP T cells is independent of MHC class II expression on the thymic epithelium. One of properties of these MP CD4 + T cells is that they can rapidly secret IFNγ after P/I stimulation. To determine if these MP CD4 + T cells that developed in MHC Class II -/- host are the same population observed in the WT and Itk -/- mice, we stimulated the splenocytes from recipient mice and examined IFN-γ secretion by the donor MP CD4 + T cell. The results showed that a large proportion of the donor CD4 + CD44 Hi population produce IFNγ in response to PMA and Ionomycin stimulation (Figure 4-11b). These results indicate that MP CD4 + T cells develop in the absence of thymic epithelia MHC Class II molecule expression exhibit effector function to secrete effector cytokines ex vivo The development of MP CD4 + T cells is dependent on bone marrow expression of MHC class II We have demonstrated that development of memory phenotype CD4 + T cells is independent of expression of MHC Class II on the thymic epithelium. Since the bone marrow chimeras still contain the MHC class II molecules expressed on hematopoietic cells, it remains unclear if these cells require MHC class II on hematopoietic cells for their selection. To address this question, we generated bone marrow chimeras that express MHC class II only on thymic stromal cells by transferring bone marrow cells from MHC class II -/- mice into WT mice. In this situation, MHC class II is only expressed

90 77 on the radio-resistant thymic stroma, not on hematopoietic cells. Analyses of these chimeras revealed that MP CD4 + T cells could not develop in mice that had received bone marrow from MHC class II deficient mice (Figure 4-12a). Although a small percentage of CD4 + T cells with apparent MP were observed, these cells could not response to PMA/Ionomycin stimulation, which is the main characteristic of these MP CD4 + T cells (Figure 4-12b). These data indicate that the development of MP CD4 + T cells is dependent on expression of MHC Class II molecule on hematopoietic cells. These data also suggest that these MP CD4 + T cells are only selected on MHC Class II on hematopoietic cells and not the thymic epithelium cells. By contrast the development of the NP CD4 + T cells is selected by MHC class II on thymic epithelium, not on hematopoietic cells The development of MP CD4 + T cells is thymus independent It is well accepted that the thymus is the organ that supports the activation, differentiation and selection of T cells, and that T cell development requires signals from nonhematopoietic stromal cells including various thymic epithelial cells and mesenchymal fibroblasts (4-6). It is also well known that precursors for conventional T cells arise from the hemotopoietic stem cells in the bone marrow and migrate to the thymus to mature (2, 3). We have shown that the requirements for development of MP CD4 + T cell are different from those needed for the development of NP CD4 + T cells. We have also shown that MP CD4 + T cells can develop in the thymus. However, it remains uncertain if these cells require the thymus for their development. To address this question, we generated bone marrow chimeras that lack thymi by transferring bone marrow cells from

91 78 WT mice into nude mice. In this situation, the recipient mice are athymic due to defective development of the thymic epithelium. Analyses of these chimeras revealed that both CD4 + and CD8 + T cells can develop in the recipient nude mice and all CD4 + T cells developed show memory phenotypes and could produce IFN-γ in response to PMA/Ionomycin stimulation (Figure 4-13a, b). These data indicate that the development of MP CD4 + T cells is thymus independent. These data also suggest that although the development of MP CD4 + T cells are thymus independent, the development of the NP CD4 + T cells is thymus dependent Itk is required for TCR induced but not innate signal induced elaboration of MP CD4 + effector function Itk regulates signals emanating from the T cell receptor (166, , ). To determine if the MP CD4 + T cells require Itk for their proliferation through the TCR, we analyzed purified CD4 + CD62L Lo and CD4 + CD62L Hi T cells for proliferative responses to anti-cd3 or anti-cd3/28 stimulation. The results show that both CD4 + CD62L low and CD4 + CD62L hi populations from Itk -/- mice had less proliferation in response to anti-cd3 and anti-cd3/28 stimulation (Figure 4-14), which indicated that both populations of cells are dependent on Itk for TCR induced proliferation. Similar results were found when we examined IFNγ secretion, with only WT CD4 + CD62L low, but not Itk -/- CD4 + CD62L low MP cells making this cytokine, although at much lower levels than seen with figure 4-3a (data not shown). A role for MP CD8 + T cells in the early innate response following infection with L. monocytogenes has been reported ( ). Infection of macrophages with L.

92 79 monocytogenes results in the secretion of IL-12 and IL-18, which together can induce the rapid secretion of IFNγ from MP CD8 + T cells. We have shown that MP CD8 + T cells respond to IL-12/IL-18 stimulation by rapidly secreting IFNγ, and can also respond to infection with L. monocytogenes by rapidly secreting IFNγ (224). We therefore determined if MP CD4 + T cells could also respond to IL-12/IL-18 stimulation to secrete IFNγ. Analysis of IFNγ production in cells from WT and Itk -/- mice revealed that MP CD4 + T cells produced significant levels of IFNγ, and similar percentages of WT and Itk null CD4 + CD44 hi cells responded (Figure 4-15a, note that PMA/Ionomycin stimulated cells, shown in Figure 4-3a, reveal similar responses). Analysis of MP CD4 + T cells revealed that these cells (and a similar percentage in WT and Itk -/- mice) could also rapidly secrete this IFNγ during infection with L. monocytogenes (Figure 4-15b). These data suggest that Itk is required for TCR stimulation of MP CD4 + T cells, but not for their development or elaboration of innate immune function.

93 DISCUSSION In this chapter, we show that development of a population of CD4 + T cells that carry memory markers CD44 Hi CD62L Lo are independent of Itk expression, while the naïve population of CD4 + T cells, CD44 Lo CD62L Hi, are dependent on Itk for their development. Our data also suggest that these MP CD4 + cells develop very early in the thymus, and are not dependent on active Itk kinase mediated signals. These MP CD4 + T cells carry preformed message for IFNγ and T-bet, and rapidly secrete this cytokine upon stimulation with IL-12 and IL-18. More importantly, this population rapidly secretes IFNγ upon infection with L. monocytogenes, suggesting that they may participate in the early innate immune response. These data suggest that these cells represent an apparently separate subpopulation of CD4 + cells from those with the naïve phenotype. The data in this chapter provides compelling evidence to support the existence of two independent subpopulations of CD4 + T cells: CD62L Hi CD44 Lo which are an apparent naïve phenotype and CD62L Lo CD44 Hi, which are an apparent memory phenotype. We use the term apparent as these markers have traditionally been used to refer to these two populations, however, it is clear that they include distinct populations that have different requirements for development. The CD4 + CD62L Hi CD44 Lo T cells resemble the NP T cell population, and lack preformed message for IFNγ and T-bet, and do not rapidly secrete IFNγ upon stimulation with P/I, IL-12/IL-18 or Listeria infection. This population is dependent on Itk expression and activity for its development. By contrast, the CD4 + CD62L Lo CD44 Hi T cell population develops in an Itk independent manner. Our data also shows that the development of these two populations of CD4 + T cells is T cell

94 81 intrinsic since, since Itk null bone marrow also gave rise to these two populations in a cell intrinsic manner when transferred into WT mice. The independence of these two cell populations is supported by the fact that they appear very early in T cell development. These two populations of cells were not significantly different in their expression of TCR Vβ 3, 5, 6 and 8, suggesting that they are not oligoclonally selected or expanded in vivo (data not shown), as in the case of NKT cells (230). These populations also carry different cell surface markers that separate them phenotypically. Some of these markers can be clearly tied to their function, such as the expression of CD122 in CD4 + CD44 Hi T cells, which allows responsiveness to IL-15 ( ). Indeed, IL-15 has been shown to be critical for the expansion of similar populations of CD4 + CD62L Lo CD44 Hi memory phenotype T cells, as well as in generating effective memory T cells following exposure to antigen (234, 235). These CD4 + CD62L Lo CD44 Hi T cells also express lower levels of the chemokine receptor CCR7, suggesting that their trafficking may different from the CD4 + CD62L Hi CD44 Lo naïve population which express higher levels of this receptor. In addition to their phenotypic characteristics, CD4 + CD62L Lo CD44 Hi T cells are distinct from CD4 + CD62L Hi CD44 Lo T cells in their effector functions, the CD4 + CD62L Lo CD44 Hi T cells resemble previously activated/memory T cells and produce IFNγ directly ex vivo in response to stimulation. These features are shared by some nonconventional T cell lineages that can develop effector function prior to antigen encounter such as NKT cells (236). We note that it is unlikely that the cytokine secretion response that we observed is due to NKT cells, since these latter cells comprise at most 10% of this MP CD4 + T cell population, but we get up to 45% of these MP CD4 + T cells secreting IFNγ upon

95 82 stimulation. In addition, the Itk -/- have reduced percentage and numbers of NKT cells (data not shown, (228, 229)). On the basis of these characteristics, CD62L Lo CD44 Hi CD4 + T cells should likely be included amongst these types of innate T cells, and we suggest the term innate memory phenotype CD4 + T cells. The CD4 + CD62L Lo CD44 Hi T cells with innate function are also distinct from the CD4 + CD62L Hi CD44 Lo T cells in that they have different intracellular signaling requirements for development. As we show here, Itk deficiency greatly decreases the number of CD4 + CD62L Hi CD44 Lo T cells, while having no effect on the number of CD4 + CD62L Lo CD44 Hi T cells, suggesting that Itk is not required for the development of the CD4 + CD62L Lo CD44 Hi T cells. Although the development of CD4 + CD62L Lo CD44 Hi T cells is Itk independent, our data also shows however, that Itk is still required for functional TCR signaling in these cells. Thus development and functional TCR activation of these cells have different signaling requirements. We and others have previously reported that the development of conventional CD8 + CD44 Lo CD122 Lo T cells is abolished in the absence of Itk ( , 224). Our data shows that the development of a similar population of CD4 + T cells is also affected in the absence of Itk, suggesting that Itk plays a critical role in the development of both CD8 + as well as CD4 + T cells that have a naïve CD44 Lo phenotype. We have also shown that the MP CD8 + T cells observed in the absence of Itk share the same properties with a population in normal WT mice, and more importantly can function in an innate manner to rapidly secrete IFNγ during infection with Listeria (224). Together, our data suggest that the memory phenotype CD44 Hi CD8 + and CD4 + T cell populations that develop in an Itk independent manner both seem to be able to function innately.

96 83 CD62L and CD44 are used as markers to distinguish naïve and memory T cell in many studies (237). The CD62L Lo CD44 Hi CD4 + T cells described in this chapter are defined as MP T cells and these cells can arise spontaneously in normal mice, which is different from the antigen-specific memory T cells generated by antigen administration (235). It is assumed that memory T cells having this phenotype are generated by antigen activation, expansion and differentiation, and that MP T cells found in normal mice reflect the fact that T cells are exposed to various environmental antigens leading to the development of these memory T cells (234, 235). However, our data suggest that at least some T cells with the CD4 + CD62L Lo CD44 Hi MP pool can be generated during thymic development and exist very early in the life of the animal. Our data therefore suggest that some or perhaps most CD4 + CD62L Lo CD44 Hi MP T cells are not descendants of naïve T cells that have responded to foreign antigens, but are a unique population of T cells that have innate function and behave like traditional memory T cells. It is possible that this T cell population develops in order to rapidly respond to antigen or innate signals until naïve T cells can differentiate and participate in the immune response.

97 84 Fig. 4-1 a) b) Figure 4-1: Increased percentage of CD4 + CD62L Lo CD44 Hi MP T cells in mice lacking Itk. (a) Spleen cells from WT and Itk -/- mice were stained for CD4, CD44, CD62L expression and analyzed by FACS. The percentages of CD4 + CD62L Lo CD44 Hi and CD4 + CD62L Hi CD44 Lo T cells in WT and Itk null mice are shown. (b) Splenocytes from WT and Itk -/- mice were analyzed for CD4 and CD62L expression over the indicated time frame. The percentage of CD4 + CD62L Lo population is shown (n=4-5, *p<0.05).

98 85 Fig. 4-2 a) b) Figure 4-2: Homeostasis of naïve and memory phenotype CD4 + T cells. (a) WT and Itk -/- mice were treated with BrdU containing water for 9 days, and splenocytes collected and analyzed for CD4 and CD62L expression along with analysis for BrdU. The percentage of BrdU + cells on gated CD4 + CD62L Hi or CD4 + CD62L Lo cells were analyzed. (b) WT and Itk -/- mice were treated with BrdU containing water for 9 days, and then placed on normal water for the indicated days prior to analysis of their splenocytes for BrdU incorporation in gated CD4 + CD62L Lo T cells as a percentage of total CD4 + T cells (n=3).

99 86 Fig. 4-3 a) b) Figure 4-3: MP CD4 + rapidly secrete IFNγ upon stimulation and carry high levels of preformed message for IFNγ and T-bet. (a) Splenocytes from WT and Itk -/- mice were stimulated with PMA/Ionomycin followed by analysis for intracellular IFNγ in gated CD4 + T cells (data representative of at least 3 experiments with the same result). (b) CD4 + CD62L Lo and CD4 + CD62L Hi T cells from WT and Itk -/- mice were sorted and mrna for IFNγ and T-bet were analyzed by Q-RT-PCR. Data are corrected for GAPDH expression and expressed as fold over the WT CD4 + CD62L Hi populations, which was set at 1 (n=3, *p<0.05, there was no statistical difference between WT and Itk -/- CD4 + CD62L Lo cells).

100 87 Fig. 4-4 Figure 4-4: Surface phenotype of CD4 + CD62L Lo CD44 Hi and CD62L Hi CD44 Lo T cells from WT and Itk -/- mice. Splenocytes from WT and Itk -/- mice were stained for CD4, CD62L, CD44 and the indicated surface markers. FACS profiles shown are gated on CD62L Lo CD44 Hi and CD62L Hi CD44 Lo CD4 + T cells (percentages were not statistically different between the two types of cells from WT and Itk -/- mice except NK1.1 and α- galcer/cd1d Tetramer staining in the CD62L Lo CD44 Hi cells (n=3). Data representative of 2 experiments with the same result).

101 88 Fig. 4-5 a) b) Figure 4-5: Memory phenotype of CD4SP thymocytes. (a) Splenocytes from WT and Itk -/- day 2, day 4 and day 7 old mice were stained for CD4, CD44 and CD62L and analyzed by FACS. The percentages of CD44 Hi CD62L Lo populations on gated CD4 + T cells were analyzed (n=4, *p<0.05). (b) Thymocytes harvested from 6-8 week old agematched WT and Itk -/- mice were stained for CD4, CD8 and CD44 and CD122 and analyzed by FACS. The histograms show gated CD4 + and CD8 + T cells. WT (solid line), Itk -/- (dashed line), Isotype staining control (Shaded). Arrow indicates increased percentage of CD4 SP CD44 Hi and CD4 SP CD122 Hi cells in Itk null thymus.

102 89 Fig. 4-6 Figure 4-6: MP CD4 + T cells can develop in the thymus. Fetal Thymic Organ Cultures from embryonic day 16 of WT and Itk null mice incubated in vitro for the indicated days and analyzed for CD4 and CD8 (top panel) or for CD44 and CD122 expression in the CD4 single positive population (data representative of 3-4 mice).

103 90 Fig. 4-7 a) b) Figure 4-7: Altered CD4 + T cell development in the absence of Itk is intrinsic to bone marrow-derived cells. (a) Bone marrow from Thy1.1 congenic WT and Itk -/- mice were injected into irradiated Thy1.2 WT mice, followed by analysis 8 weeks after reconstitution. The percentages of CD4 + CD44 Hi CD62L Lo and CD4 + CD44 Lo CD62L Hi populations of gated Thy1.1 + (donor) cells are shown (data representative of 6 mice with the same result). (b) The numbers of donor derived Thy1.1 + CD4 + CD44 Hi CD62L Lo and CD4 + CD44 Lo CD62L Hi T cells were determined and plotted (n=3, p<0.05).

104 91 Fig. 4-8 a) b) Figure 4-8: The development of memory phenotype CD4 + T cells is Itk independent. (a) A 1:1 mixture of bone marrow from Thy1.2/CD45.1 congenic WT and Thy1.2/CD45.2 congenic Itk -/- mice were injected into irradiated Thy1.1 WT mice, followed by analysis of donor derived T cells 6 weeks after reconstitution. The percentages of CD44 Hi populations of gated donor WT and Itk null CD4 + cells are shown (data representative of 7 mice with the same result). (b) The numbers of donor derived WT or Itk null CD4 + CD44 Hi CD62L Lo and CD4 + CD44 Lo CD62L Hi T cells were determined and plotted (n=3, repeated twice, *p<0.05).

105 92 Fig. 4-9 a) b) Figure 4-9: The development of MP CD4 + but not NP CD4 + T cells is independent of active Itk signaling. (a) Spleens from WT, Itk -/-, Tg(CD2-Itk tg )Itk -/-, and Tg(Lck- ItkΔKin)Itk -/- mice were stained for CD4, CD44, CD62L expression and analyzed by FACS, gating on CD4 + populations. The percentages of CD4 + CD62L Lo CD44 Hi and CD4 + CD62L Hi CD44 Lo populations are shown (data representative of 6 mice with the same result). (b) Total numbers of splenic CD4 + CD62L Lo CD44 Hi and CD4 + CD62L Hi CD44 Lo T cell populations from the same mice as Figure 6a. (n=3, *p<0.05 vs. Itk -/- CD4 + CD62L Hi CD44 Lo T cells; **p<0.05 vs. Itk -/- CD4 + CD62L Hi CD44 Lo T cells).

106 93 Fig Figure 4-10: Txk cannot replace Itk in the development of NP CD4 + T cells. Spleens from WT, Itk -/-, Tg(CD2-Itk tg )Itk -/-, and Txk tg /Itk -/- mice were stained for CD4, CD44, CD62L expression and analyzed by FACS, gating on CD4 + populations. The percentages of CD4 + CD62L Lo CD44 Hi and CD4 + CD62L Hi CD44 Lo populations are shown (data representative of 6 mice with the same result).

107 94 Fig a) b) Figure 4-11: The development of MP CD4 + T cells is independent of MHC Class II expression on the thymic epithelium. a) Bone marrow from Thy 1.1 congenic WT mice were injected into irradiated Thy 1.2 WT or MHC Class II -/- mice, followed by analysis 8 weeks after reconstitution. The percentages of CD4 + CD44 Hi CD62L Lo MP and CD4 + CD44 Lo CD62L Hi NP populations of gated Thy1.1 + (donor) cells are shown (data representative of 6 mice with the same result). (b) Splenocytes from recipient mice were stimulated with PMA/Ionomycin for 6 hours and followed by analysis for intracellular IFNγ on gated donor CD4 + T cells. Percentages shown indicate those MP CD4 + T cells secreting IFNγ upon stimulation (data representative of 6 mice with the same result).

108 95 Fig a) b) Figure 4-12: The development of MP CD4 + T cells is dependent on expression of MHC Class II on bone marrow derived cells. a) Bone marrow from Thy 1.2 congenic WT and Class II -/- mice were injected into irradiated Thy 1.1 WT mice, followed by analysis 8 weeks after reconstitution. The percentages of CD4 + CD44 Hi CD62L Lo MP and CD4 + CD44 Lo CD62L Hi NP populations of gated Thy1.2 + (donor) cells are shown (data representative of 6 mice with the same result).(b) Splenocytes from recipient mice were stimulated with PMA/Ionomycin for 6 hours and followed by analysis for intracellular IFNγ on gated donor CD4 + T cells. Percentages shown indicate those MP CD4 + T cells secreting IFNγ upon stimulation (data representative of 6 mice with the same result).

109 96 Fig a) b) Figure 4-13: The development of MP CD4 + T cells is thymus independent. a) Bone marrow from CD45.1 congenic WT mice were injected into irradiated CD45.2 WT and Nude mice, followed by analysis 8 weeks after reconstitution. The percentages of CD4 + and CD8 + T cells of gated CD (donor) cells are shown (left). CD4 + CD44 Hi CD62L Lo MP and CD4 + CD44 Lo CD62L Hi NP populations of gated CD (donor) cells are on the right (data representative of 4 mice with the same result).(b) Splenocytes from recipient mice were stimulated with PMA/Ionomycin for 6 hours and followed by analysis for intracellular IFNγ on gated donor CD4 + T cells. Percentages shown indicate those MP CD4 + T cells secreting IFNγ upon stimulation (data representative of 4 mice with the same result).

110 97 Fig a) b) Figure 4-14: TCR activation of MP CD4 + TCR is Itk dependent. (a) CD4 + CD62L Lo MP and CD4 + CD62L Hi NP T cells from WT or Itk -/- mice were stimulated with anti-cd3 for 3 days. Thymidine uptake was determined as a measure of proliferation in the last 18 hrs (n=3, *p<0.05). (b) CD4 + CD62L Lo MP and CD4 + CD62L Hi NP T cells from WT or Itk -/- mice were stimulated with anti-cd3/28 for 3 days. Thymidine uptake was determined as a measure of proliferation in the last 18 hrs (n=3, *p<0.05).

111 98 Fig a) b) Figure Innate activation of MP CD4 + TCR is Itk independent. (a) Splenocytes from WT and Itk -/- mice were stimulated with IL-12 and IL-18 followed by analysis for intracellular IFNγ on gated CD4 + T cells. Percentages shown indicate those MP CD4 + T cells secreting IFNγ upon stimulation (data representative of 6 mice with the same result). (b) WT and Itk -/- mice were infected with CFU L. monocytogenes. Twenty-four hrs later, splenocytes were harvested and CD4 + CD44 Hi T cells were analyzed for intracellular IFNγ. Percentages shown indicate those MP CD4 + T cells secreting IFNγ upon infection (data representative of 6 mice with the same result).

112 99 Chapter 5 Itk derived signals regulate the expression of Th-POK and controls CD4/CD8 lineage decisions

113 INTRODUCTION The development of mature T cells takes place in the thymus and is critically dependent on both the environment and signals through the TCR. During the T cell development, TCR signals generated by interaction with major histocompatibility complex class (MHC)-II peptide complexes are required for differentiation of CD4 + T cells, while TCR signals generated by interaction with class I MHC-peptide complexes are required for differentiation of CD8 + T cells. This process, referred as CD4 and CD8 commitment, is a major developmental process after positive and negative selection. The signals that determine this lineage commitment are not completely understood, but a role for TCR signaling in CD4/CD8 lineage commitment is supported by a body of experimental evidence (22-24). These observations suggest that high signaling activity generated by the tyrosine kinase Lck or MAP kinases ERK1/2 enhances CD4 SP development (159, 160), while low activity of Lck, ZAP70 or ERK1/2 leads to CD8 SP development ( ). These findings support the idea that attenuating TCR signaling could redirect thymocytes with class II restricted TCRs from CD4 to the CD8 lineage, while the enhanced signaling could redirect thymocytes with class I specific TCRs from CD8 to CD4 lineage. Other evidence indicates that the duration of signaling as well as the number of TCRs triggered are key factors in determining CD4/CD8 T cell fate decision (27, 28). A role for TCR affinity has not been implicated in these events, but may play a role dependent on whether TCR affinity affects these implicated signaling events. The IL-2-inducible T cell kinase (Itk ), a Tec family kinase, regulates the TCR signals and is considered as an amplifier of these signals in T cells (131, 135, 136, 138, 238). Itk is directly activated by Lck and in turn plays a role in the activation of PLCγ1,

114 101 Ca 2+ mobilization and activation of ERK/MAPK (138). Itk-deficient mice had decreased numbers of mature thymocytes (136). The loss of Itk may also affect most stages of thymopoiesis, including pre-tcr signaling, as well as positive and negative selection (124, 146, 154). Based on these observations and the important roles of other TCR signaling proteins such as Lck and ZAP70 in the CD4/CD8 commitment, it seems likely that the Itk may also influence CD4/CD8 commitment(155). By contrast Berg and colleagues have examined this issue by crossing Itk deficient mice to mice carrying the AND MHC class II restricted TCR, along with different MHCs that had varying affinities, and observed no difference on the development of CD4 or CD8 SP thymocytes, suggesting that the Itk signaling is not involved in the lineage commitment (154). However, they did not rule out the possibility that TCR signal strength in the AND TCR transgenic system may be above a certain threshold, and even in the absence of Itk, the signal may still be above the threshold that influences CD4 and CD8 lineage commitment. We and others have recently reported that large majority of CD8 + T cells in mice lacking Itk have the characteristics of memory cells and innate immune cells (109, ). These cells have been referred to as non-conventional CD8 + T cells or innate memory phenotype cells. By contrast, cells carrying the surface phenotype of naïve CD8 + T cells have the characteristics of conventional CD8 + T cells, and are drastically reduced in the absence of Itk. These data suggest that signals regulated by Itk may regulate the development of specific subpopulations of CD8 + T cells. However, it is not clear if these CD8 + T cells develop because they are destined to become CD8 + T cells, and the Itk signals regulate whether they develop into conventional or non-conventional CD8 + T

115 102 cells, or whether Itk signals regulate the development of these cells regardless of their initial cell fate choice. Here, we have analyzed mice lacking Itk and carrying a low affinity TCR transgene specific for a class II restricted peptide derived from Ovalbumin (OVA, OT-II mice), to determine if cells destined to become class II restricted CD4 + T cells are affected by the absence of Itk (243). Our results show that thymocytes in these OT-II/Itk -/- mice receive reduced TCR signals, with subsequent development of significant numbers of peptide specific MHC class II restricted TCR transgenic CD8 + T cells. Furthermore, a large majority of these cells have the characteristics of nonconventional or innate memory phenotype CD8 + T cells. These data indicate that the absence of Itk allows the development of non-conventional CD8 + T cells even if they carry a class II restricted TCR, and suggest that the development of these cells is regulated in part by altered signals from this low affinity TCR. This altered development in the absence of Itk may be a consequence of reduced TCR signaling, resulting in reduced expression of the transcription factor, Th-POK, a master regulator of CD4 commitment, with accompanying changes in cell fate decision (43-45).

116 RESULTS The absence of Itk results in reduced development of OT-II transgenic CD4 + T cells and enhanced development of OT-II transgenic CD8 + T cells To determine if reduced TCR signals due to the absence of Itk can influence the development of cells into CD8 lineage, we crossed Itk -/- mice to TCR transgenic OT-II mice. OT-II mice carry a transgenic αβ TCR (Vα2, Vβ5) that recognizes ovalbumin in the context of MHC class II I-A b (243). Greater than 95% of TCR transgene positive T cells are CD4 + T cells (Figure 5-1, (243)). However, the absence of Itk in OT- II mice dramatically blocked the development of CD4 single-positive (SP) thymocytes and lead to the development of a significant number of CD8SP thymocytes that were positive for the transgenic TCR (Figure 5-1a, Figure 5-2). In the lymph node, the ratio of the CD4/CD8 that was TCR transgene positive was changed from more than 50 to 2.5 in the OT-II/Itk -/- mice (Figure 5-1b, Figure 5-2). These data suggest that Itk may be essential for the development of OT-II thymocytes into the CD4 lineage, and that CD8 lineage development is more likely to occur in the absence of Itk. Although the OT-II/Itk -/- mice showed a 3-4 fold reduction in total thymocytes that were TCR transgene positive compared to OT-II mice (data not shown), the absolute number of TCR transgene positive CD8SP thymocytes was increased 2-3 fold, while that of CD4 SP thymocytes was reduced 5-6 fold (Figure 5-2b). This reduction in TCR transgene positive CD4 + T cells was more exaggerated in the lymph node, where we observed a fold difference. This could be the result of reduced homeostatic expansion in the absence of Itk after T cells have migrated to periphery, which was observed in the non-

117 104 transgenic mice when naïve T cells from Itk -/- mice were transferred into RAG -/- mice (unpublished data). In addition, the increase in TCR transgene positive CD8 + T cells was less pronounced in the lymph node, with a ~1.5 increase compared to WT OT-II mice (similar results were seen in the spleen) Reduced TCR signaling in the absence of Itk during T cell development Previous analysis of Itk -/- mice suggests that T cells from these mice receive weak signals through their TCR, and that Itk may act as an amplifier of T cell receptor signals (131). CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the signaling intensity received by developing thymocytes (244). Indeed, as shown in figure 5-3a, CD5 expression was significantly lower in non-transgenic total DP thymocytes from Itk -/- mice compared to WT mice, while CD5 levels were only slightly reduced in the TCR hi DP thymocytes, CD4SP and CD8SP thymocytes. These data confirmed that developing DP thymocytes receive weak signals from the TCR in the absence of Itk. To test if T cells from OT-II/Itk -/- also receive weak signals, we analyzed the surface marker CD5 on the OT-II and OT-II/Itk -/- mice thymocytes. CD5 expression on DP and SP cells from the TCR transgenic OT-II and OT-II/Itk -/- mice and similar to non-tcr transgenic mice, thymocytes from transgenic mice lacking Itk had lower levels of CD5 on their total DP thymocytes, and slightly lower CD5 on transgenic TCR hi CD8SP thymocytes. However, unlike the non-tcr transgenic Itk -/- mice, transgenic TCR hi DP and CD4SP thymocytes from OT-II/Itk -/- mice continued to show significantly reduced levels of CD5 expression (Figure 5-3). These data indicates that the strength of the signal through the TCR is reduced in the absence of Itk, that this reduction is more

118 105 severe if the thymocytes carry a low affinity TCR such as the OT-II TCR, and that the CD4 population receive weaker signals than the CD8 population. Alternatively, it is possible that DP thymocytes that receive weak signals can differentiate into CD8SP T cells, while CD4 SP development requires stronger signals Normal survival of CD4SP and CD8SP TCR transgenic thymocytes in the absence of Itk OT-II/Itk -/- mice showed altered CD4 and CD8 ratios and numbers in both thymus and periphery compared with OT-II mice. We therefore determined if the absence of Itk alters the survival of CD4 + thymocytes compared to CD8 + thymocytes. Since Bcl-2 is a prominent survival factor that has been implicated in preventing programmed cell death in T cells, and is required for prolonged lymphocyte survival following maturation (245), we examined the expression of Bcl-2 in thymocytes from OT-II and OT-II/Itk -/- mice. We found that the levels of Bcl-2 was low in total OT-II DP thymocytes, and was upregulated in TCR hi DP thymocytes, remaining high in TCR hi CD4 and CD8 SP thymocytes (Figure 5-4a). Similarly, Bcl-2 was up regulated in transgenic TCR hi DP and SP thymocytes from OT-II/Itk -/- mice to the same levels as that seen in OT-II thymocytes. These data indicate that OT-II/Itk -/- thymocytes may have similar ability to survive as OT-II thymocytes. This conclusion was confirmed by analysis of Annexin V/7AAD staining of these thymocyte populations (Figure 5-4b) Transgenic TCR hi CD8 + T cells respond to antigen specific stimulation to make IFN-γ

119 106 As shown in figure 5-1, transgenic DP thymocytes developed into transgenic TCR hi CD8 + T cells in the absence of Itk, and we wanted to determine if these transgenic TCR hi CD8 + T cells could respond to the MHC class II restricted antigen that is recognized by the transgenic TCR (OVA peptide). Splenocytes from OT-II and OT-II/Itk -/- mice were therefore cultured in the presence of the OVA peptide (total splenocytes were used due to the difficulty of isolating the small number of TCR hi CD8 + T cells from these TCR transgenic mice). After 7 days of culture cytokine secretion from the transgenic TCR hi CD8 + cells was analyzed. The transgenic TCR hi CD8 + cells from OT-II/Itk -/- mice, but not the cells from OT-II mice, produced IFN-γ after MHC class II-restricted OVA stimulation (Figure 5-5). These data indicate that the transgenic TCR hi CD8 + T cells that developed in OT-II/Itk -/- mice are MHC class-ii restricted and can be activated by class II-restricted peptide The development of transgenic TCR hi thymocytes into CD8SP cells is bone marrow cell intrinsic To ensure that the development of transgenic TCR hi class II-restricted CD8 SP cells is cell intrinsic and not affected by other environmental factors in the Itk null mice, we generated bone marrow chimeric mice in which OT-II and OT-II/ITK -/- (CD45.2) bone marrow was injected into lethally irradiated WT (CD45.1) congenic mice. After 12 weeks of reconstitution, the percentage of donor derived TCR hi transgenic CD8 SP cells in the thymus was clearly higher in mice reconstituted with OT-II/Itk -/- bone marrow compared to those reconstituted with OT-II bone marrow (Figure 5-6). We also observed that the donor TCR hi transgenic CD4 SP thymocytes were much lower in OT-II/Itk -/-

120 107 reconstituted chimeric mice. These data suggest in the absence of Itk, thymocytes destined to become MHC class- II CD4 SP T cells can develop into MHC class II restricted CD8 SP cells, and that this is cell intrinsic Transgenic TCR hi CD8 + T cells that develop in OT-II/Itk -/- mice have a memory phenotype and innate function We and others have recently shown that CD8 + T cells develop in Itk -/- mice have a memory phenotype and show innate function (239, 240, 242). To determine if the transgenic TCR hi CD8 + T cells developed in OT-II/Itk -/- have this phenotype, we examined the transgenic TCR hi CD8 + thymocytes and TCR hi CD8 + T cells in the spleens of OT-II/Itk -/- for expression of various markers that differentiate these cells from regular CD8 + T cells. Our results show that TCR hi CD8 + SP thymocytes from OT-II/Itk -/- mice show phenotypes similar to CD8 + SP thymocytes of non-transgenic Itk -/- mice, including low levels of HSA, and high levels of CD44 and CD122, not seen in CD8 SP thymocytes from WT mice (Figure 5-7a,b). To determine if these memory phenotype CD8 + T cells from OT-II/Itk -/- mice exhibit similar functional behavior to CD8 + T cells with memory phenotype (characterized by high levels of CD44 ((242)) from WT and Itk -/- mice in vitro, we analyzed IFN-γ production following PMA/Ionomycin stimulation of these cells. The experiments revealed that the OT-II/Itk -/- CD8 + memory phenotype T cells behave similar to the equivalent populations from WT and Itk -/- mice in rapidly producing IFN-γ (Figure 5-8). These data indicate that the transgenic TCR hi CD8 + T cells that develop in OT-II/Itk - /- mice have a memory phenotype and innate function in rapidly producing IFNγ after stimulation.

121 Down-regulation of the CD4 lineage commitment factor, Th-POK, in Itk null DP thymocytes A large body to evidence supports the view that the amount of TCR signaling determines the lineage decisions for development of the CD4 and CD8 T cells, and the nature of the signals that affect changes in gene expression that specify the CD4/CD8 T cell lineage commitment has become a major goal of research in this area. Several transcription factors essential for CD4/CD8 T cell lineage commitment have been identified, including the Runx factors, as well as TOX, GATA3 and Th-POK (33, 34, 36-38, 40, 41). Among these transcription factors, Th-POK has been identified as a master regulator of CD4 commitment (43-45). Th-POK (T help inducing POK factor), a Zn finger transcription factor, is expressed specifically in the CD4 lineage and enforced constitutive expression of Th-POK not only restores normal development of MHC class II-restricted T cells to the CD4 lineage but also causes redirection of MHC class I-restricted cells to the CD4 lineage (45, 46). Notably, however, signals that regulate the expression of Th-POK and thus CD4 lineage commitment are less clear. Our data suggest that the absence of Itk affects the strength of signal through the TCR and affects the commitment of DP thymocytes to CD4 and CD8 in an environment of enforced MHC class II-restriction. To determine if these reduced signals regulate the transcription factors that modulate CD4/CD8 lineage commitment, we analyzed sorted transgenic TCR hi DP thymocytes from OT-II and OT-II/Itk -/- mice and determined the expression levels of different transcription factors using quantitative real-time RT-PCR. As shown in Figure 5-9, Th- POK expression was significantly lower in OT-II/Itk -/- mice compared with those from OT-II mice. Given the role of this factor in regulating the development of CD4 SP

122 109 thymocytes, these data suggest that signals regulated by Itk may regulate the expression of Th-POK, and thus CD4 T cell development. We also analyzed the levels of Runx3, which has been shown to bind to the CD8 enhancer and contribute to CD8 expression in CD8SP and mature CD8 lineage T cells (36). Figure 5-9 shows that the level of Runx3 is higher in OT-II/Itk -/- DP thymocytes compared with cells from OT-II, which is consistent with previous reports that Th-POK expression and genetic programming for CD4 T cell development is inhibited by Runx-dependent silencer activity (246). Similarly, OT-II/Itk - /- DP thymocytes expressed significantly lower levels of GATA3, another factor that has been shown to regulate CD4 T cell development (40, 41). By contrast, no significant difference was observed in levels of TOX, although TOX has been suggested to regulate CD8 commitment.

123 DISCUSSION This study was designed to examine whether CD4/CD8 lineage choices are affected by the reduced TCR signals in the absence of Itk. We analyzed Itk -/- mice crossed to mice carrying the low affinity MHC class II-restricted transgenic TCR (OT-II). Our results show that class II-restricted OT-II T cells destined to develop into CD4 + T cells can develop into CD8 T cells in the absence of Itk, indicating the important role of Itk in the CD4 and CD8 lineage commitment. Our work also suggest that in the absence of Itk, non-conventional or innate memory phenotype CD8 + T cells develop despite the presence of a MHC class II restricted transgenic TCR that normally drives CD4 + T cell development. Finally, our work suggests that this choice of CD8 + T cell development is controlled in part by the expression of the CD4 T cell master regulator Th-POK, whose expression is regulated by Itk derived signals. Berg and colleagues have previously used a TCR transgenic approach to analyze the role of Itk in CD4 and CD8 T cell commitment (154). They analyzed three different lines of MHC class II-restricted TCR transgenics, 2B4, 5C.C7 and AND, all lacking Itk and surprisingly, found no significant difference in the number and development of CD4 + or CD8 + T cells in these systems. One potential explanation for this difference between our findings and the findings of Berg et al is that the affinity of the TCRs analyzed (2B4, 5C.C7 and AND) are higher than that of the OT-II transgenic system, and even in the absence of Itk, the signal strength may be high enough for specific thresholds to be reached for CD4 lineage commitment. Similarly, when Lck was analyzed for its role in CD4/CD8 commitment, Nakayama and colleagues did not detect altered CD4 and CD8

124 111 generation in the absence of active Lck using the DO11.10 MHC class II-restricted TCR. Note that while DO11.10 TCR recognizes the same peptide as that recognized by OT-II TCR, the restriction element is H-2 d, and the affinity of DO11.10 TCR for the OVA peptide is more than 1000 fold higher than that of the OT-II TCR (247). However, Alerola-Ila and colleagues showed that functional CD8 + T cells develop in mice transgenic for the MHC class II-restricted AND TCR when Lck activity was reduced (156, 160). We and others have previously shown that Itk is required for the development of conventional CD8 + T cells and in its absence, the CD8 + T cells that develop have a memory phenotype and exhibit innate function (239, 240, 242). In this chapter, we show that the memory phenotype CD8 + T cells can still develop even in the presence of a fixed MHC class II restricted TCR transgene that drives CD4 + T cell development, suggesting that the development of these CD8 + MP cells may be a consequence of impaired TCR signaling. Indeed, MHC class I restricted OT-1/Itk -/- mice develop normal conventional CD8 + T cell due to efficient selection in the thymus of T cells carrying this transgenic TCR ((240) and data not shown). Alberola-Ila and colleagues previous work on the influence of Lck and Ras/MAP kinase pathways on CD4 and CD8 T cell commitment suggested that MHC class II-restricted CD8 + T cells that develop in an environment of reduced Lck signals behave as competent cytotoxic T cells, able to kill target cells (160). It was not determined whether the CD8 + T cells that developed were all conventional CD8 T cells. The CD8 + T cells that develop in OT-II/Itk -/- mice also express high levels of perforin and granzyme B, and can degranulate following stimulation with the MHC class II-restricted peptide stimulation (data not shown), all characteristics of cytotoxic

125 112 CD8 + T cells. It is possible that the CD8 + T cells that develop under conditions of reduced Lck activity are also innate memory phenotype cells. Our analysis of genes that regulate the expression of CD4 and CD8 lineage commitment in double positive thymocytes revealed that Itk mediated signals regulate the expression of Th-POK, which has been identified as a master regulator of CD4 T cell commitment [24-26]. Similarly, the expression of GATA3, another regulator of CD4 T cell commitment was significantly reduced in TCR transgenic DP thymocytes [34, 35]. However, the expression of TOX, a regulator of CD8 T cell commitment was not altered. These data suggest that perhaps, there is an absolute amount of signal that is required to induce or maintain the expression of Th-POK, following which CD4 lineage commitment is fixed. However, in the presence of a low affinity TCR and reduced signals in the absence of Itk, Th-POK expression is reduced. This results in a default differentiation program to CD8SP cells, since TOX expression is not changed. In addition, since Itk derived signals are also required for the development of conventional CD8SP T cells, CD8 T cells can develop in the OT-II/Itk -/- background even when they carry the MHC class II restricted TCR, and they develop into non-conventional or innate memory CD8 + T cells. Although the discovery of Th-POK as a master regulator of CD4 lineage commitment represented a huge advance in our understanding of CD4 T cell development, signals that regulate the expression of the factor are unknown. Our work reported here shows that Itk plays a role in regulating this factor, and in its absence CD8 + T cells can develop, perhaps as a default pathway due to continued TOX expression. In addition, our work connects signals emanating from the TCR, via Itk, and the expression of specific genes that

126 113 regulate lineage commitment, suggesting a possible mechanism by which TCR signals can regulate CD4/CD8 commitment. High levels of Lck and ERK MAPK has been shown to lead to enhanced CD4 T cell development to the detriment of CD8 T cell development (159, 160), while reduced activity of Lck, ZAP70 and ERK leads to reduced CD4 T cell development, leaving CD8 T cell development intact ( ). Our data here suggest that a Lck-Itk-MAP kinase signaling pathway in double positive thymocytes modulate the development of CD4 + T cells, with perhaps default development of CD8 T cells (with of course consequences for the development of conventional vs. non-conventional or innate memory phenotype CD8 T cell development). Our data also suggest that components of the TCR signaling pathway may affect the lineage choices of T cells dependent on the affinity of the TCR. Final decisions on lineage choice may thus be determined by the combination of TCR affinity and strength of the TCR signals with absolute levels of signaling determining lineage commitment choices.

127 114 Fig. 5-1 a) b) Figure 5-1: Development of MHC class II restricted TCR hi CD8SP cells in a CD4/MHC-II restricted environment in the absence of Itk. Thymocytes (a) and LN cells (b) from OT-II and OT-II/Itk -/- mice were stained for CD4, CD8α and TCRVβ5 expression. Cells were analyzed by flow cytometry as described in experimental methods. The percentages of CD4 + and CD8 + cells of total and transgenic TCR hi cells are shown. A minimum of 10 mice of each OT-II and OT-II/Itk -/ weeks of age were analyzed, and representative flow profiles are shown.

128 115 Fig. 5-2 a) b) Figure 5-2: The percentage and absolute number of MHC class II restricted TCR hi SP cells. (a) The percentage of transgenic TCR hi CD4 + and transgenic TCR hi CD8 + T cells in thymus and lymph nodes from OT-II and OT-II/Itk -/- mice are presented. Values are the mean of 3-5 animals for each genotype and represent 3-4 experiments. (b) The absolute numbers of transgenic TCR hi CD4 + and CD8 + T cells in thymus and lymph nodes are presented from the same mice analyzed in (a).

129 116 Fig. 5-3 a) b) Figure 5-3: The strength of the TCR signal is reduced in the absence of Itk. Thymocytes from non-transgenic WT, Itk -/- mice (a), or TCR transgenic OT-II and OT- II/Itk -/- mice (b) were stained with CD4, CD8α, TCRVβ5 and CD5. Histograms of CD5 expression on DP, transgenic TCR hi DP, transgenic TCR hi CD4SP and transgenic TCR hi CD8SP from WT (solid line), Itk -/- (dashed line), OT-II (solid line) and OT-II/Itk -/- (dashed line) mice are shown. A minimum of 6 mice of each genotype with 6-12 weeks of age were analyzed, and representative flow profiles are shown.

130 117 Fig. 5-4 a) b) Figure 5-4: Normal survival of TCR transgenic CD4 and CD8 SP thymocytes in the absence of Itk. (a) Thymocytes from TCR transgenic OT-II and OT-II/Itk -/- were stained with CD4, CD8α, TCRVβ5 and intracellular Bcl-2. Histograms of Bcl-2 expression on DP, transgenic TCR hi DP, transgenic TCR hi CD4SP and transgenic TCR hi CD8SP from OT-II (solid line) and OT-II/Itk -/- (dashed line) are shown (filled histogram: nonspecific isotope staining). A minimum of 10 mice of each genotype with 6-12 weeks of age were analyzed, and representative flow profiles are shown. (b) Thymocytes from transgenic OT-II and OT-II/Itk -/- were stained with CD4, CD8α and TCRVβ5, along with 7AAD and Annexin V. Histograms of Annexin V expression on 7AAD - DP, 7AAD - transgenic TCR hi DP, CD4SP and CD8SP are shown. The percentage of Annexin V + cells for each subset is presented. A minimum of 4 mice of each genotype with 6-12 weeks of age were analyzed, and representative flow diagrams are shown.

131 118 Fig. 5-5 Figure 5-5: Transgenic TCR hi CD8 + T cells produce IFN-γ upon MHC class IIrestricted OVA-peptide stimulation. Total splenocytes were cultured in the presence of OVA peptide for 7 days, followed by restimulation with OVA peptide, and IFNγ and IL- 4 production by transgenic TCR hi CD8 + T cells analyzed as described in the experimental methods section. The percentage of TCR hi CD8 + T cells producing IFN-γ and IL-4 is shown. The data was representation of 2 independent experiments.

132 119 Fig. 5-6 Figure 5-6: Development of MHC class II-TCR restricted thymocytes to CD8SP lineage in the absence of Itk is cell intrinsic. Bone marrow cells from transgenic OT-II and OT-II/Itk -/- mice (both CD45.2) were reconstituted into irradiated non-transgenic CD45.1 WT mice. Thymocytes were analyzed 12 weeks after reconstitution. The percentages of donor derived (CD ) CD4 + SP and CD8 + SP cells or donor derived TCR hi cells are shown. A minimum of 6 mice of each genotype were analyzed, and representative flow cytograms are shown.