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The role of Cav1.4 calcium channel in T cell activation, proliferation, effector functions and death Wang, Teresa Yi Wen 2010

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The Role of Cavi .4 Calcium Channel in T Cell Activation, Proliferation, Effector Functions and Death by Teresa Yi Wen Wang B. Sci. Hon., The University of Toronto, Saint George Campus, 2006  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2010 ©Teresa Yi Wen Wang, 2010  ABSTRACT T lymphocytes are an important part of the immune system that identify and destroy foreign antigens in the body as well as activate and deactivate other immune cells. In T lymphocytes, calcium is a secondary messenger that regulates activation and proliferation, effector function, survival and death. Although calcium release from the intracellular stores within T lymphocytes is well characterized, the calcium entry pathway from extracellular sources into T lymphocytes is unclear, despite contributing to the majority of elevated intracellular calcium ions during T lymphocyte activation. Preliminary studies have shown that L-type calcium channels play significant roles in the calcium influx pathways, mediating T lymphocyte activation and proliferation in vitro. Cavi .4 L-type calcium channel has been found to be expressed in both mouse and human T lymphocytes. To date, three Cay 1.4 calcium channel splice variants have been identified with differential expression throughout the T lymphocyte proliferation process.  I hypothesized that pore forming subunit of a L-type calcium channel, Cavl.4, regulates T lymphocyte activation and proliferation in vivo. To test this hypothesis, I used loss of function L type calcium channel knock-out (KO) mice lacking the entire gene coding for this calcium channel and its splice variants to study its effects on T cell activation, proliferation, death, calcium uptake, and effector responses. From these studies, I predict that the lack of L-type calcium channels will cause T lymphocyte activation, proliferation and development to be impaired. These studies shed light on the mechanism of T lymphocyte activation and also enabled us to better understand how antagonistic drugs such as nifedipine may cause immunosuppressive effects.  1%  TABLE OF CONTENTS. Abstract  .  ii  Table of Contents  iii  List of Diagrams  V  List of Figures  v  List of Abbreviations  vi  Acknowledgements Dedication Chapter one:  x Introduction  1  1.1  Calcium ion channel functions in physiological events  1  1.2  Calcium ion channel functions in the immune system  1  1.3  Candidates of SOCs  7  1.3.1 1P 3 receptor (IP R) calcium channels 3  8  1.3.2 STIM/ORAI  8  1.3.3 Mammalian homologues of transient receptor potential (TRP) calcium channels 10 1.3.4 P2X receptors  11  1.3.5 L-type voltage dependent calcium channels (VDCCs)  12  Chapter two: Materials and Methods  17  2.1  Mice  17  2.2  Cell line and culture conditions  17  2.3  PCR  17  2.4  Isolation of splenocytes, thymocytes, and peripheral blood for T cell subset distribution analysis 18  2.5  Isolation of splenocytes, thymocytes, and peripheral blood for T cell surface marker analysis 18  11)  2.6  Calcium phenotype cell sorting  2.7  VSV infection for tetramer staining and CTL assay  19  2.8  Bone marrow transfer experiments  20  2.9  NF- KB mobilization is modulated in the Cacnalf knockout mice  20  2.10  Statistical analysis  21  .18  Chapter three: Results  22  3.1  Genotyping  3.2  Isolation and flow cytometry of T cell subset analysis of T lymphocytes from spleen, thymus, lymph node and peripheral blood 23  3.3  Calcium influx  28  3.4  Activation and maturation markers  35  3.5  Bone marrow transfer experiment  42  3.6  Tetramer analysis  44  3.7  Chromium release assay  45  3.8  NF-icB nuclear mobilization expression  46  22  Chapter four: Discussion  48  References  52  Appendix I: Ethics Approval  59  iv  LIST OF DIAGRAMS  Diagram 1  T lymphocyte activation signaling cascade  3  Diagram 2  T lymphocyte survival signaling cascade  6  V  LIST OF FIGURES  Figure 1  PCR indicating Cavi .4 identity of individual mice  23  Figure 2  T cell subset profiles  27  Figure 3  T cell subset cell number  28  Figure 4  Resting and activated splenocyte  29  Figure 5  Splenocyte calcium influx profile  30  Figure 6  CD3+ T lymphocyte gate  30  Figure 7  T lymphocyte calcium influx profile  31  Figure 8  T lymphocyte subset accounts for most of the calcium influx profile observed in splenocytes 31  Figure 9  7 week old Cavi .4 KO splenocytes shows severe reduction in extracellular and intracellular calcium influx 32  Figure 10  7 week old Cavi .4 KO T lymphocytes shows more reduction in extracellular and intracellular calcium influx 33  Figure 11  12 week old Cavl.4 KO mice I lymphocytes show faster and reduced calcium 33  Figure 12  Higher concentration of IonomycinlPMA causes moderate amount of increase in calcium influx in splenocytes 34  Figure 13  Higher concentration of IonomycinlPMA causes moderate amount of increase in calcium influx in T lymphocytes  Figure 14  Lack of Cavl .4 calcium channel favours CD8 SP T lymphocyte development... .37  Figure 15  Lack of Cavl .4 calcium channel causes a chronic activated phenotype  39  Figure 16  Cay 1.4 is important for T cell development and lineage commitment  41  Figure 17  Cay 1.4 calcium channel is important for peripheral T cell expansion and survival of T lymphocytes 43  Figure 18  Cavi .4 calcium channel is important in generating antigen specific CTLs  45  Figure 19  Cay 1.4 calcium channel is important in CTL killing  46  Figure 20  Cavl .4 calcium channel is important for NF-icB localization  47  vi  LIST OF ABBREVIATIONS  2-APB ADPR AIDS APCs ATCC ATP BAK BAX Bcl BCR BM cADPR CaMK CCE CFSE ConA COS 1 CRAC CREB CTL DAG DHPs DN DP EDTA ER FBS FcR FITC FHLH HEPES 1CM IL-2 IL-7 3 1P R 3 IP KO MAP MEF2 MHC NAADP NAD NFAT NF-icB NK cells P2X receptors PBS  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  2-aminoethoxydiphenyl borate ADP-ribose acquired immune deficiency syndrome antigen presenting cells American type culture collection adenosine triphosphate bcl-2 homologous antagonist/killer protein bcl-2-associated x protein B-cell lymphoma B cell antigen receptor bone marrow cyclic ADPR calmodulin-dependent kinases capacitative calcium entry carboxyfluorescein succinimidyl ester concanavalin A African green monkey kidney cell line 1 calcium release activated calcium target cyclic AMP-responsive element-binding protein cytotoxic T lymphocyte diacyglycerol 1, 4-dihydropyridines double negative double positive diaminoethane-tetraacetic acid endoplasmic reticulum fetal bovine serum Fc receptors fluorescein isothiocyanate familial hemophagocytic lymphohistiocytosis 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid immunofluorescence confocal microscopy interleukin-2 interleukin 7 inositol 1 ,4,5-trisphosphate 3 receptor 1P knockout mitogen activated protein myocyte enhancer factor 2 major histocompatibility complex nicotinic acid adenine dinucloetidue phoaphate nicotinamide adenine dinucleotide nuclear factor of activated T cells nuclear factor kappa-light-chain-enhancer of activated B cells natural killer cells purinoreceptors phosphate buffered saline vi  PBTs PCR PHA 2 PIP PLC-y PMA RBL PBMCs RPM! RT-PCR Ry Soc SP SCID STIM/ORAI Te cells TCR TRP Th cell Treg cells VDCC VSV  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  -  peripheral blood T lymphocytes polymerase chain reaction phytohemagglutinin phosphoatidylinositol 3 ,4-bisphosphate phospholipase C gamma 1 2-O-tetradecanoylphorbol- 13-acetate rat basophilic leukemia human peripheral blood mononuclear cells Roswell Park Memorial Institute reverse transcription PCR ryanodine store-operated calcium channels single positive severe combined immunodeficiency stromal interaction molecules! cytotoxic T cells T cell antigen receptor mammalian homologues of transient receptor potential T helper cell regulatory T cells L-type voltage dependent calcium channel vesicular stomatitis virus  viii  ACKNOWLEDGEMENTS I offer my sincerest gratitude to the faculty, staff and my fellow students at the UBC, who have tirelessly inspired and assisted me in my work in this field. I owe particular thanks and gratitude to Dr. Wilfred A. Jefferies, for his biological insight, for his patience, and for supporting me through this degree with clear guidance, direction and compassion. I thank Dr. Xiaoxi Chen for continuing to push me to think and master my scientific mind and skills. I thank Dr. D Waterfield for going above and beyond to help me finish my work. I thank Dr. M Horwitz for providing vision and answers to my questions always. I would also like to thank Dr. Cheryl Pfeifer for never her unfaltering faith in me to succeed. I would like to thank Andy Johnson for facilitating work with FACS, I would like to thank Kyung Bok Choi for training me and assisting me with molecular biology work, Kyla Omilusik, Anna Reinicke, Robyn Seipp, Kaan Biron and Genc Basha for training me and assisting me with numerous experiments. Ray Gopaul for caring for the mice. Taka Murakami for genotyping work. I thank Professor Bech-Hansen for providing the Cavi .4 knockout mouse for which none of the studies conducted here could have been possible without. Special thanks are owed to my parents, who have supported me throughout my years of education, both morally and financially.  ix  DEDICATION This work is dedicated to my parents, who have been there to support me through everything in life. This work is only possible with help from Xiaoxi Chen, Kyla Omilusik, Kyung Bok Choi, Rayshad Gopaul, Hung-Sia Teh, N. Torben Bech-Hansen, and Wilfred A. Jefferies  CHAPTER ONE: INTRODUCTION 1.1 Calcium ion channels functions in physiological events Calcium ion acts as a secondary messenger to regulate many physiological events such as motility (1), photoreceptor function (2), skeletal muscle functions (3), lymphocyte activation (4), proliferation (5), differentiation (4), effector functions (6, 7), transcription (8-10), survival (11), anergy and death in many cell types (12, 13).  1.2 Calcium ion channelfunctions in the immune system  Calcium signaling is important in immune responses, including those associated with autoimmunity. Calcium signals act as an important secondary messenger for many immune cells such as T cells, B cells, mast cells and natural killer NK) cells (4). In immune cells, calcium signals are tightly regulated by membrane receptors, signaling molecules, and ion channels.  Immune cells use calcium signals as a secondary messenger to induce signaling transduction pathways (Diagram I), leading to such diverse functions as activation, proliferation, effector function, anergy, and death. Lymphocytes in their resting state have a calcium concentration of approximately 1 00-200nM. During activation, these intracellular stores of calcium are mobilized through a series of coordinated events. Antigen presenting cells (APC5) present antigen to the immunoreceptors: T cell antigen receptor (TCR) in T cells, B cell antigen receptor (BCR) in B cells, and Fc receptors (FcR) on mast cells and NK cells. Once the corresponding immunoreceptor recognizes the specific antigen, immunoreceptor ligation takes place, causing non-receptor tyrosine kinases to phosphorylate and activate phospholipase C gamma (PLC-y) (PLC-yl in T lymphocytes, and PLC-y2 in B cells) (14-17-19). PLC-y cleaves phosphatidylinositol, 3,4-bisphosphate (PIP ) from plasma membrane phospholipids to generate 2  1  diacyglycerol (DAG) and inositol 1 ,4,5-trisphosphate (1P ) (4). The released 1P 3 3 binds to 1P 3 receptor in the membrane of endoplasmic reticulum (ER), which leads to the release of intracellular calcium ions from the ER through store-related Ins( 1, 4, 5)P3 receptor calcium channels, ryanodine (Ry) receptors and nicotinic acid adenine dinucleotide phosphate (NAADP) receptors (20, 21). The entire process from immunoreceptor ligation to intracellular calcium release takes place in tens of seconds. The decrease in the ER calcium level causes an opening of the store-operated calcium channels (SOCs) on the plasma membrane, which causes a sustained calcium influx from the extracellular space (22, 23). Calcium influx through the SOCs activates calmodulin dependent serine/threonine phosphatase calcineurin, calmodulin-dependent kinases (CaMK) and downstream transcription factors such as nuclear factor of activated T cells (NFAT), CaMK and its target cyclic AMP-responsive element-binding protein (CREB), myocyte enhancer factor 2 (MEF2). Sustained calcium signal ranging from a concentration of approximately 20 nM to 1 1 iM for up to 48 hours is required for the phosphorylation and translocation of NFAT into the nucleus. Elevated levels of NFAT in the nucleus causes various downstream signals to be transcribed, such as interleukin-2 (IL-2) (24). Similar calcium signaling pathway is observed for cytotoxic T (Tc) cells and T helper (Th)-cell activation and proliferation (5).  2  Anbgmn ee.nhin eII  Lewis, RS (2001) Annu. Rev. Immunol. 19. 497-521. (24) Diagram 1. T lymphocyte activation signaling cascade  Immediate effects of calcium signaling, independent of gene transcription, are within minutes. Examples include regulation of lymphocyte motility (4), and granule exocytosis in allergen-sensitized mast cells (4). Long term effects of calcium signaling may take several hours to days. Utilizing downstream gene transcription, a plethora of immunological functions take place, including T and B lymphocyte activation, cytokine and chemokine production, lymphocyte proliferation, lymphocyte development, differentiation, effector function, anergy establishment and death (4).  Calcium signaling is also pivotal for downstream effector functions, such as perform dependent cytotoxic T lymphocyte (CTL) killing (6) and helper T cell (Th cell) cytokine production. Perform dependent killing is utilized by CD3 CD8 CTLs, NK cells (25, 26), some subsets of CD3+ CD4+ effector cells (7), including some regulatory T (Treg) cells (27).  3  CD3 CD8 CTL perform dependent killing is a crucial effector function used to kill virus infected cells, transformed cancerous cells, non-self cells in allografted tissues and organs (3 0-34), and also cells infected by intracellular bacteria such as Salmonella typhimurium (35) and Mycobacterium tuberculosis (36).  In Th cell cytokine production, calcium influx in Th cells occurs primarily via capacitative calcium entry (CCE), where calcium channel at the plasmic membrane is triggered by depletion of intracellular calcium stores (37). The specific channel responsible has been coined as the calcium release activated calcium (CRAC) channel (14, 22, 3 8-40), similar to the channel used for T cell activation.  In perform-dependent CTL target exocytosis, the process is initiated when CTL adheres to a target. When its TCR recognizes the specific antigen, an immunological synapse is formed (41-43). This triggers a downstream signaling transduction pathway which results in the polarization of the microtubule organizing center, Golgi apparatus and lytic granules towards the target. Exocytosis then occurs at the site of contact. (44-46). The entire perform-mediated target killing process takes minutes to tens of minutes (30, 47-50) and does not require new gene transcription (31, 32, 5 1-52).  Calcium signaling is also utilized in controlling whether T cells undergo pathways leading to survival, anergy or cell death. Extremely high levels of calcium influx from the extracellular matrix disrupt homeostasis and activate calcium-sensitive proteases, leading to cell death (49).  4  The fate of the T lymphocyte depends significantly on calcium signaling. The survival of naive T cells is dependent on both low-grade TCR signaling upon encounter with self peptides/self-MHC molecules and exposure to the cytokine interleukin 7 (IL-7) (54). Together, TCR and IL-7 receptor signaling promote T cell survival by influencing the balance and function of pro-survival and pro-apoptotic proteins. Moreover, a critical function of IL-7 receptor signaling has been postulated to be the up-regulation of the anti-apoptotic protein Bcl-2 since enforced Bcl-2 expression can rescue T cell development in IL-7 receptor-deficient mice (5556). Important pro-survival transduction mechanisms involve promoting the expression and function of members of the Bcl-2 family or activating the phosphoidylinositol kinase-AKT pathway. Either of these singlet pathways represses the function of Bcl-2-associated x protein (BAX) and Bcl-2 homologous antagonist/killer protein (BAK) that are required for release of lymphocyte mitochondrial cytochrome c (57). Bcl-2 is a potent inhibitor of apoptosis and is an integral membrane protein located on the ER and mitochondria. Bcl-2 inhibits calcium release from the ER (5 8-62) while another member of the family Bcl-2 family, Bc!, inhibits mimchondrial calcium waves initiated by inositol-P3-mediated calcium signals (61, 63). Furthermore, Bcl-2 binds calcineurin and thereby inhibits NF-AT activation by calcium (64).  _____  kpn31R  scpi1 !3t4et  t  R42  t  fl_+44+  Cb.  1pror2  0•  .,.,.  44  CDZ 44  p-T3)  (rT4.  () ......  •  F—  I42.  Y  c*  //  ,/ &14 44. (rI’  tj)  TCR  VD448  CP4 $ 4 ”  p*h  aun  /  \  —•  .—  CD4dy pttr’r  TR peaw  Shibasaki, F., Kondo, E., Akagi, T. & McKeon, F.  Diagram 2. T lymphocyte survival signaling cascade  Calcium signaling is also important in anergy. When self reactive lymphocytes escape the thymus during negative selection and becomes distributed in the periphery, several mechanisms are used to inactivate or eliminate the presence of these self reactive lymphocytes. The mechanisms employed include induction of anergy, dominant suppression by regulatory T cells, and peripheral deletion of self-reactive T cells. Anergy is used to cause self reactive lymphocytes to be functionally inactivated. It has been found that calcium signaling pathways such as calcium/calcineurin pathways and calciumINFAT pathways directly control anergy induction (64).  Calcium signaling is also significant for programed cell death. Also termed apoptosis, it is a process where cells shrink and dissociate from their surrounding neighbours, their organelles retain in size, and in the nucleus chromatin forms dense aggregates on the nuclear membrane and eventually undergoes fragmentation (65). The lack of apoptosis leads to autoimmune diseases or  6  cancer (65); whereas an overreactive amount of apoptosis leads to chronic pathologies such as neurodegenerative disease like Alzhemier type dementia or immune deficiencies such as acquired immune deficiency syndrome (AIDS) (66). The induction of apoptotic pathway by different stimuli such as caspases and B-cell lymphoma (Bcl) family of proteins eventually converge to lead to an increase in calcium signaling. Therefore, calcium signal is a general mediator of apoptotic events (65). Whether T lymphocytes undergo activation, proliferation and sustainance or follow the pathway of anergy or cell death depends significantly on calcium signaling.  1.3 Candidates ofSOCs  Although the mechanisms of calcium release from the intracellular stores is well characterized, the calcium signaling pathway from extracellular space into T lymphocytes through SOC is unclear, despite contributing to the majority of the calcium signaling in the T lymphocyte signaling pathway (67). Downstream effects of SOC channel may provide vital information on how T lymphocyte activation, proliferation, death is controlled as well as providing potential insights and therapeutic functions to disease phenotypes associated with T lymphocyte responses. One form of severe combined immunodeficiency (SCID) is a result of severe defects in SOC channel function, which leads to severe defects in cytokine impression and lymphocyte functions (41, 68, 69). Various SOC channel candidates have been put forward including IP R calcium channels (70), STIM/ORAI (7 1-73), TRP calcium channels (74-76), P2X 3 receptors (77-79), and L-type voltage dependent calcium channels (80-87).  The availability of electrophysiology enabled whole-cell patch clamping profile of the calcium channel for a cell type to be elucidated. Electrophysiological profile of the calcium  7  channel responsible for calcium influx from the extracellular matrix is therefore defined. Originally identified in hematopoietic cells in a basophil-mast-cell line, the as yet unindentified calcium channel responsible for the observed calcium influx in T lymphocytes is termed calcium release activated calcium (CRAC) channels (88-89 into 84-85). CRAC calcium current is found to be inwardly rectifying, highly selective for calcium ions over other cations (except barium ions) and with an extremely low calcium ion conductance.  Identification of CRAC candidate has been difficult as it is likely multimeric and no candidate has expressed the exact electrophysiological profiles of CRAC without lacking certain properties associated with CRAC or containing additional properties separate from what is observed in CRAC. Potentially, several calcium channels may be taken together to be responsible for the observed CRAC current (86).  1.3.1 1P 3 receptor calcium channels  3 receptor (IP 1P R) calcium channels (70) were initially found as intracellular calcium 3 channels on the ER (91). Studies have shown that T lymphocytes express three isoforms of the R channels as integral plasma membrane proteins (92). However, functional redundancies of 3 IP the three isoforms have rendered it difficult to define the contribution of these channels to calcium signaling in T lymphocyte (93).  1.3.2 Stromal interaction molecules  One candidate of CRAC channel is stromal interaction molecules (STIM 1). Found with the use of RNA interference (RNAi) screening in Drosophila and human, STIM1 has been  8  identified as the calcium sensor within stores. (71-73). Using the same approach, a pore forming subunit of CRAC has been identified as ORAl 1 (also named CRACM 1 or TMEM 1 42A) (94-96).  Upon MHC engagement, downstream tyrosine phosphorylation of kinases and adaptor molecules take place. This facilitates STIM1 and ORAI1 to colocalize to the site of MHC engagement (97, 98), forming a cap-like structure at the distal pole of the cell. This cap moves from the distal pole to an existing or a newly formed immunological synapse. It is postulated that the cap may contain preassembled calcium channel components that can be delivered to newly formed immunological synapses (98).  Evidence indicates that both STIM1 and ORAI1 knockout mice exhibit T lymphocyte abnormalities. ORAl 1 knockout mice exhibits partial decrease of calcium influx (99, 100); STIMI -/- mice were found to exhibit abolishment of peripheral T lymphocytes and double positive (DP) thymocytes (4, 101).  However, it remains elusive whether STIM 1/ORAl I composes the sole calcium channel responsible for directing calcium influx through the extracellular matrix. STIM1 -I- mice demonstrated no developmental defect in al3 TCR T cells (4, 101). This may be due to the rescue effect from STIM2; however, CD4Cre knockout of both STIM1 and STIM2 loci did not show a phenotype that affects thymocyte development (102). In addition, in ORAI1 -I- mice, calcium influx was not completely abolished, 10-15% of calcium influx remained to be observed in ORAI1 deficient DP thymocytes as compared to wildtype (99, 100). Currently, no ORAI2 mouse model is available to demonstrate whether ORAI2 alone or the combinations of the two are used as the CRAC channel. But ORAI2 is found to be more highly expressed in the thymus as compared to ORAl 1, and it has been shown to conduct CRAC currents in in vitro over-  9  expression systems (103, 104). Furthermore, CRAC channel inhibitors such as SKF96365, 2aminoethoxydiphenyl borate (2-APB) and lanthanum ion do not affect thymocyte motility (105). Most importantly, it has been found that human patients with mutations in STIM1 or ORAI1 are found to develop mature T cells in the periphery (68, 69, 106). This evidence taken together, indicate that although further work is required, it is plausible that other calcium channels may be present in addition to STIMI/ORAII.  1.3.3 Mammalian homologues of transient receptor potential (TRP) calcium channels  There has been accumulating evidence indicating that store-independent calcium channels may contribute to calcium signaling in T lymphocyte (74-76). One candidate for the CRAC channel is the transient receptor potential (TRP) calcium channel. It was originally found in Drosophila melanogaster (107). TRP calcium channel is a six-transmembrane cationpermeable channel. It regulates intracellular concentrations of sodium, calcium and magnesium ions. Twenty-eight TRP channel proteins are grouped into six sub-families based on sequence homology: TRPC1-6, TRPV1-6, TRPM1-8, TRPA, TRPML1-3, and TRPP2,3,5. They are found to be widely expressed in mammalian tissues (108). In particular, in Jurkat and human T lymphocytes, TRPC1, TRPC3, TRPC6, TRPM2 and TRPV6 are found to be expressed and functional (74, 109). TRPM2 has been extensively studied in T lymphocyte cell lines.  TRPM2 has been found to be activated by intracellular second messengers such as ADP ribose (ADPR), nicotinamide adenine dinucleotide (NAD), H , and cyclic ADPR (cADPR) 0 2 (110-112). When TCR engagement takes place, it activates soluble ADP-ribosyl cyclase which increases intracellular cADPR. In T lymphocyte lines, when ADPR and cADPR are directly  10  applied to cells, calcium signaling was induced through TRPM2 channels; therefore implying that TRP channels may contribute to calcium signaling in T lymphocytes (4).  Another channel, TRPV6, also termed CaT1 and ECaC2, exhibits many electrophysiological properties associated with CRAC when over-expressed in Jurkat T lymphocytes.  However, there are arguments against TRP channel being the CRAC channel. First, CaT1 does not exhibit all the electrophysiological properties associated with CRAC current (75, 77). It has also been found to exhibit additional electrophysiological properties not found in CRAC (113). Second, TRP channels are only partially regulated by store-depletion (75), contrary to what is observed for CRAC. Furthermore, major abnormalities in T cell development and function have not been reported in TRP mouse models (114).  1.3.4 P2X receptors  Another candidate for the CRAC channel is the P2X receptor. P2X receptors are adenosine triphosphate (ATP)-gated channels with high calcium permeability, which are activated by the increase in extracellular ATP, causing an influx of calcium signal upon activation (115). As it has been found that TCR engagement triggers a rapid release of ATP, it’s plausible that the release of ATP may activate P2X receptors (78). P2X receptors form homo or heteromeric P2X receptors. There are 7 P2X receptors ) 7 1 (P2X P2X . Evidence indicate that , P2X 1 P2X , P2X 2 , P2X 6 7 subunits are found to be expressed in lymphocytes. However, major abnormalities in T cell development and function have not been reported in P2X mouse model (79).  11  1.3.5 L-type voltage dependent calcium channels (VDCCs)  There has been accumulating evidence that a store independent L-type voltage dependent calcium channel (VDCC) may participate in T lymphocyte functions as the CRAC channel candidate. L-type VDCCs are heteromultimeric proteins containing a channel forming c subunit, a  13 subunit, and a cty subunit. It is voltage gated as it responds to changes to the electrical  potential across the plasma membrane and thereby change its conformation to become activated (82).  Four subtypes of L-type voltage dependent calcium channels are found in T and B lymphocytes: Cavl.1, Cavl.2, Cavl.3, Cavl.4 (83, 84, 87). Studies done on Jurkat T lymphocytes by Densmore et al. (83, 84) identified an electrically responsive current in the plasma membrane of T lymphocytes which had different electrophysiological properties from CRAC but was activated through TCR’CD3 complex and calcium store depletion (83, 84). Reverse transcription (RT)-PCR showed Cavl.l  (ii)  and Cavi .2 (cLic) pore forming subunits of  L-type calcium channels are expressed in Jukat T lymphocytes (85). Additionally, Savignac et al. demonstrated that 2G12. I murine T cell hybridoma line expresses L-type calcium channel mRNA and protein, and modulate calcium-dependent IL-4 gene transcription (86). Cavi .2 mRNA and protein as well as the auxiliary 13-subunit were also found to be expressed in various human B and T cell lines (87). In human L3055 B cell line, antibodies against extracellular region of Cavi .2 L-type calcium channel caused sustained calcium influx (87).  12  There are also pharmacological evidences supporting the function of VDCCs in T lymphocytes as calcium signal modulators. 1, 4-dihydropyridines (DHP5), a class of synthetic derivatives that selectively binds to L-type voltage-dependent calcium channels, are found to modulate calcium signaling in lymphocytes (80, 86, 87), whereas synthetic 1, 4-dihydropyridine (DHP) L-type calcium channel antagonist, nifedipine, is a potent suppressor of T lymphocyte proliferation. Birx  et  at. demonstrated that using an  in vitro  [3Hjthymidine uptake assay, that  0.001 -1 00iM nifedipine prevents the proliferation of human T lymphocytes in response to the mitogens phytohemàgglutinin (PHA) and concanavalin A (ConA) (116). Another study shows that human peripheral blood mononuclear cells (PBMCs), stimulated with PHA cannot proliferate in the presence of 1 0-200uM nifedipine, whereas IL-2 addition restores the proliferative response in the nifedipine-treated cells (117). Nifedipine is found to be a dosedependent inhibitor for T lymphocyte proliferation when added in combination with immunosuppressive agent cyclosporine A, as demonstrated in  in vitro  proliferation assays (118,  119).  Previous work in our laboratory has shown that a subtype of VDCCs, Cavl.4  (alF),  may  be responsible for the calcium influx through the plasma membranes in T lymphocytes (80, 81, 120).Cavl.4 L-type calcium channel is coded by the CACNA1F gene. It was initially cloned from human retina (121). In photoreceptors, it modulates calcium signal (122). A knockout of Cavl.4 calcium channel causes night blindness (123). Our laboratory has used PCR to prove that the mRNA of pore forming  alF  —subunit L-type calcium channel transcript is expressed in human  T lymphocytes (80). This was confirmed by McRory  et  at., that Cavi .4 is found to be expressed  in human spleen, thymus and bone marrow as well as in the retina (124). In addition, (+1-) Bay K 8644 (agonist) and nifedipine (antagonist) were found to modulate early T lymphocyte activation  13  through calcium signaling in a dosage dependent manner in both Jurkat T cell leukemia line and in human peripheral blood T lymphocytes (PBTs) (80). Furthermore, (+1-) Bay K 8644 and nifedipine were additionally found to modulate late T lymphocyte activation signals such as phospho-p44/42 mitogen activated protein (MAP) kinase activation, NFAT transcription, IL-2 secretion, and splenocyte proliferation during T lymphocyte activation and proliferation in Jurkat T cells and in human PBTs (80).  Although VDCCs are typically observed in excitable cell types such as photoreceptors and skeletal muscle, they may still function in non-excitable cell types such as I lymphocytes. It’s possible that a mechanism such as alternative splicing is used to generate splice variants expressing calcium channels. These alternatively spliced calcium channels may share common structural similarities to VDCCs, but are not gated by changes in membrane potential. This has been previously demonstrated in our laboratory by Koturri et al (81), Using nested RT-PCR, we identified two novel alternative splice variants of the Cay 1.4 in human spleens with alternative splicing in their carboxy termini. Cavl .4a variant had a deletion in the IVS4 voltage sensor domain; Cavi .4b variant had a deletion of the IVS3-S4 interlinker domain. This could explain the feature of T lymphocyte insensitivity to membrane depolarization as well as its low affinity for DHP as compared to electrically excitable cell types. In Jurkat cells, Cavi .4a and Cavl .4b mRNA expression following TCR engagement are found to be differentially expressed in  different splenocytes and temporally regulated (81). This has also been confirmed by Badou et al. (125). Our laboratory also found that Cavi .4a expression is limited to lymphocytes; whereas Cavi .4b expression was also found in monocytes. To establish CavI .4 protein expression in T lymphocytes, Cavi .4 protein was found in both Jurkat T lymphocytes as well as human PBTs. In Jurkat T lymphocytes, Cavi .4 protein expression is increased following TCR engagement,  14  suggesting alternative functional expression patterns (81). However, all work on VDCCs have been done on cell lines, no work has been done to characterize the expression and function of voltage dependent L-type calcium channels in mouse models.  We propose that an L-type voltage dependent calcium channel may be at least partially responsible for the extracellular influx of calcium signal associated with downstream T lymphocyte functions. Previously, mRNA from two splice variants of L type calcium channel Cavl.4 has been identified (81). The original Cavi .4 gene, which was cloned from retina, is 5.8 kb in length, with 48 exons. The Cavl .4 protein has 5 5-62% amino acid sequence identity to other Cavi L-type calcium channels. The retinal Cavl.4 channel mediates calcium signal entry into the photoreceptors to promote tonic neurotransmitter release. Mutations in the CACNA1F gene causes night blindness.  Since very little is understood about the molecular structure of the Cavl.4 channel in lymphocytes, we demonstrated that the full length Cay 1.4 mRNA is present in T cells and gave rise to a functional protein. The two splice isoforms were found to be expressed in T lymphocytes, but not in the human retina. cDNA sequencing showed that these two splice variants (Cavl .4a and Cavl .4b) had alternatively spliced out exons 31, 32, 33, 34 and 37. This caused deletion in the transmembrane segments S3, S4, S5 and half of S6 in motif IV of the spleen Cavi .4a channel. Transmembrane segment IVS4 (exon33) deletion caused the removal of a voltage sensor domain; whereas IVS6 (exon 37) deletion caused the removal of the DHP binding site and an EF-hand 2 Ca binding motif. Cavi .4b splice variant alternatively spliced out only exon 32 and 37, and retained the voltage sensor domain in IVS4. Exon 32 deletion removed an extracellular loop between segments IVS3-S4. The removal of either the IVS4 voltage sensor  15  domain or the IVS3-S4 interlinker may prevent Cavl.4 splice isoforms from being gated by membrane depolarization. At the 3 terminus region of both splice isoforms, deletion of exon 37 caused a frameshift and led to a premature termination of channel protein translation at the carboxy-terminus and caused the amino acid sequence downstream of the frameshift to lose 55% of the amino acid sequence identity compared to the human Cavl.l L-type Ca 2 channel wild type found in skeletal muscle (GenBank accession number XPOO191O). The frameshift is not a PCR artifact or a sequencing error since this unique sequence has been repeatedly isolated and sequenced not only from human spleen, but also from many different human, rat and mouse T cell lines, mouse splenocytes and thymocytes, and naïve human PBTs. Both splice isoforms had 99% nucleotide and 95% amino acid sequence identity to the Cavi .4 channel from human retina. There were no additional differential splice sites found upstream of exon 31 in Cavi .4a and Cavl .4b. In summary, the Cavi .4 splice variants expressed in T lymphocytes contain novel structural features at the carboxy-terminus which may have a unique impact on the Ca 2 kinetics gated by these channels in T lymphocytes.  The work presented in this thesis characterizes the function of Cavl .4 in T lymphocyte activation, proliferation and death by using a Cavl .4 knockout mouse model, lacking the entire gene sequence coding for the Cavl .4 L-type voltage dependent calcium channel. These studies will shed light on the mechanism of Cavi .4 calcium channel in T lymphocyte activation, proliferation, effector function, anergy, survival, and death.  16  CHAPTER TWO: MATERIALS AND METHODS 2.1 Mice Cavl .4 knockout heterozygous female and homozygous knockout male were provided -  by Dr. Bech-Hansen at the University of Calgary. As described in Mansergh et al. (126), Cavl.4 calcium channel is coded by the Cacnalf gene. Cacnalf knockout mice were derived by a targeted disruption strategy with a 7Obp insertion in exon 7 of the Cacnalf gene which created an in-frame (TAA) stop codon to be placed in position 305, which resulted in the premature termination of Cacnalf translation. The 7Obp insertional mutation transmission is X-linked. PCR, RT-PCR and RNA isolation conducted by Bech-Hansen’s laboratory confirmed the existence of the 7Obp mutation. In our laboratory, Cacnalf knockout mice and C57B1/6 wildtype mice were housed at the animal facilities at the University of British Columbia. Mice were kept in specific pathogen-free conditions, and all animal experiments were conducted according to institutional guidelines and animal care regulations. All mice were used between 6 to 12 weeks of age. All mice studies were approved by the Committee on Animal Care at the University of British Columbia using the guidelines set out by the Canadian Council on Animal Care. 2.2 Cell line and culture conditions The human T cell leukemia line Jurkat clone E6-l was -  obtained from American Type Culture Collection (ATCC, Manassas, VA) maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 20mM 4-(2-hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES), and 1mM sodium pyruvate. African green monkey kidney cell line 1 (COS1) was also cultured under the same medium condition and used as a control.  2.3 PCR  —  PCR reactions were performed using the following primers: exon 7F: ATA TGG  AAG CAG AGG AGG ACC and exon 7R: CCA GTA GAG GAC GTC TGT CCA. PCR Taq  17  polymerase (Invitrogen) was used and the PCR reaction was conducted in a Whatman Biometra Unoll Thermocycler at 94°C for 30 sec, followed by a 10-mm extension at 72°C. PCR fragments were resolved on a 1.4% agarose gel and visualized by staining with CyberSafe.  2.4 Isolation of splenocytes, thymocytes and peripheral bloodfor T cell subset distribution analysis Whole blood (10-50m1), thymus and spleen were collected from three C57B116 and -  Cacnlf males and females. Thymus and spleen were homogenized. Thymocytes, splenocytes and peripheral blood were separated by centrifugation at 900  x  g for 30 mm at 4°C over a ficoll  paque PLUS (Amersham Biosciences) gradient. The resulting mono-nuclear cell layer was washed and resuspended in FACS buffer (5% FBS in Phosphate buffered saline (PBS)), then stimulated for 20 mm with anti-CD3, CD4, and CD8 monoclonal antibodies, and resuspended in FACS buffer. Thymocytes, splenocytes and PBLs were analyzed on a FACSCalibur cytometer (BD Biosciences) with fluorescein isothiocyanate (FITC)-conjugated CD3(BD Pharmingen), PE conjugated CD4(BD Pharmingen) and PE-conjugated CD8(BD Pharmingen). 2.5 Isolation of splenocytes, thymocytes and peripheral bloodfor T cell surface marker analysis Monoclonal antibodies (Abs) against CD4 (GK1.5), CD8a (53-6.7), CD8b (53.38) -  TCRb (H57-597), CD25 (PC6I.5) CD44 (1M7), CD62L (MEL-14), CD69 (H1.2F3), Cd127 (A7R34), Thy 1.1 (HIS5 1), CD45.2 (104) were purchased from eBioscience. Data were acquired using either a FACSCalibur/Cell Quest software or LSRII/FACSD1va software (BD Biosciences). Data were analyzed with FlowJo software.  2.6 Calcium phenotype cell sorting  —  Splenocytes were extracted from 7 or 12 week old  C57B1/6 and Cacnalf male and females. Spleens were homogenized, splenocytes were separated by centrifugation at 900  x  g for 20 mm at 4°C over a ficoll-paque PLUS gradient. The resulting  18  ficoll layer was washed and resuspended in optiMEM buffer, then stained for 20 mm with anti CD3 FITC and Endo-1 dye for intracellular calcium measurement. Splenocytes were analyzed on a FACS cell sorter. Intracellular calcium level was measured upon T cell specific mitogen stimulation for 7 minutes.  2.7 VSVlnfection for tetramer staining and CTL Assays  —  C57B1/6 or CACNA1F knockout  mice were infected by intraperitoneal injection of VSV virus at 2x10 5 TCID5O (50% tissue culture/infectious dose). At 7 days post viral infection, mice were sacrificed and spleens were collected. Splenocytes were cultured for 5 days in RPMI 1640 complete medium (RPMI 1640 supplemented with 10% FBS, 0.1 mM nonessential amino acids, 1mM sodium pyruvate, 50 uM 2-mercaptoethanol, 2 mM L-glutamine) and 1 uM of the H2Kbrestricted peptide, VSV N(5259), RGYVYQGL. To identify the CD8 cells specific for the VSV-N(52-59) peptide complexed with the H2Kb allele, splenocytes were double stained with anti-CD8 specific monoclonal antibody (BD Pharmingen) conjugated to FITC and anti-H-2K-VSV-NP52-59 iTAgTM Tetramer Streptavidin-Phycoerythrin (SA-PE, immunomics-Beckman CoulterTM) for 30 mm at 4°C. The cells were examined using a FACSCalibur flow cytometer (Beckton Dickinson) and analyzed using FlowJo software to assess the percentage of VSV N(52-59) specific CD8 cells. Cytotoxicity was assessed with a standard Chromium 51 release assay. Target cells (H2Kb transfected L cell fibroblasts were incubated with 1 uM VSV N(52-59) peptide, labeled for 1.5 hours with sodium chromate (100 jiCi; Amersham), then washed and resuspended in RPMI 1640 complete medium. CTLs were incubated for 4 h at 37°C with target cells (lx  cells per well in  96-well plates) at various effector/target ratios. Spontaneous Chromium release by labeled cells was measured in the absence CTL and maximum release was quantified by lysing target cells in  19  2.5% Triton X- 100 detergent. All experiments were performed in triplicates and virus-specific Chromium release was calculated using the below formula.  % Specific 51 Cr release  =  Experimental release Maximum release  —  —  Spontaneous release x 100%  Spontaneous release  2.8 Bone marrow transfer experiments Bone marrow (BM) cells were prepared from thigh -  bone extracts of Thyl.1 wild type (Thy1.1 CD45.2 or Cavl.4 -I- (Thy1.2 CD45.2). Wild type and mutant BM cells were then mixed 50:50 based on number and confirmed by flow cytometry (Thy l.1:Thy1.2=1:1) before being transferred intravenously into sub-lethally irradiated (1000 rads) CD45.1 hosts (Thy1.2 CD45.1). Splenocytes were extracted 30 days after adoptive transfer; Thy 1.1 and CD45 .2 were used as markers to discriminate wildtype and mutant donors. CD4 and CD8 T cell purification and in vitro proliferation assay: Single cell suspensions from lymph nodes and spleens ofC57Bl/6 wildtype or Cavl.4 knockout mice were prepared and then incubated with biotinylated anti-CD4 (GK1 .5) or anti-CD8 (53-6.7) mAb on ice for 30 mm, followed by positive selection using the MiniMACs system (Miltenyi Biotec), according to the manufacturer’s specifications.  2.9 NF-KB mobilization is modulated in the Cacnalf knockout mice Single cell suspensions -  from spleens of C57 wildtype or cacnaif knockout mice were prepared and T lymphocytes were positively selected using CD9O MACS beads (Miltenyi Biotec), according to the manufacturer’s specifications. Following selection, lxi 06 cells were activated with ionomycin and ConA for 120 minutes. The cells were fixed in 4% formaldehyde for 30 mill then permeabilized with 0.2% Triton X-100 detergent for 2 mm. Non-specific binding was blocked by 30 mm incubation in 3% BSA and 5% normal goat serum. The cells were stained with a goat anti-NF-icB (nuclear factor  20  kappa-light-chain-enhancer of activated B cells) antibody (1:200 dilution) followed by a rabbit anti-goat antibody conjugated to Alexa fluor 555 (1:600 dilution). The cells were counterstained with Hoechst stain solution for 5 mm, and fluorescence was visualized by immunofluorescence confocal microscopy (1CM). Data were analyzed using Image J. 1 to select single slices and Adobe Photoshop 7.0 to merge images.  2.10 Statistical analysis statistical significance was determined by the student’s t test, using -  two factorial design without replication. For all tests, p statistical significance.  21  0.01 was considered to indicate  CHAPTER THREE: RESULTS 3.1 Genotyping  In order to study the effect of Cay 1.4 calcium charmel on T lymphocyte activation, proliferation, effector functions, survival and death, we have obtained a Cay 1.4 knockout mouse model from the Bech-Hansen laboratory at the University of Alberta. One heterozygous female and one homozygous male were provided. Mice were backcrossed 7-10 times with C57B1/6 wildtype mice prior to arriving to our laboratory. The Cavl .4 knockout mouse model was derived by a targeting disruption strategy where a 7Obp insertion in exon 7 of the Cavi .4 gene (CACNAIF) was used to create a in frame TAA stop codon at position 305. This stop codon results in the premature termination of CACNA 1 F translation. The 7Obp insertional mutation is X-jinked. The Cavl.4 knockout mice were further backcrossed with C57B1/6 for seven generations in our laboratory prior to this study. Age and sex matched homozygous mutants were used in our study. In order to confirm the insertional mutation in the Cavi .4 knockout mouse model, PCR with primers flanking the region of insertion and 200bp upstream of the insertion were used. Lane 1 represents the 1kb ladder. Lane 2 represent homozygous wildtype C67B1/6 mice with the 200bp PCR product from primers flanking the region of insertion; lane 3 is the negative control; lane 4-6 represent homozygous Cavi .4 mutant with the 7Obp insertion, making the sequenced region 270bp in length. The results were identical for all mice used in the studies of more than 100 mice. Therefore, we conclude the existence of the 7Obp insertion in the Cay 1.4 knockout mouse model.  22  1  2  3  45  6  123456  I -  ‘  IQ  14O1  Fig. 1. PCR indicating Cavl.4 identity of individual mice. 1. 1kb ladder. 2. C57B1/6 WT. 3. Negative control. 4-6. Cacna if homozygous KO progeny. Figure representative of over 20 individual experiments. All mice used in these studies were genotyped to confirm for Cavl .4 identity.  3.2 Isolation andflow cytometry of T cell subset analysis ofT lymphocytes from spleen, thymus, lymph nodes andperipheral blood In order to assess whether Cavi .4 calcium channel is important in regulating T lymphocyte development and function, spleens, thymuses, lymph nodes and peripheral blood were extracted from C57B1/6 wildtype and Cavl .4 knockout mice (three replicates, experiment repeated three times) in order to analyze whether Cavi .4 calcium channel has functional significance in T lymphocyte development and maturation. Splenocytes, thymocytes, lymph node cells and peripheral blood cells were gated for analysis based on granularity and size. T cell subsets from splenocytes, thymocytes, lymph nodes and peripheral blood were stained with CD3, CD4 and CD8 antibodies to analyze the population of T cell subsets. T lymphocytes were recognized by the CD3 antibodies. DP T lymphocytes were recognized by the presence of CD3,  23  CD4, and CD8 antibodies; double negative (DN) T lymphocytes were recognized by the presence of CD3 alone, and the absence of CD4 and CD8 antibody staining. Single positive (SP) cytotoxic T (Tc) cells were recognized by both CD3 and CD8 antibodies; whereas SP helper T (Th) cells were recognized by CD3 and CD4 antibodies. All the results shown are representative FACS plots and were found to be significant with p  0.01.  Results indicated that in the thymus, there is an overall decrease in the number of T lymphocytes in the thymus (Fig. 2a); there is also a reduction in the overall number of each of DN, CD4 SP and CD8 SP cells in the Cavi .4 knockout as compared to the wildtype; but an increase in the total number of DP cells were observed (Fig. 3a). The ratio of thymic CD4 SP cells in the Cavl.4 knockout is approximately half of that in the C57B1/6 wildtype (Fig. 2a); the ratio of thymic DN cells decreased by approximately 2.5 times in the Cavl.4 knockout as compared to the wildtype; whereas the ratio of thymic CD8 SP cells is decreased by approximately 1.4 times in the Cavl.4 knockout as compared to the wildtype. The ratio of thymic CD4 SP to CD8 SP cells changed in the Cay 1.4 mutants as compared to the wildtype (Fig. 2a). In the CavI .4 wildtype thymocytes, CD4 SP to CD8 SP cell ratio is approximately 8 to 1; whereas in the Cavl .4 mutant, the ratio becomes 6 to 1 (Fig. 2a). This suggests a strong upregulation of CD8 SP T cell development in the Cavl.4 knockout mice. In the Cavl.4 knockout mice, there is a decrease in the frequency of DN thymocytes as compared to the wildtype. 1.79% of C57B 1/6 wildtype thymocytes were DN; whereas 0.77% of CavI .4 thymocytes were DN. There is an increase in the number of DP thymocytes as compared to the wildtype. Indicating that the lack of Cay 1.4 calcium channel blocks the transition of DP cells into CD4 SP and CD8 SP cells.  24  In the spleen, the physical sizes of spleens in the Cavl .4 knockout were smaller than in the C57B1/6 wildtype. There is a decrease in the number of CD4 SP in the Cavl.4 knockout as compared to the wildtype (Fig. 2b). There is also a marked decrease in the number of DN in the Cavi .4 knockout as compared to the wildtype. The total number of splenocytes in the Cavi .4 knockout was also found to be less than in the C57B1/6 wildtype (Fig.3b). There is an overall decrease in the total number of CD4 SP cells in the Cavl.4 knockout as compared to the wildtype. The total number of CD8 SP cells in the Cay 1.4 knockout is also decreased as compared to the wildtype (Fig. 3b). The frequency of CD4 SP cells decreased in the Cay 1.4 knockout as compared to the wildtype, however, the ratio of CD8 SP cells is similar to the ratio seen in the wildtype. The CD4 SP to CD8 SP ratio in the wildtype is approximately 2 to 1; however, in the Cay 1.4 knockout, the ratio becomes 1.2 to 1. In the Cay 1.4 knockouts, lymph nodes were found to be enlarged as compared to the wildtype. Interestingly, there is a large decrease in the CD4 SP population in the Cavi .4 knockouts as compared to the wildtype; however, there is a marked increase in the CD8 SP population in the Cavl .4 knockouts (Fig. 2c). There is a decrease in the total T lymphocyte number in the Cavi .4 knockout as compared to the wildtype (Fig. 3c). This indicates a chronically activated and exhausted phenotype as apoptosis takes place. The frequency of CD4 SP cells are found to be decreased in the Cavi .4 knockout as compared to the wildtype. The frequency of CD4 SP cells in the Cavi .4 knockout is approximately half that in the C57B1/6 wildtype. However, the frequency of CD8 SP cells is increased in the Cay 1.4 knockout as compared to the wildtype. The ratio of CD4 SP to CD8 SP cells changed from 1.3 to 1 as seen in the wildtype to 0.56 to 1 as seen in the Cavi .4 knockout. Indicating that in the periphery, the lack of Cavi .4 calcium channel prevents the establishment of CD4 SP cells.  25  Peripheral blood lymphocyte (PBL) T cell subset analyses showed a decrease in the total number of CD4 SP and CD8 SP cells in the periphery in the Cay 1.4 knockout as compared to the wildtype. As well, CD8 SP Tc cells (Fig. 2d) were slightly down-regulated in peripheral blood in the knockout compared to the wildtype; the CD4 SP T helper cell populations were also down regulated in the Cavl.4 as compared to the wildtype (Fig. 2d). This result indicates that although there was an up regulation of CD8 SP cells in the lymph nodes, the number of CD8 SP cells was reduced in the periphery. As well, there was a general decrease in the total number of T lymphocytes in the periphery. This indicates that Cavi .4 is important in establishing T lymphocyte survival and regulating homeostasis. Therefore, the lack of Cay 1.4 calcium channel causes T cell lymphopenia in the periphery despite the high frequency of CD8 SP cells in the thymus.  26  Fig.2  WT  KO  a THY  ,0  1* SPL IL  ) .  C) C.  LN  d. PBL  CD8 Fig. 2. T cell subset profiles. C57B 1/6 wildtype and Cavi .4 knockout thymocyte (a), splenocyte (b), lymph node (c), and peripheral blood cell (d) CD4, CD8 profiles. Results shown are representative FACS plots. Experiment was conducted using 3 mice in 3 replicates. Results were found to be significant with p 0.01.  27  ___  Fig.3  a  TIfI  b.  ‘1000  WT  DKO  WT tKO  101  —  J_____ 4  $  .1  4?  $  4?  LNS =w1-  a —  I  a  Fig. 3. T cell subset cell number. C57B1/6 wildtype (WT) in blue; Cavl.4 knockout (KO) in red. a. Thymocyte, b. splenocyte, and c. lymph node tota’ cell number profiles. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. *Results were found to be significant with p 0.01. Results without * were found to have a statistical significance of p 0.5.  3.3 Calcium Influx Splenocytes were utilized to measure calcium influx in order to assess the function of CavI .4 calcium channel in T lymphocyte. Activated and resting splenocytes exhibit different granularity and size. As shown in Fig. 4, the gate on the left (4a) captured the resting splenocytes, whereas the right hand gate (4b) captured the activated splenocytes. Splenocytes were labeled with endo 1 dye to measure intracellular calcium levels. Fig. 5 shows splenocyte calcium influx profile  28  shown by intensity of Indo-1 dye release over time. T lymphocytes were specifically labeled with anti-CD3 FITC to distinguish T lymphocytes amongst various splenocyte subpopulations (Fig. 6). IonomycinlPMA (1 2-O-Tetradecanoylphorbol- 13-acetate) in different titrations were used as an activator to trigger lymphocyte activation (data not shown). 2C 11, a hamster anti mouse CD3E antibody was used to trigger T lymphocyte specific activation. EDTA (Diaminoethane-tetraacetic acid) is a calcium chelator used to sequester free calcium in the media. This was used as a control to show that the calcium influx observed was due to an extracellular calcium influx instead of intracellular calcium release from the ER and the sarcoplasmic reticulum. Fig. 7 shows the T lymphocyte calcium influx profile. Fig. 8 shows calcium influx levels between splenocytes and the T lymphocyte subset. T lymphocyte subset can account for most of the calcium influx observed from splenocytes. Results were preliminary, later data done in our laboratory by others have confirmed these data.  1  J  Fig. 4. Resting (4a.) and Activated (4b.) splenocyte.  29  Time(s) Fig. 5. splenocyte calcium influx profile shown by intensity of Indo- 1 dye release over time.  TIu. -J U-  CD3 Fig. 6 CD3+ T lymphocyte gate.  30  I  100 80  ‘-.  0 C  ci0  200 400 lime(s)  I  600  Fig. 7. T lymphocyte calcium influx profile  [  40( 35( 3O( 25( 500  Time  iboc  ‘10o  Fig. 8. T lymphocyte subset accounts for most of the calcium influx profile observed in splenocytes. red: Splenocyte calcium influx profile upon activation with IonomycinlPMA. Blue: T lymphocyte calcium influx profile taken from the splenocyte subset. Results shown are representative FACS plots. Five mice were used in this experiment in 8 replicates. Results were found with a statistical significance of p 0.5.  EDTA, a calcium chelator, sequesters free calcium in the media, thereby eradicating calcium influx from  the extracellular matrix. Wild type mice splenocytes (Fig. 9), in the presence  of EDTA and mitogen (IonomycinlPMA) showed no extracellular calcium influx, but show a small calcium influx peak which quickly disintegrates, characteristic of intracellular calcium fluxes. In normal conditions, wildtype mice splenocytes generated normal extracellular calcium influx profile as expected. While knockout mice splenocytes exhibit marked reduction in the  31  ability to induce extracellular calcium influx in the presence of mitogen alone. They still maintained the low level intracellular calcium influx in the presence of both mitogen and EDTA. T lymphocyte results, however, indicate that while wildtype mice T lymphocytes (Fig. 10) exhibited both intracellular and extracellular calcium influx as expected. Cavi .4 knockout mice T lymphocytes do not have either intracellular or extracellular calcium influx. This finding suggests that Cavi .4 calcium channel is involved in intracellular and extracellular calcium influx in T lymphocytes, and is involved in only extracellular calcium influx in other splenocyte subsets such as B cells, NK cells & mast cells.  6  It  IflI  Fig. 9. 7 week old Cavl .4 KO splenocytes shows severe reduction in extracellular and intracellular calcium influx. Red. 7 week WT splenocytes, calcium influx profile upon activation with 50J.LM lonomycinlPMA. Blue: 7 week KO splenocytes, 5OiM Ionomycin/PMA. Green: 7 week WT splenocytes, 5OjiM lonomycinlPMA, lOp.M EDTA. Yellow: 7 week KO splenocytes, 5O.tM Ionomycin/PMA, I OjiM EDTA. Results shown are representative FACS plots. Five mice were used in this experiment in 8 replicates. Results between WT and KO were found with a statistical significance of p 0.5.  32  r  -J LL  Fig. 10. 7 week old CavI .4 KO T lymphocytes shows more reduction in extracellular and intracellular calcium influx. Red. 7 week WT T lymphocytes, calcium influx profile upon activation with 50,iM IonomycinlPMA. Blue: 7 week KO T lymphocytes, 5OuM Ionomycin/PMA. Green: 7 week WT T lymphocytes, 50iM IonomycinlPMA, 1O.iM EDTA. Yellow: 7 week KO T lymphocytes, 5OjiM IonomycinlPMA, lOj.tM EDTA. Results shown are representative FACS plots. Five mice were used in this experiment in 8 replicates. Results between WT and KO were found with a statistical significance of p 0.5.  Twelve week old knockout mice showed a 20% reduction in the amount of calcium influx observed in T lymphocytes, as well as a faster calcium influx response (Fig. 11); whereas seven week old mice showed an almost complete annihilation of calcium influx in T lymphocytes (Fig. 10).  60(  Ii  40( 20( •  100  •  Time  150  00  Fig. 11. 12 week old Cavi .4 KO mice T lymphocytes show faster and reduced calcium influx Red: Splenocytes were extracted from 12 week old wildtype mice. Calcium influx profile is induced by activation with 2OjiM IonomycinlPMA. Blue: Splenocytes were extracted from 12 week old knockout mice. Calcium influx is measured by activation with 20jiM IonomycinlPMA. Results shown are representative FACS plots. Three mice were used in this experiment in three replicates. Results were found with a statistical significance of p 0.5. .  33  5o 4O —I L1  3O 2O  rsII ItLH.  Fig. 12. Higher concentration of IonomychilPMA causes moderate amount of mcrease in calcium influx in splenocytes. Blue: 7 week WT splenocyte calcium influx profile activated with 5OjiM Ionomycin!PMA. Red: 7 week WT splenocyte calcium influx profile activated with 2OtM IonomycinlPMA. Results shown are representative FACS plots. Three mice were used in this experiment in three replicates. Results were found with a statistical significance of p 0.5.  I Time Fig. 13. Higher concentration of IonomycinlPMA causes moderate amount of increase in calcium influx in T lymphocytes. Blue: 7 week WT T lymphocyte calcium influx profile activated with 5OjiM IonomycinlPMA. Red: 7 week WT T lymphocyte calcium influx profile activated with 2OtM Ionomycin!PMA. Results shown are representative FACS plots. Three mice were used in this experiment in three replicates. Results were found with a statistical significance of p 0.5.  In both splenocytes (Fig. 12) and T lymphocytes (Fig. 13) 5O.iM of IonomycinlPMA caused a higher calcium influx than 2OiM of IonomycinlPMA. However, 1 5O i M of IonomycinlPMA also caused more apoptosis in both WT and KO.(data not shown). 34  3.4 Activation and maturation markers To further investigate the decrease of peripheral T cells in the Cay 1.4 knockout mice, the activation and maturation markers on developing thymocytes, splenocytes, and lymph node T cells were examined (Fig. 14-16). CD4 and CD8 markers were used to distinguish cytotoxic T cells and helper T cells. TCRI3 and CD69 were development markers. CD 127 is an 1L7 receptor, signaling for activation, memory and survival in T cells. CD62L is an activation and memory marker. CD44 and CD25 are T cell activation and development markers. Thymocytes, splenocytes and lymph node cells were activated and then stained with activation and maturation markers. Splenocytes, thymocytes and lymph node cells were stained against CD4, CD8, CD44, CD69, TCRI3, CD25, CD62L and CD 127 markers. In thymocytes, there is a decrease in TCRf3 in CD8 SP populations, as well as a decrease in CD69 in CD4 and CD8 SP cells; whereas there is no difference in CD44, CD25 and CD62L markers between C5 7B 1/6 wildtypes and Cay 1.4 knockouts(Fig. 4a, 4b). TCRI3 and CD69 are development markers, appearing during CD4 CD8 DP stage, before T lymphocytes transition into CD4 and CD8 SPs. This suggests that the lack of Cay 1.4 calcium channel interferes with development. This aligns with our T lymphocyte subset analysis, where development seems to be blocked from the double positive stage to CD4 and CD8 SP stage. The decrease in TCRI3 and CD69 signaling is particularly obvious with CD8 SP cells. According to the strength of signaling hypothesis (120), weak TCRJMHC engagement leads to CD8 SP T cell development; whereas moderate/strong TCRJMHC engagement leads to CD4 SP T cell development. Therefore, the decreased frequency of CD4 SPs as observed in T cell subset analysis could be due to the lack of calcium signaling in the Cay 1.4 calcium channel knockout mice. This led to weak downstream signaling, which favors the development of CD8 SP T cells in the thymus of Cavi .4 knockout mice.  35  Furthermore, there is decrease in CD 127 (also termed IL7RcL) signaling in both CD4 SP, CD8 SP and double negative cells in the thymus. CD 127 is a pro-survival 1L7 receptor. As 1L7 signaling is important for positive selection of thymocytes, this suggests that the developmental defects of T cells in the thymus of Cavi .4 knockout mice is due to the lack of 1L7 receptor signaling resulting from dysregulated 1L7 receptor. Therefore, it is postulated that Cay 1.4 calcium channel provides calcium signals which either directly or indirectly regulate 1L7 receptor expression.  36  THY  Fig. 14a TCR  CD69  CD25  CD4SP  CD8SP  008  DP  DN  DN  Fig. 14a. Lack of Cavl.4 calcium channel favours CD8 SP T lymphocyte development. CD4, CD8, DN and DP profiles of thymocytes stained with TCRj3, CD69 and CD25. Wildtype in red; Cavl.4 knockout in blue. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. TCR43, CD69 results were found to be significant with p <0.01. CD25 results were found to be significant with p 0.5.  37  THY  Fig. 14b CD44  CD62L  CDI 27  100  100  100  80  80  80  60  60  00  40  40  40  20  20  CD4SP  38  11.3 20  100  10’  108  101  4 1o  FL2-H C04  0io”’\___. i FL2-H  ,90  10  C04  101  102 F [2-H  10  ¶94  COO  100  100  A )A\  60  00  62.9  00  62.1  [j  379  CD8SP  00  11.4  84.6  212  40  20  ,  100 C08  10’  102 FL2-H  io  100  10  101  102 F12-6  io  1o  IC  l’  io  102  FL2-H  COO  C08  DP  DP  OP  Op  ON  ON  COO  DO  Fig. 14b. Lack of CavI .4 calcium channel favors CD8 SP T lymphocyte development. CD4, CD8, DN and DP profiles of thymocytes stained with CD44, CD62L and CD127. Wildtype in red; Cavl.4 knockout in blue. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. CD127 results were found to be significant with p <0.01. CD44, CD62L results were found to be significant with p 0.5.  38  To investigate the effects of the knockout of Cavi .4 calcium channel on T lymphocytes in the periphery, splenocytes and lymph node cells were extracted and activation and maturation markers were analyzed post activation. In splenocytes, TCRI3, CD69, CD62L and CD 127 expression showed no statistically significant difference between C57B1!6 wildtype and Cavi .4 knockout (Fig. 1 5a, 1 5b). However, there is down regulation of CD25 in the Cay 1.4 knockout compared to the wildtype; there is also a distinct population of up regulated CD44 signaling in the Cavl .4 knockout as compared to the wildtype, exhibiting a more activated phenotype in the Cavi .4 knockout. Since acute activation markers such as CD44 is heightened in the Cavi .4 knockout as compared to the wildtype, this indicates the chronically activated phenotype.  Fig. 15a  SPL TCRI3  CD69  CD25  CD4SP  CD8 SP  100 CD8  Fig. 15a. Lack ofCavl.4 calcium channel causes a chronic activated phenotype. CD4, CD8, DN and DP profiles of splenocytes stained with TCRI3, CD69 and CD25. Wildtype in red; Cavi .4 knockout in blue. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. CD25 results were found to be significant with p <0.01. TCRf3, CD69 results were found to be significant with p 0.5.  39  Fig. 15b  SPL CD62L  CD44  CDI 27  CD4SP  DI’  CD4  DP  CD8 SP  ON  008  ON  Fig. 1 Sb. Mice lacking Cavi .4 calcium channel exhibit a chronic activated phenotype. CD4, CD8, DN and DP profiles of splenocytes stained with CD44, CD62L and CD 127. Wildtype in red; Cavl.4 knockout in blue. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. CD44 results were found to be significant with p <0.01. CD62L and CD127 results were found to be significant with p 0.5.  In lymph node cells (Fig. 1 6a, 1 6b), TCRf3, CD25 and CD69 expression showed no statistically significant difference between C57B/6 wildtype and Cavi .4 knockout. However, there is a distinct population of up regulated CD44 signaling in the Cavl.4 knockout as compared to the wildtype, exhibiting a more activated phenotype in the Cay 1.4 knockout. There is also a down regulation of CD62L marker in the Cay 1.4 knockout. There is a down regulation of CD127, which is an important signal for survival and the generation of memory T cells. Since  40  acute activation markers such as CD25 and CD69 expression were comparable to the wildtype, and CD127 is a memory marker; taken together, the CD44 high, CD62L low, CD127 low phenotype is indicative of the chronic activation effects of the knockout of Cay 1.4 calcium channel. This effect is even more prominent for CD4 SP cells. This suggests that Cavi .4 is important for T cell development and lineage commitment. This explains why the lack of Cav.4 calcium channel causes T cell lymphopenia, chronic activation of CD4 SP cells, and the decrease in CD8 SP cells that exhibit memory T cell phenotypes.  LN  Fig. 16a TCR  CD69  CD25  CD4SP  CD8SP  Fig. 16a. CavI .4 is important for T cell development and lineage commitment. CD4, CD8, DN and DP profiles of lymph node cells stained with TCRI3, CD69 and CD25. Wildtype in red; CavI .4 knockout in blue. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. TCRI3, CD69 and CD25 results were found to be significant with p 0.5.  41  Fig. 16b  LN  CD44  CD62L  CDI 27 CD4SP  C04  CD4  CO4  100  60.6.  80  60 42.8 40  40  i 1 c  CD8SP jH  1  20  lo CD8  /1  10’  io2 FL2.H  1o  100  4 1o  101  102 FL2-H  108  io  COO  COO  Fig. 16b. Cavl.4 is important for T cell development and lineage commitment. CD4, CD8, DN and DP profiles of Lymph node cells stained with CD44, CD62L and CD 127. Wildtype in red; Cavl .4 knockout in blue. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. CD44, CD62L and CDI 27 results were found to be significant with p < 0.01.  3.5 Bone Marrow Transfer experiment To further investigate whether Cavi .4 knockout mice exhibit defects in development and  peripheral T cell expansion, bone marrow transfer experiment was performed (Fig. 17). Thigh bone marrow was extracted from Thyl .lpositive, CD45.2 positive wildtype mice, and Thyl .2 positive, CD45.2 positive Cavl.4 knockout mice. Mixed in 50:50 portions, they were injected into Thy 1.2 positive, CD45.1 positive irradiated congenic hosts. The spleens of these recipient mice were analyzed one month post injection. Results indicate that the ratio of T lymphocytes in Thy 1.1 positive, CD45 .2 positive C57B 1/6 wildtype mice to Thy 1.2 positive, CD45 .2 positive Cavi .4 knockout mice were 44 to 1 (Fig. 17b), suggesting that Cavl.4 calcium channel is important for peripheral T cell expansion and survival of T lymphocytes.  42  Furthermore, in the congenic host, while 68% of the total wildtype donor T lymphocytes were expressing CD4 marker; only 22.4% of the Cavi .4 knockout I lymphocytes were CD4 positive. Taken together, the bone marrow transfer experiment indicates that Cavl .4 calcium channel is important for T cell development and peripheral T cell expansion. In addition, in the congenic host, 23.6% of wildtype donor T lymphocytes were expressing CD8 marker; and only 14.1% of the Cavl.4 knockout T lymphocytes were expressing CD8 marker, showing a slight reduction in the number of CD8 cells in its ability to develop and expand in the periphery.  Fig. 17  a.  Icoc.  5X  Cl) Cl)  .3D  IC  I-. 2D0•  £1 0  2)0  ‘00  SOD  5)0  10  lOX  FSC C.  •‘  10’  Ly5.2  ID,  10  0 C)  ID  CD8  —  r  Fig. 17. Cavl.4 calcium charmel is important for peripheral T cell expansion and survival of T lymphocytes. Bone marrow transfer experiment, a. showing granularity and size of recipient splenocytes, b. showing gating of the C57B 1/6 wildtype and Cavi .4 knockout T lymphocytes. C. showing the CD4 and CD8 distribution of C57B 1/6  43  wildtype T lymphocytes d. showing the CD4 and Cd8 distributioi ofCavl.4 knockout T lymphocytes. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. Results were found to be significant with p <0.01.  3.6 Tetramer analysis To further investigate on the effects of the Cavl.4 calcium channel knockout on cell mediated immune responses, we analyzed the ability of Cavi .4 knockout mice to generate vesicular stomatitis virus (VSV) specific cytotoxic T lymphocytes (CTLs) by in vivo priming followed by in vitro re-stimulation (Fig. 18). The results indicate that the number of VSV specific CTLs generated by Cavi .4 knockout mice is 4 times less than that generated by C57B1/6 wildtype mice. This demonstrates a severe impairment in the ability of Cavl.4 knockout mice to generate antigen specific CTLs, illustrating the severe immunodeficiency that the Cavi .4 knockout mice demonstrates.  44  Fig. 18  WT  KO  a.  102  C, 10’  100 100  WT  KO  NoTetramer  C. G)  E  102  10’  100  10’  102  2 1o  io  100  102  10’  100  0 1o  100  10’  102  3 1o  4 io  CD8 Fig. 18. Cavl .4 calcium channel is important in generating antigen specific CTLs. Tetramer analysis. a. showing CD4, CD8 profile ofC57Bl/6 wildtype mice. b. showing CD4, CDd8 profile of Cavl.4 knockout mice. c. showing VSV-tetramer specific cytotoxic T cells of wildtype mice .d. showing VSV-tetramer specific cytotoxic T cells of knockout mice. e. control with no tetramer. Results shown are representative FACS plots. Three mice were used in this experiment in 2 replicates. Results were found to be significant with p <0.01.  3.7 Chromium release assay In order to establish whether the limited repertoire of antigen specific cytotoxic T lymphocytes are able to mount appropriate cytolytic effector functions, a standard chromium release assay was conducted. Chromium release assay uses the amount of radioactive Chromium that the target cells release when killed, to measure the ability of the CTLs to perform target killing response. Results indicate that while C57B1/6 wildtype mice mounted appropriate CTL target killing response as expected, Cavi .4 knockout mice wasn’t able to effectively mount effector functions to effectively kill target cells on a cell per cell basis in comparison to the  45  wildtype (Fig. 19). These results indicate that Cay 1.4 knockout mouse model generates reduced number of CD8 SP and CD4 SP cells, which are chronically exhausted and have reduced effector function.  Fig. 19  CTL WT pep •-* WT+ pep KO-.pep 1(0 + pep -.-  L 1  0  Effecior: Target Ratio Fig. 19. Cavl.4 calcium channel is important in CTL killing. CTL assay. Yellow and dark blue line representing wildtype and Cay 1.4 knockout control, respectively, without peptide. Pink line representing wildtype incubated with peptide, showing percentage killing rate, and knockout, in light blue, showing percentage killing rate. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. Results were found to be significant with p <0.01 between WT and KO samples under the same conditions.  3.8 NF-KB nuclear mobilization expression In order to assess whether downstream T cell activation and proliferation is affected by the lack of Cay 1.4 calcium channel, NF-KB nuclear mobilization was analyzed using confocal microscopy. C57B1/6 wildtype and Cavl.4 knockout mice T lymphocytes were purified with magnetic beads and stained with anti-NF-KB monoclonal antibody and counterstained with nuclear stain at 0 minute and 120 minutes post activation with lonomycin and ConA Blue color represents the nucleus, red color represents NF-KB dye. Pink colour indicates co-localization. Results are quantified by counting the number of cells showing pink representing colocalization.  46  As shown in figure 20, following activation, while the localization of NF-icB in the nucleus was seen in the wild type T lymphocytes; the localization of NF-icB in the nucleus was largely absent in Cavi .4 knockout mice T lymphocytes. In addition, co-localization at time zero in Cavi .4 knockout mice seem higher than the corresponding wildtype mice, which suggests that T lymphocytes of the Cavl.4 knockout mice are in a state of exhaustion prior to encountering stimulation.  Fig. 20  0Mm  120 Mm  WT  KO  Fig. 20. Cay! .4 calcium channel is important for NF-icB localization. NF-icB confocal staining. Blue color representing nuclear dye, red color representing NF-icB dye. Pink colour indicating co-localization. Two graphs on top showing wildtype expression, two panels on the bottom showing Cavi .4 knockout expression. Two panels on the left indicating co-localization at time 0 minutes, and two panels on the right indicating co-localization at time 120 minutes post activation. Results shown are representative FACS plots. Three mice were used in this experiment in 3 replicates. Results were found to be significant with p 0.01.  47  CHAPTER FOUR: DISCUSSION In this study, a knockout mouse model lacking Cavi .4 calcium channel was used to elucidate the effects and biological functions of Cay 1.4 calcium channel in a physiological setting. The identity of the Cal.4 knockout mice was confirmed using PCR. Results showed that the DNA of Cavl.4 knockout mice contained the 70 bp insertion as compared to the C57B1/6 wildtype. Cavi .4 calcium channel is found to be important for both T lymphocyte extracellular and intracellular calcium influx; whereas in total splenocyte population, Cavi .4 calcium channel is only important for extracellular calcium influx but not intracellular calcium influx. We have also found older CavI .4 KO mice showed faster but reduced calcium influx profile. We have found that Cavi .4 calcium channel is important for T cell development. In the thymus, CD8 SP cells are preferentially selected. In the wildtype, the ratio of CD4 SPs to CD8 SP cells are 3 to 1; however, in the knockout, the ratio becomes 1.3 to 1. Furthermore, in the thymus, the number of CD8 SP cells in Cay 1.4 knockout are approximately 1.5 times that of the wildtype. In the thymus, there is an increase in the number of double positive T lymphocytes in the Cavi .4 knockout as compared to the wildtype, suggesting that Cavl .4 deficiency prevents double positive cells to progress into single positive cells. To further investigate on the effect of Cay 1.4 calcium channel knockout on development, thymocytes were stained with surface markers. In the thymus, a general trend of decrease in TCRf3 and CD69 were seen in the Cavi .4 knockout. This corresponds to the previous T lymphocyte subset analysis, where thymocyte development was blocked from the double  48  positive stage to CD4 or CD8 SP stage. The decrease in TCR3 and CD69 is especially severe in CD8 positive cells, confirming that development is hindered. As well, CD 127, which is an 1L7 marker that regulates survival, has shown marked decrease in the Cavi .4 knockout compared to the wildtype. One hypothesis to explain the preferential selection of CD8 SP cell development in thymus is that weak TCRIMHC engagement signals for CD8 SP cell development; whereas strong TCRJMHC engagement signals for CD4 SP cell development, the lack of Cay 1.4 calcium channel causes the calcium signaling downstream of TCRIHMC engagement to be hindered, resulting in weak downstream signaling, which eventually leads to the preferential development of CD8 SP cells instead of CD4 SP cells. In conclusion, CavI .4 calcium channel is important for the development, lineage commitment and survival of T lymphocytes. To elucidate whether the developmental defect observed in Cavi .4 calcium channel knockout mouse model stems from defective developmental conditions or whether they stem from the lack of Cay 1.4 calcium channel in these specific T cells, a bone marrow transfer experiment was conducted. Thyl .1 positive, CD45.2 positive wildtype mice bone marrow were extracted and mixed 50:50 with the bone marrow of Thyl.2 positive, CD45.2 positive Cavl.4 knockout mice bone marrow. This mixture was injected into previously eradiated Thyl .2 positive, CD45. 1 positive recipient mice. One month post injection, the spleen of the recipient mice was analyzed. It was found that the ratio of surviving wildtype to Cay 1.4 knockout T lymphocytes in the recipient mice were 44 to 1, indicating that despite normal developmental environment, T lymphocytes from Cavi .4 knockout mice were unable to survive and proliferate. Alternatively, this could indicate that the Cay 1.4 knockout T lymphocytes were outcompeted by the wildtype T lymphocytes. Furthermore, the percentage of wildtype CD4 SP cells were  49  approximately three times that of the Cay 1.4 knockout, reconfirming our previous observations on T cell lineage commitment. The effect of Cay 1.4 calcium channel on T lymphocytes in the periphery was also analyzed. The total number of T lymphocytes in the periphery was decreased in the Cavi .4 knockout as compared to the wildtype. It is also found that periphery T cells in the Cavl.4 knockout exhibits a CD44 high, CD62L low, CD 127 low phenotype, which is typically associated with chronic activation. This profile is more prominent in CD4 SP cells, which is in line with our developmental data. Whereas CD44 high, CD62L high, CD 127 high phenotype is associated with what is observed in memory T cells, we can conclude that the lack of Cavi .4 calcium channel causes lymphopenia, chronic activation of CD4 SP T cells, and a decrease in CD8 SP memory T cells. In order to elucidate the importance of Cay 1.4 calcium channel in T cell effector functions, we conducted a tetramer analysis to elucidate whether Cay 1.4 knockout mice were able to generate antigen specific cytotoxic T cells. Our results indicate that the ability of Cavi .4 knockout mice to generate VSV specific cytotoxic T cells were 3.4 times less than that of the C57B1/6 wildtype, demonstrating that Cavl.4 calcium channel is important in regulating T lymphocyte effector functions. In order to further investigate whether the limited antigen specific cytotoxic T cells were also functionally defective, we conducted a standard chromium release assay to test the ability of these cytotoxic T lymphocytes to kill target cells. Using equivalent numbers of wildtype and Cavl.4 knockout antigen specific cytotoxic T cells, it was found that while C57B1/6 wildtype antigen specific cytotoxic T cells were able to kill target cells rigorously as expected, the ability of Cavl.4 knockout antigen specific cytotoxic T cells to kill target cells is reduced. This could be  50  due to the chronically activated phenotype observed in the Cavi .4 knockout, which causes the Cay 1.4-I- cytotoxic T cells to be chronically exhausted, thereby, hamper its effector functions. Alternatively, as calcium signaling is a critical secondary messenger in the signaling transduction pathways used to initiate effector functions, it is possible that Cay 1.4 calcium channel plays crucial roles in signaling for cytotoxic killing, causing the Cavi .4-I- cytotoxic T cells unable to effectively perform cytotoxic effector functions. In order to demonstrate that the knockout of Cavi .4 calcium channel causes downstream signaling transduction pathways to be affected, we tracked the nuclear mobilization of NF-kB into the nucleus. NF-KB translocation is one of the major initial steps in T cell activation, proliferation, effector function and death. At time zero, it was observed that the Cavi .4 knockouts exhibited slightly more NF-icB translocation as compared to the wildtype. This could be due to the observed chronically activated/exhausted phenotype we have found to be associated with Cavi .4 knockout mice. At time 120 minutes, we can see a clear contrast in the number of wildtype T lymphocytes exhibiting NF-icB translocation as compared to the Cavl.4 knockout. Indicating that the lack of Cay 1.4 calcium channel induced calcium signaling causes downstream signaling transduction essential for T cell functioning to be reduced. In conclusion, Cay 1.4 is important for T cell development, lineage commitment, survival, establishing homeostasis, generating functional effector and memory T cells. It would be important to determine the function of Cay 1.4 calcium ëhannel in cell death. As well, electrophysiological profiles of the Cay 1.4 knockout mice would provide important insight in whether Cavi .4 contributes at least in part to the proposed CRAC channel electrophysiology.  51  REFERENCES 1. 2. 3. 4. 5. 6. 7.  8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.  Levitan ES. Signaling for vesicle mobilization and synaptic plasticity. Mol Neurobiol. 2008;37(1 ):39-43. 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Birx. D. L., Berger, M., and Fleisher, T. A. The interference of T cell activation by calcium channel blocking agents. (1984) J Immnol. 133, 2904-2909 Gelfand, E. W., Cheung, R. K., Grinstein, S., and Mills, G. B. Characterization of the role for calcium influx in mitogen-induced triggering of human I cells. Identification of calciumdependent and calcium-independent signals. (1986) Eur. J. Immunol. 16, 907-9 12 Padberg, W. M., Bodewig, C., Schafer, J., Muhrer, K. H., and Schwemmle, K. Synergistic immunosuppressive effect of low-dose cyclosporine A and the calcium antagonist nifedipine, mediated by the generation of suppressor cells. (1990) Transplant Proc. 22, 2337 Marx, M., Weber, M., Merkel, F., Meyer zum Buschenfelde, K. H., and Kohler, H. Additive effects of calcium antagonists on cyclosporin A-induced inhibition of I-cell proliferation. (1990) Nephrol. Dial. Transplant. 5, 1038-1044  57  120. 121.  122.  123.  124.  125.  126.  Kotturi MF, Hunt SV, Jefferies WA. Roles of CRAC and Cay-like channels in T cells: more than one gatekeeper? Trend Pharmacol Sci. 2006;27(7):360-7. Fischer, S. E., Ciccodicola, A., Tanaka, K., Curci, A., Desicato, S., D’Urso, M., Craig, I. W., 1997. Sequence-based exon prediction around the synaptophysin locus reveals a gene-rich area containing novel genes in human proximal Xp. Genomics 45, 340-347. Strom, T.M., Nyakatura, G., Apfelstedt-Sylla, E., Hellebrand, H., Lorenz, B., Weber, B.H., Wutz, K., Gutwillinger, N., Ruther, K., Drescher, B., Sauer, C., Zrenner, E., Meitinger, T., Rosenthal, A., Meindl, A., 1998. An L-type calcium-channel gene mutated in incomplete X linked congenital stationary night blindness. Nat. Genet. 19, 260—263. Boycott, K. M., Pearce, W. G. & Bech-Hansen, N. I. Clinical variability among patients with incomplete X-linked congenital stationary night blindness and a founder mutation in CACNA1 F. Can J Ophthalmol 2000;35: 204-13. McRory, J.E., Hamid, J., Doering, C.J., Garcia, E., Parker, R., Hamming, K., Chen, L., Hildebrand, M., Beedle, A.M., Feldcamp, L., Zamponi, G.W., Snutch, T.P., 2004. The CACNA1F gene encodes an L-type calcium channel with unique biophysical properties and tissue distribution. J. Neurosci. 24, 1707—1718. Jha MK, Badou A, Meissner M, MeRory JE, Freichel M, Flockerzi V, Flavell RA. Defective survival of naïve CD8+ I lymphocytes in the absence of the beta 3 regulatory subunit of voltage-gated calcium channels. Nat Immuno. 2009; 1275-82. Kappes DJ, He X, He X. CD4-CD8 lineage commitment: an inside view. Nat Immunol. 2005 ;6(8):76 1-766.  58  Appendix I: Ethics Approval  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE BREEDING PROGRAMS  Application Number: A07-0373 Investigator or Course Director: Wilfred A. Jefferies Department: Michael Smith Laboratories Animals:  Mice CacnalF 100 Mice Cacnalc 100  Approval Date: April 9, 2009 Funding Sources:  Unfunded title:  Breeding L-Type Calcium Channels and T-Lymphocytes -  The Animal Care Committee has examined and approved the use of animals for the above breeding program. This certificate is valid for one year from the above approval date provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  59  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  60  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE BREEDING PROGRAMS  Application Number: A09-0824 Investigator or Course Director: Wilfred A. Jefferies Department: Medical Genetics Animals:  Mice CaV1.4 (CACNA1F -I-) 20  Mice E3 6.7K Tg 20 Mice H2K-/- H2D-/- 100 Mice ABCF1 +1- (XK097B6) 50 Mice All Listed 0 Mice Lysine Transgenics 75 Mice Delta Yl TG, Delta 7 56.1 TG, KbWt Kb3A6 TG 150 Mice HIV nef TG 100 Mice B2M -1-, ClIta -1-, B2MXCHta -1-60 Mice Tapi -I-, Tap2 -I-, TapL -I-, Tapasin -I- 345 Mice ABCF1 -i-I- (XK097B6) 50 Mice OT—1, OT-Il, h-CAR TG 175 Mice C57B1I6, C3H, CBA, BalbIC, ICR 100 Mice Ii -I- (Invariant chain) 45  Approval Date: May 6, 2010  61  Funding Sources: Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR)  Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency: Funding Title:  Unfunded title:  Regulation of antigen processing machinery in carcinomas  The function of a rheumatoid arthritis-associated gene during mouse development and immune responses  Dissecting the molecular mechanism of HIV nef immunosubersion Canadian Institutes of Health Research (CIHR) Characterization of functional L-type calcium channels in lymphocytes US Department of Defense Examination of epigenetic activators that enhance breast tumor cell recognition Canadian Institutes of Health Research (CIHR) Regulation of antigen processing machinery in carciomas  Canadian Institutes of Health Research (CIHR) Breeding: Studies on antigen presentation in dendritic cells  N/A  62  The Animal Care Committee has examined and approved the use of animals for the above breeding program. This certificate is valid for one year from the above approval date provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102,6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  63  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE BREEDING PROGRAMS  Application Number: A04-0332 Investigator or Course Director: Wilfred A. Jefferies Department: Michael Smith Laboratories Animals:  Mice 720  Approval Date: February 3,2009 Funding Sources: Funding Agency: Funding  Unfunded title:  National Cancer Instftute of Canada Breeding: Studies on exogenous pathways of antigen presentation  N/A  The Animal Care Committee has examined and approved the use of animals for the above breeding program.  64  This certificate is valid for one year from the above approval date provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-8275 111 Fax: 604-8225O93  65  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A07-0270 Investigator or Course Director: Wilfred A. Jefferies Department: Michael Smith Laboratories Animals:  Mice C57B1/6 background carrying nef gene 105 Mice XK097 on C57B1/6 background 105 Mice C57B116 210 Mice deltaY, delta7, ABCB9, TAP1, TAP2 450  Start Date:  July 18, 2007  Approval Date:  November 5, 2009  Funding Sources: Funding Agency: Funding Title: Funding Agency: Funding Title: Funding Agency:  Canadian Institutes of Health Research (CIHR) Regulation of antigen processing machinery in carciomas  National Institutes of Health Transgenic modeling the dendritic cell cross-priming pathway  Canadian Institutes of Health Research (CIHR)  66  Funding  Regulation of antigen processing machinery in carcinomas  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Studies on Antigen Presentation in Dendritic Cells  Funding Agency:  Canadian Institutes of Health Research (CIHR) The role of HIV nef protein in AIDS cognitive disorder  Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Dissecting the molecular mechanism of HIV nef immunosubversion  Unfunded title:  N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-8275 111 Fax: 604-822-5093  67  The University of British Columbia UBC  9  Biohazard Approval Certificate  PROTOCOL NUMBER: B06-0040 INVESTIGATOR OR COURSE DIRECTOR: Wilfred A. Jefferies DEPARTMENT: Michael Smith Laboratories Facility and Room Number Building Biomedical Research Centre Michael Smith Laboratories Zoology South Campus  Other Building  Room Number 200, 300 255 141  PROJECT OR COURSE TITLE: Aspects of Antigen Presentation by Breast and Lung Carcinomas and Dendritic Cells APPROVAL DATE: December 11, 2009  START DATE: January 27, 2006  APPROVED CONTAINMENT LEVEL: 2 Genus Species and Strain Genus Species There are no items to display  Strain  FUNDING TITLE: Epigenetic regulation of antigen processing machinery in prostate carcinomas FUNDING AGENCY: Prostate Cancer Canada FUNDING TITLE: Studies on antigen presentation by dendritic cells in immune responses FUNDING AGENCY: Canadian Cancer Society Research Institute FUNDING TITLE: Regulation of antigen processing machinery in prostate carcinomas FUNDiNG AGENCY: Prostate Cancer Canada  68  FUNDING TITLE: Dissecting the molecular mechanism of HIV nef immunosubersion FUNDING AGENCY: Canadian Institutes of Health Research (CIHR) FUNDING TITLE: Characterization of functional L-type calcium channels in lymphocytes FUNDING AGENCY: Canadian Institutes of Health Research (CIHR) FUNDING TITLE: Regulation of antigen processing machinery in carciomas FUNDING AGENCY: Canadian Institutes of Health Research (CIIIR) FUNDING TITLE: Aspects of antigen presentation by breast and lung carcinomas and dendritic cells FUNDING AGENCY: Canadian Institutes of Health Research (CIIIR)  UNFUNDED TITLE: Aspects of Antigen Presentation by Breast and Lung Carcinomas and Dendritic Cells The Principal Investigator/Course Director is responsible for ensuring that all research or course work involving biological hazards is conducted in accordance with the University of British Columbia Policies and Procedures, Biosafety Practices and Public Health Agency of Canada guidelines. This certificate is valid for one year from the above start or approval date (whichever is later) provided there are no changes. Annual review is required. A copy of this certificate must be displayed in your facility. Office of Research Services 102,6190 Agronomy Road, Vancouver, V6T 1Z3 Phone: 604-827-5111 FAX: 604-822-5093  69  

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