MODULATION OF T CELL FUNCTION BY COAGULATION FACTOR Xa. Kaustav Chatterjee

Transcription

1 MODULATION OF T CELL FUNCTION BY COAGULATION FACTOR Xa by Kaustav Chatterjee A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Medical Biophysics University of Toronto Copyright by Kaustav Chatterjee 2011

2 Modulation of T Cell Function by Coagulation Factor Xa Master of Science, 2011 Kaustav Chatterjee Department of Medical Biophysics, The University of Toronto ABSTRACT The serine protease factor Xa (FXa) plays an integral role in the coagulation cascade and has recently been implicated in a variety of proinflammatory roles, establishing it as a link between coagulation and inflammatory processes. In this thesis, I elaborate on previous literature by characterizing further the response of primary human T lymphocytes to FXa. Building on previous literature that describes the effect of FXa on whole T cell populations, I describe here the effect of FXa on both antigen-independent and antigen-dependent proliferation and costimulation of primary CD4 + and CD8 + T cells, thereby establishing an immunological role for FXa. Further, I show that FXa elicits an immediate and direct effect on T cells demonstrated by the rapid upregulation of the signalling cascade kinases, ERK1 and ERK2. Lastly, I demonstrate that the protease activated receptor 2 (PAR2) is involved in the mediation of this direct FXa effect. ii

3 ACKNOWLEDGEMENTS This thesis is the culmination of years of work and it would not have been possible without the help and support of many individuals. Firstly, I would like to thank my supervisor, André Schuh, for giving me the opportunity to work on such an interesting project and for all the support and guidance throughout the years, including the critical reading and editing of my thesis. I wish you good luck in the future! To Wendy, Joe, and Xiang-Fu, thank you for all of the support, guidance, and camaraderie, not to mention your numerous and generous donations of blood, without which the project would have come to a grinding halt. You made working in a confined space day in and day out enjoyable. I wish you every success! To all of my close friends, I thank you for being there for me all of these years through the good times and especially the bad. Lastly, to my family (my father, Probir, my mother, Chandra, and my sister, Kisha), to whom I dedicate this thesis, thank you for your undying and unconditional love and support through the years, and for your continued faith in me. This thesis truly would not have been possible without you. iii

4 TABLE OF CONTENTS Abstract... ii Acknowledgements... iii Table of Contents... iv List of Figures... vii List of Abbreviations... viii Introduction... 1 Hemostasis and the coagulation cascade... 1 Extrinsic pathway... 2 Intrinsic Pathway... 4 Regulation of procoagulant activity... 5 Fibrinolytic pathway... 6 Summary of process... 7 Factor Xa... 8 Inflammation, immunity and coagulation Immunomodulatory roles of FXa Protease activated receptors (PARs) T cell activation and function Relevance of work Materials and Methods Cell culture Peripheral Blood Mononuclear Cell (PBMC) purification Primary T cell culture for ERK-Phosphorylation assays Flow Cytometry T cell purity HLA-A2 typing Influenza A MP58-66 response iv

5 PAR1 and PAR2 expression Antigen-Dependent Stimulation Tetanus Toxoid Assays Influenza A Matrix Peptide Assay Antigen-Independent Stimulation Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) ERK Phosphorylation Assays SDS-PAGE Western Blotting Data Analysis Results FXa enhances the antigen-independent proliferation of both human primary CD4 + and CD8 + T cells.. 33 FXa enhances the antigen-dependent proliferation of human primary CD8 + T cells FXa enhances the antigen-dependent proliferation of human primary CD4 + T cells PAR1 and PAR2 are expressed by T cells PAR1 and PAR2 mrna expression is activation-independent FXa enhances ERK1/2 phosphorylation in T cells PAR2 likely mediates FXa signalling in T cells Discussion FXa enhances the proliferation of both CD4 + and CD8 + T cells FXa provides a costimulatory signal to T cells PAR2 is involved in FXa signalling in T cells Role of FXa in Innate/ Adaptive Immunity Conclusions Future Work References v

6 LIST OF FIGURES Figure 1: The Coagulation Cascade... 3 Figure 2: Structure, synthesis and activation of FX to FXa Figure 3: Structure and mechanism of activation of PARs Figure 4: The T cell synapse Figure 5: FXa enhances the proliferation of CD4+ primary human T cells, in vitro Figure 6: FXa enhances the proliferation of CD8+ primary human T cells, in vitro Figure 7: FXa enhances a primary human CD8+ T cell response to Flu MP58-66 antigen, in vitro Figure 8: FXa enhances the proliferation of primary human CD4+ T cells in response to DTT Figure 9: PAR1 and PAR2 are expressed on the surface of human primary T cells and JE6.1 cells Figure 10: Activation of JE6.1 cells does not modify PAR2 surface expression Figure 11: PAR1 mrna expression in primary human T cells and JE6.1 cells is not activation-dependent, in vitro Figure 12: PAR2 mrna expression in primary human T cells and JE6.1 cells is not activation-dependent, in vitro Figure 13: 18s rrna and ABL mrna expression in primary human T cells and JE6.1, in vitro Figure 14: FXa induces ERK1/2 phosphorylation in human primary T cells and JE6.1 cells, in vitro Figure 15: PAR2 mediates FXa signal in T cells and JE6.1 cells Figure 16: PAR1 transactivation of PAR vi

7 LIST OF ABBREVIATIONS 2-ME AA Ab ABL AICD AP APC AT-III BSA Ca 2+ CD cdna CPM DNA DTT 2-β-mercaptoethanol Amino acid Antibody Abelson murine leukemia viral oncogene homolog Activation induced cell death Activation peptide Antigen presenting cell Anti-thrombin III Bovine serum Albumin Calcium ions Cluster of differentiation Complementary deoxyribonucleic acid Counts per minute Deoxyribonucleic acid Diptheria/ Tetanus toxoid EBV Epstein Barr virus EDTA Ethylenediaminetetraacetic acid EGF Epidermal growth factor EPCR Endothelial cell Protein C receptor EPR-1 Effector cell protease receptor 1 ER Endoplasmic reticulum ERK Extracellular signal-regulated kinase FACS FBS FITC FII/ FIIa FV/ FVa FVII/ FVIIa FVIII/ FVIIIa FIX/ FIXa FX/ FXa FXI/ FXIa FXII/ FXIIa FXIII/ FXIIIa Gla GC Fluorescence-activated cell sorting Fetal bovine serum Fluorescein isothiocyanate Factor II (prothrombin)/ Factor IIa (thrombin) Factor V/ Factor Va Factor VII/ Factor VIIa Factor VIII/ Factor VIIIa Factor IX/ Factor IXa Factor X/ Factor Xa Factor XI/ Factor XIa Factor XII/ Factor XIIa Factor XIII/ Factor XIIIa Glutamic Acid domain Germinal centre vii

8 GPCR h HCEC HK HLA HRP HUVEC G-protein coupled receptor Hour Human conjunctival epithelial cell High molecular weight kininogen Human leukocyte antigen Horseradish peroxidase Human umbilical vein endothelial cell ICAM-1 Intracellular adhesion molecule 1 IFN-γ Interferon-γ IgG Immunoglobulin-G IL Interleukin IU International units JE6.1 Cells kda Jurkat E6.1 cells Kilodalton L Litre LFA-1 Lymphocyte function-associated antigen 1 M Molar mab Monoclonal antibody MAC-1 Macrophage 1 antigen MAPK Mitogen-activated protein kinase MCP-1 Monocytic chemoattractant protein 1 mg Milligram MHC Major histocompatibility complex min Minute ml Millilitre mm Millimolar MP58-66 Matrix protein peptide, residues mrna Messenger ribonucleic acid NFκB NK Cells nm PAF PAI PAR PBMC PC/ PCa PDGF PE perk PHA Nuclear factor κb Natural killer cells Nanomolar Platelet activating factor Plasminogen activator inhibitor Protease Activated Receptor Peripheral blood mononuclear cell Protein C/ Protein Ca Platelet-derived growth factor Phycoerythrin Phosphorylated extracellular signal-regulated kinase Phytohemagglutinin viii

9 PK PL PP PS PVDF RNA rrna RT-PCR SD SDS-PAGE SE sirna SMC SP Prekallikrein Phospholipid Pro-peptide Protein S Polyvinylidene fluoride Ribonucleic acid Ribosomal ribonucleic acid Reverse transcriptase polymerase chain reaction Standard deviation Sodium dodecyl sulphate polyacrylamide gel electrophoresis Standard error Short interfering ribonucleic acid Smooth muscle cell Signal peptide TCR T cell receptor TBS Tris buffered saline TBST TBS with Tween 20 TET Tetracycline TF Tissue Factor TFPI Tissue factor pathway inhibitor T h 1/T h 2 T helper cell 1/2 TM Thrombomodulin TNF-α Tumor necrosis factor α t-pa Tissue-type plasminogen activator Tris tris(hydroxymethyl)aminomethane μg μl μm u-pa vwf Microgram Microlitre Micromolar Urokinase-type plasminogen activator von Willebrand Factor ix

10 INTRODUCTION In this thesis, I elaborate on recent literature by further characterizing the response of enriched human T lymphocytes to the serine protease coagulation factor Xa (FXa). Building on previous literature, I describe here the effect of FXa on both antigen-independent and antigendependent proliferation and costimulation of enriched CD4 + and CD8 + T cells, thereby establishing a more physiologically significant immunological role for FXa. Further, I demonstrate that this effect is immediate and direct, and that the protease activated receptor 2 (PAR2) is involved. I will begin by describing the coagulation system and components of the immune system, and how the two are intimately linked, focusing on the significance of FXa. In subsequent sections, I will detail my methods, the results of my experiments that establish a clearer immunomodulatory role for FXa, and the significance of the work. Literature suggests that the coagulation system is intimately linked with immune and inflammatory responses. For example, it is now known that the initiation of coagulation is not only a hemostatic response, but also a key event in the local and systemic response to inflammatory stimuli [1-4]. I will later discuss several molecular links and evidence of crosstalk between the coagulation and inflammation systems that have also been described recently. HEMOSTASIS AND THE COAGULATION CASCADE Hemostasis involves the maintenance of the balance between procoagulant responses to prevent excessive bleeding at sites of vascular injury and the maintenance of circulation, by keeping blood in a fluid state. This process involves the regulation of the pro-coagulant 1 I n t r o d u c t i o n

11 cascades and their anti-coagulant counterparts, as well as of the fibrinolytic pathway and its regulatory elements, which are all linked via complex feedback loops and parallel pathways. The coagulation system can be viewed as a cascade of proteolytic reactions in which zymogens are cleaved to produce active proteins [5-7]. Historically, coagulation was thought to proceed via two distinct pathways; the extrinsic pathway, following damage to the blood vessel resulting in the exposure of tissue factor (TF), and the intrinsic pathway, initiated by the activation of platelets and the release of platelet granules. We now understand that the two are part of a single cascade with multiple feedback loops (Figure 1). EXTRINSIC PATHWAY Typically upon vascular injury, TF, a subendothelial/endothelial membrane glycoprotein, which is normally present in the circulation at very low levels [8], is exposed to the circulation, and initiates blood coagulation [10]. The vitamin-k dependent proenzyme, FVII, binds to the extracellular portion of TF via γ-carboxyglutamic acid residues and calcium bridges, which in turn converts it into an active serine protease (FVIIa). The TF-VIIa complex activates FIX, and subsequently FX into their active serine-protease counterparts, FXa and FIXa, respectively. FXa, along with its cofactor FVa, in the presence of calcium and phospholipids, form a prothrombinase complex that converts prothrombin (FII) to its active serine protease counterpart thrombin (FIIa). Thrombin activates platelets, and converts circulating fibrinogen monomers into insoluble fibrin polymers, both of which serve as the foundation for a blood clot. Furthermore, the generation of thrombin leads to amplification of the system, as thrombin feedback activates factor V. 2 I n t r o d u c t i o n

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13 Figure 1: The Coagulation Cascade The intrinsic cascade is initiated when contact is made between blood and exposed negatively charged endothelial cell surfaces. The extrinsic cascade is initiate upon vascular injury, which leads to the exposure of TF and the activation of FVII to FVIIa. The two pathways converge at the activation of FX to FXa. The activation of FVII by FXa (red arrow), and of FIX by TF-VIIa (orange arrow), further links the two pathways. FXa hydrolyzes and activates prothrombin to thrombin, which then converts fibrinogen to fibrin and activates FXIII to FXIIIa. In a positive feedback loop (green arrows), thrombin also activates FXI, FVIII and FV, thereby amplifying the coagulation cascade. Finally, FXIIIa crosslinks fibrin polymers, thereby solidifying the clot. Ca, Calcium ions, HK, high molecular weight kininogen; PK, prekallikrein; PL, phospholipid. 3 I n t r o d u c t i o n

14 INTRINSIC PATHWAY Thrombin can also be generated through the initiation of the intrinsic pathway [reviewed in 5-7]. Upon vascular damage, FVIII-von Willebrand factor (vwf) multimer complexes, and collagen-containing subepithelial structures, are exposed to the circulation. This allows circulating platelets to bind to the collagen and FVIII-vWF multimer complexes, undergo a morphological change, release the contents of their granules, and aggregate at the site. Simultaneously, the exposure of negatively charged phospholipids and subendothelial structures leads to the adsorption of FXII and kininogen (with bound prekallikrein and FXI). Upon binding, FXII is activated partially to its protease counterpart, FXIIa. FXIIa then cleaves and activates prekallikrein and FXI into kallekrein and FXIa, respectively. This process is amplified by the conversion of partially activated FXIIa by kallekrein into an even more kinetically potent protease. FIX, which binds to the surface of aggregated platelets via γ-carboxyglutamic acid residues that interact with calcium bridges, is activated by FXIa and remains bound to the platelets. Here, it interacts with FVIII, which in turn is activated by thrombin (which exists in trace amounts in the circulation) or by FXa (via feedback amplification). Using FVIIIa as a cofactor, in the presence of calcium and phospholipids, FIXa forms a ten-ase complex that activates circulating FX, which also binds to the surface of platelets via γ-carboxyglutamic acid residues. FXa remains bounds to the surface where it attaches to its cofactor FVa. FV is either adsorbed from plasma and cleaved by thrombin into FVa, or is released as FVa from platelet α-granules. The FXa-FVa complex then binds to, cleaves and activates circulating prothrombin into thrombin, which is 4 I n t r o d u c t i o n

15 released into the circulation. The generation of thrombin once again leads to amplification of the system as thrombin feedback activates factors V, VIII and XI. Downstream of both pathways, thrombin induces local platelet aggregation and produces fibrin monomers from plasma fibrinogen molecules. Further, it cleaves and activates FXIII to FXIIIa, which covalently links fibrin monomers into fibrin polymers that form a mesh over the adherent, aggregated platelets. Platelet contractile activity draws the polymerized fibrin mesh tightly over the injured vascular surface and away from the luminal flow of blood. Once unwanted blood flow is stopped, the fibrin polymers are gradually lysed to dissolve the clot. Both FXa and thrombin are multi-directional in their reactions between the coagulation complexes, and it is now recognized that both the extrinsic and the intrinsic pathways are complementary and are both essential for the formation of FXa and thrombin. However, the VII-TF complex is able to directly convert FIX to FIXa, and subsequently, FX to FXa, allowing the extrinsic pathway to bypass the early initiating events of the intrinsic cascade. This, and the fact that all events downstream of both pathways beginning with the formation of FXa are common to both pathways, highlights a central and critical role for FXa in the process. REGULATION OF PROCOAGULANT ACTIVITY The regulatory mechanisms, which keep procoagulant activity under tight control, serve two main functions: to limit the size of the fibrin clot thereby protecting the nearby tissue from ischemia, and to localize clot formation to the site of injury, thereby preventing systemic thrombosis. Tissue factor pathway inhibitor (TFPI) in plasma, becomes active when trace amounts of FXa are produced during the initiating events of the extrinsic pathway. When active, 5 I n t r o d u c t i o n

16 it binds directly to the TF-VIIa complex mentioned above, and slows down the activation of FIX and FX [10-13]. Another inhibitor, anti-thrombin III (AT-III), directly inactivates thrombin and several other serine proteases (factors IXa, Xa, XIa, XIIa, and TF-VIIa) [14,15]. The actions of both TFPI and AT-III are greatly enhanced in the presence of heparin and similar glycosaminoglycans [11, 16]. Normal endothelial cells express heparin sulphate (a sulphated glycosaminoglycan), which binds to and increases the activity of AT-III, thereby preventing clot formation in undamaged areas. Two other vitamin K-dependent inhibitors, Protein C (PC) and Protein S (PS), work together to inactivate FVa and FVIIIa [17, 18]. PC is converted to its active serine protease form, PCa, upon the binding of thrombin to thrombomodulin (TM) [19]. TMbound thrombin then undergoes a conformational change, loses its potent procoagulant activity, and activates PC readily. Furthermore, the activation of PC by thrombin-tm is enhanced dramatically when it is bound to the endothelial cell protein C receptor (EPCR)[20], and its activity is enhanced in the presence of PS [18]. The thrombin to thrombin-tm switch is important in the normal physiological state, as normal endothelial cells express TM that bind any circulating thrombin, thereby preventing clot formation in undamaged areas. FIBRINOLYTIC PATHWAY Following coagulation, once excessive bleeding at the site of injury has stopped, vascular repair ensues. Platelet derived growth factor (PDGF), which is released from platelet α-granules, induces proliferation of vascular smooth muscle cells and fibroblasts. The fibrin mesh then undergoes lysis under the action of plasmin, to prevent blood vessel obstruction. The zymogen, plasminogen, which circulates in the blood bound to fibrinogen, is proteolysed partially into its 6 I n t r o d u c t i o n

17 active form, plasmin, by the serine proteases, tissue-type plasminogen activator (t-pa) and urokinase-type plasminogen activator (u-pa) [21,22]. Activated plasmin remains bound to fibrin and degrades it into large soluble fragments that are released into the circulation. In addition, kallikrein generated during the initiation of the intrinsic pathway, may also convert some plasminogen to plasmin. As well, proteases released by neutrophils also degrade the fibrin polymers which are then removed by phagocytosis [23]. Since plasmin can inactivate fibrinogen, FVIII and FV (which are needed for coagulation), the plasmin inhibitor α 2 -Antiplasmin, and to a lesser degree, the non-specific protease α 2 - Macroglobulin, function to inactivate circulating free plasmin [17]. Plasminogen activator inhibitors (PAIs) in the plasma, such as PAI-1 also regulate plasminogen activation by forming complexes with unbound t-pa, and removing it rapidly from circulation. The balance of t-pa and PAI-1 is essential for the modulation of fibrinolytic activity [24,25]. SUMMARY OF PROCESS The key events of coagulation and fibrinolysis can be summarized as follows. The binding of FVIIa to TF converts FX to FXa, which using factor FVa as a cofactor, converts prothrombin to thrombin. Thrombin induces fibrin polymerization and platelet activation, and in trace amounts (in areas that are undamaged) can bind to TM ensuring proper blood flow. Once blood loss ceases, the fibrin mesh is gradually dissolved, and vascular repair ensues under the activity of PDGF. 7 I n t r o d u c t i o n

18 FACTOR XA The vitamin K-dependent serine protease FXa plays a central role in coagulation since components of the coagulation downstream of it are common to both coagulation pathways [5]. The inactive form of FXa, FX, is synthesized in the liver and requires several posttranslational modifications during biosynthesis before it can be released into the plasma as a two-chain polypeptide (Figure 2). Following synthesis of the protein and transport to the endoplasmic reticulum (ER), the signal peptide and adjoining propeptide are removed. This targets a vitamin K-dependent carboxylase to the amino terminus where it carboxylates the γ- carbons of glutamic acid residues in a glutamic acid rich domain (Gla domain). This process occurs, as well, with other vitamin-k dependent proteases such as thrombin, FVII,FIX, PC and PS [17]. Following γ-carboxylation, the single chain precursor is converted into a two-chain molecule consisting of a 17kDa light chain and a 45kDa heavy chain held together by a disulphide bridge. This γ -carboxylation is essential for procoagulant activity as it allows the protein to bind calcium, which then mediates binding to a phospholipid surface. Furthermore, the protein is glycosylated before being released into the bloodstream as the zymogen, FX. The light chain of FX contains two epidermal growth factor (EGF) homology domains, the first of which contains a Ca 2+ binding site that acts as a hinge to fold the EGF and Gla domains together. This allows FX to recognize and bind to cellular membranes. The heavy chain contains the activation peptide, which is cleaved during coagulation to induce a conformational change in the molecule that renders the adjacent latent serine protease catalytic domain active. Activation of FX is mediated by the TF-FVIIa and FVIIIa-FIXa complexes during coagulation, as 8 I n t r o d u c t i o n

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20 Figure 2: Structure, synthesis and activation of FX to FXa. The mature FX molecule is a two-chain polypeptide consisting of a 17-kDa light chain joined to a 45-kDa heavy chain by a disulfide bond (-S-S-). The light chain contains a cluster of glutamic acid residues (EE) in its amino terminus (Gla domain) and two epidermal growth factor (EGF) domains (inter-egf domain). The first EGF homology domain contains a Ca 2+ binding site that acts as a hinge to fold the EGF and Gla domains toward one another, thereby forming a region involved in the recognition of cellular membrane binding epitopes. The carboxy-terminal heavy chain contains most of the carbohydrate moieties, as well as the latent serine protease catalytic domain and the activation peptide. The synthesis of FX involves several post-translational processing events (green arrow heads). Following synthesis of the protein and transport into the endoplasmic reticulum, the signal peptide (SP) is removed. Subsequent endopeptidase cleavage releases the adjoining propeptide (PP), thereby targeting the Gla domain for γ- carboxylation (+) by a vitamin K-dependent carboxylase, and converts the single-chain precursor into a two-chain molecule. The protein is also glycosylated prior to being secreted into the bloodstream as a mature, inactive zymogen. During blood coagulation, activation of FX by the FVIIa-TF or FIXa-FVIIIa complexes results in a proteolytic cleavage at Arg42-Ile53 (red arrow head) of the heavy chain, thereby releasing a 52 aa activation glycopeptides from its amino terminus, and yielding the proteolytically active molecule, FXa. 9 I n t r o d u c t i o n

21 well as by Cathepsin G or CD11b while bound to the CD11b/CD18 complex (Mac-1) on monocytes [28]. Activation by the extrinsic and intrinsic complexes proceeds via proteolytic cleavage at Arg 52 -Ile 53 of the heavy chain [5], resulting in the release of a 52aa activation glycopeptide and the formation of an active two-chain peptide. FXa can now associate with its cofactor, FVa, in the presence of Ca 2+ and phospholipids, and can rapidly convert prothrombin to thrombin. Since thrombin can mediate a variety of procoagulant activities, the generation of thrombin is a key step, and hence FXa plays a pivotal role in blood coagulation. As I will discuss below, however, FXa has also been shown to have an effect on a variety of cells that are not a part of normal hemostasis, alluding to a potential immunomodulatory role. INFLAMMATION, IMMUNITY AND COAGULATION Inflammation is the first reaction the body has to foreign pathogens or irritants. The response is largely dominated by components of the innate immune response that act quickly, in a relatively non-specific fashion, to neutralize infection. If the infection persists, a more specific adaptive immune response ensues that specifically targets the infectious agent, eventually resulting in immunity. Inflammation consists of both humoral and cellular components. Humoral elements include complement proteins, interferons, platelet activating factor (PAF), histamine, serotonin, vasoactive amines and products of the kinin system, arachadonic acid derivatives (prostaglandins and leukotrienes), and cytokines. Cellular elements include neutrophils, eosinophils, mast cells, monocytes/macrophages, natural killer (NK) cells, platelets, and endothelial cells [29-36]. An inflammatory stimulus triggers the release and involvement of these elements, which act together or in sequence, to amplify the inflammatory response and 10 I n t r o d u c t i o n

22 to modify and regulate it, until the inflammatory agent is removed, whereby the inflammatory mediators dissipate, and are catabolyzed or are inhibited. There is a growing body of evidence suggesting interaction at multiple levels among the various elements of coagulation and innate immune processes. These include interactions at the cellular and humoral levels. The simultaneous activation of these two processes is a highly preserved survival strategy tracing back to early eukaryotic evolution before the separation of plants and invertebrate animals [37]. Structural homology between various components of the two processes provides evidence that there exists tight evolutionary linkage between coagulation and immunity [reviewed in 4]. For example, TF has structural homology to cytokine receptors [38], the lectin domain of TM has homology to selectins involved in leukocyte adhesion [39], and the structure of EPCR is very similar to the major histocompatibility complex (MHC1)/CD1 family of molecules [40]. Therefore, it is likely that components of the two processes evolved from common ancestors in eukaryotic development, and furthermore, that these two systems have maintained a high level of integration to protect the host following tissue injury and microbial invasion. Examples of this integration are discussed below. First, there exists a third alternative pathway of coagulation initiation that involves cellular components of the immune system. Monocytes can mediate an alternative procoagulant response after the binding of FX to the integrin Mac-1 on the monocyte surface [28]. This process is independent of the initiating events of both the extrinsic and intrinsic pathways, and instead is triggered by the proteolytic cleavage of monocyte-bound FX [41,42]. This occurs in a three step process; inflammatory stimuli or ligand binding to Mac-1 induce the release of 11 I n t r o d u c t i o n

23 granule proteases [43], released Cathepsin G subsequently cleaves and activates membranebound FX at Leu 177 -Leu 178 in the zymogen s activation peptide, and the newly generated FXa remains bound to the monocyte surface and promotes procoagulant activity and thrombin formation [28]. Since TF, the main initiator of coagulation, is undetectable on normal monocytes and undisturbed endothelium [44], the assembly of procoagulant components on the surface of monocytes may be important as an initiating event for coagulation [45] and anticoagulation [6] in protected membrane microenvironments, and protease-dependent mechanisms of vascular cell activation and signal transduction. The endothelium is also a very important link between coagulation and inflammation as damaged endothelium serves as an interface for both procoagulant and proinflammatory events. For example, following vascular injury, cytokines such as interleukin-1 (IL-1), IL-6 and tumor necrosis factor α (TNF- α), three of the most important initiators of inflammation, induce the expression of TF on endothelial surfaces [46], that in turn initiates the extrinsic pathway of coagulation. Platelets also serve as an important link between the two processes. Following vascular injury and binding to subendothelial structures, activated platelets release from their granules a number of elements that modify tissue integrity [47,48]. For example, platelets secrete the cytokine IL-1, which activates leukocytes and induces neutrophil and monocyte adherence (a precursor for diapedesis, a key step in targeting immune cells to extravascular sites of infection) [49]. They are also a major source of soluble CD40 ligand [50], which upregulates cytokine and 12 I n t r o d u c t i o n

24 chemokine expression on vascular smooth muscle cells, and upregulates TF synthesis on macrophages [51]. Upon the onset the inflammation, neutrophils and monocytes rapidly migrate to the target site, where among other proinflammatory events, the expression of TF on monocytes is greatly enhanced [9, 52]. These TF-expressing monocytes can generate thrombin locally, which can then bind to TM on endothelial surface, providing another link between inflammation and coagulation [53]. There exists interaction between the two processes at the humoral level as well, whereby biochemical mediators of one process enhance, modify, or regulate the function of the other process. For example, complement activation can facilitate the assembly and release of procoagulant enzyme complexes on the surface of platelet microparticles [54]. An increase in the level of the C4 binding protein (a component of complement activation), as well as proteolytic cleavage by proteases release by neutrophils, can downregulate the activity of PS [55]. The inflammatory cytokines TNF-α and IL-1 can downregulate EPCR and TM gene transcription, that in turn downregulates the PC/PS pathway of anticoagulant activity [56], and IL-6 can enhance the responsiveness of platelets to thrombin [57]. Acute phase proteins can inhibit anticoagulant function and increase TF production [55]. As well, inflammation promotes fibrinogen synthesis [58]. Similarly, components of pro- and anticoagulant processes affect inflammatory and immune responses. For example, the TF-VIIa complex can directly activate endothelial cells, platelets and leukocytes and induce the production of proinflammatory cytokines IL-6 and IL-8 via PAR2 13 I n t r o d u c t i o n

25 [37, 59]. The PARs are also another important link between coagulation and inflammation (as discussed below, see Protease Activated Receptors). Activated protein C (PCa) can downregulate NF κ B signalling and mrna synthesis in monocytes [60-63] and endothelial cells [64], respectively, leading to the downregulation in the expression of cell surface molecules and cytokine formation. Thrombin has a multitude of effects on inflammatory processes. It is a potent chemoattractant for neutrophils [65,66] and monocytes [67], induces mast cell degranulation [68], and activates the endothelium and enhances the expression of platelet activating factor (PAF) that stimulates neutrophils [69]. Activation of platelets by thrombin also increases the expression of CD40 [70,71] ligand that in turn increases the expression of TF and subsequently the expression of IL-6 and IL-8 [50,72]. Thrombin can also induce the mitogenesis of a variety of cell types including T cells [73-77]. IMMUNOMODULATORY ROLES OF FXA Of importance to this thesis, is the observation that FXa has been implicated in a variety of immunomodulatory roles as well. For example, in addition to causing mast cell degranulation [78], it can induce the production of the proinflammatory cytokines monocytic chemoattract protein 1 (MCP-1), IL-6 and IL-8 by endothelial cells and dermal fibroblasts [79,80], as well as of IL-1 by macrophages [81]. Furthermore, when injected into the paw of rats, it induces an acute inflammatory response [78], and has also been implicated in a human mesangioproliferative glomerulonephritis model [82]. These data, together with the observation that FXa directly stimulates PBMCs and lymphocytes in vitro [83], and has a significant proinflammatory effect in vivo [78], have implicated FXa as a key link between coagulation and inflammation. 14 I n t r o d u c t i o n

26 Although membrane association of FXa occurs during coagulation and is essential for its procoagulant activity [84, 85], several lines of evidence suggest, however, that this is insufficient for its proinflammatory action, and that rather, a specific FXa receptor(s) must mediate these effects [86,87]. The discovery of a putative lymphocyte FXa receptor, deemed effector cell protease 1 (EPR-1), seemed to resolve this question [88,89]. Reports suggested that FXa binds to EPR-1 via the FXa inter-egf repeat domain, and mediates a proinflammatory effect independent of its serine protease activity [79, 83, 90-92]. However, recent studies have brought the nature and very existence of EPR-1 under suspicion [80, 93,94]. As well, previous work done in our lab and by others, demonstrates that the serine protease activity of FXa is necessary for its cellular effects, and that the inter-egf repeat domain was likely not involved in FXa membrane/receptor association [80, 87, 93, 94]. Since thrombin, another serine protease, has been shown to mediate its proinflammatory effects via the protease activated receptors (PARs), and especially via PAR1, in a protease dependent manner [75, 77, 95-98], it was speculated that FXa might mediate its cellular effects through these receptors as well [84, 87, 93, 94, ]. Consistent with this notion are recent studies that demonstrate that FXa induces Ca 2+ release and induces ERK1/2 phosphorylation via PAR1 and/or PAR2 in several nonlymphoid cells such as human umbilical vein endothelial cells (HUVECs), smooth muscle cells (SMCs), fibroblasts, human mesangial cells, osteoblasts, keratinocytes and kidney epithelial cells [82, 97, 100, ]. 15 I n t r o d u c t i o n

27 PROTEASE ACTIVATED RECEPTORS (PARS) The PARs are a family of homologous seven transmembrane, G-protein coupled receptors. To date, four members have been described in mouse and in human - PAR1, PAR2, PAR3 and PAR4. Unlike most G-protein coupled receptors, activation occurs via proteolytic cleavage of the extracellular N-terminus, rather than via ligand binding (Figure 3) [ ]. This cleavage results in the production of a novel N-terminus that acts as a tethered ligand for the receptor, with the extracellular portions of the receptor forming the ligand binding site. Upon ligand binding, a conformation change in the receptor occurs that leads to guanine nucleotide exchange on associated G proteins and initiates intracellular signalling, the downstream components of which include Ca 2+ release from the ER, and extracellular response kinase 1 and 2 (ERK1/2) phosphorylation. PARs are disposable, single-use receptors that are rarely recycled to the surface. As such, PAR-activated cells become unresponsive to further proteolytic signalling, until protein synthesis occurs to replenish the cell membrane [ ]. Of the four PARs, three are activated by thrombin (PAR1, PAR3 and PAR4) [96-98, 109], whereas PAR2 can be activated by trypsin [110] and mast cell tryptase [111], and to a lesser degree by the FVIIA/TF complex [84] and FXa [84, 86, ], but importantly, not by thrombin. Trypsin can also activate PAR4 [112, 113]. This indicates protease selectivity within the family. PARs also comprise an important link between coagulation and inflammation, as they serve as cellular sensors for serine proteases involved in coagulation and anticoagulation. For instance, the TF-VIIa bound FXa generated during initiating events of the extrinsic pathway activates 16 I n t r o d u c t i o n

28

29 Figure 3: Structure and mechanism of activation of PARs. A. Proteolytic cleavage of the extended N-terminus by a protease such as thrombin or FXa results in a neo-n-terminus that serves as a tethered ligand for the receptor. B. Binding of this ligand to its binding site (formed by the extracellular domains of the protein) is believed to trigger a conformational change in the receptor that in turn leads to guanine nucleotide exchange on associated G proteins (Gαβγ), C, and initiates intracellular signalling. 17 I n t r o d u c t i o n

30 endothelial cells via PAR1 or PAR2 [7]. In the anticoagulant PC pathway, thrombin-tm activates PC bound to EPCR, which functions as a required coreceptor for PCa-mediated signalling through endothelial cell PAR1 [40]. PARs are expressed in a variety of tissue and cell types, including skeletal muscle vascular and non-vascular smooth muscle, fibroblasts, endothelial cells, epithelial cells, exocrine glands, keratinocytes, osteoblasts, astrocytes, neurons, platelets, monocytes, and mast cells [114]. T CELL ACTIVATION AND FUNCTION In order to appreciate the potential contribution of FXa mediated signalling in T cells to the overall understanding of immune processes, it is necessary to understand T cell activation. Armed effector T cells are crucial to almost all adaptive immune responses. Adaptive immune responses are initiated when naive T cells encounter foreign peptides or antigens, through the T cell antigen receptor (TCR). These antigens are presented by a cell surface protein of the major histocompatibility complex (MHC) on the surface of an antigen-presenting cell (APC) [115], that also expresses the co-stimulatory molecules CD80 and CD86 (B7.1 and B7.2, respectively; Figure 4) [116, 117]. This engagement initiates a series of biochemical events that can induce the naive T cell to 1) enter into a pathway leading to generation of effector T cells, with the rapid onset of proliferation and production of effector cytokines (which have a variety of immunological roles) 2) enter into an antigenically non-responsive state known as anergy, or 3) undergo apoptosis by a process known as activation induced cell death (AICD). In most cases, these first encounters with antigen are thought to occur with a dendritic cell that has taken up antigen at a site of infection, migrated to local lymphoid tissue and matured to become a potent activator of naive 18 I n t r o d u c t i o n

31

32 Figure 4: The T cell synapse. Molecules on the surface of a T cell and counterstructures on an APC involved in T cell activation form the T cell synapse. The characteristic distribution of cell surface molecules, which form a supramolecular activation complex (SMAC) is shown. The TCR occupies a central region known as the csmac; a peripheral region, known as the psmac, contains adhesion molecules. Upon adhesion of the T cell to APCs, antigenic remnants are presented to the TCR via either an MHC Class I molecules or an MHC Class II molecule, that interact with either the CD8 costimulatory molecule (shown in the diagram) or with the CD4 costimulatory molecule (not shown), respectively. CTLA4, cytotoxic T lymphocyte antigen 4; ICAM1, intercellular adhesion molecule 1; LFA, lymphocyte-function associated antigen 1; MHC, major hsitocompatibility complex; PI(3)K, phosphatidylinositol-3-oh kinase; SHP2, SH2-domain containing protein tyrosine phosphatase; ZAP70, ζ-chain (TCR)-associated protein kinase, 70 kda; lck, lymphocyte protein tyrosine kinase; CD, cluster of differentiation. 19 I n t r o d u c t i o n

33 T cells. The type of response mounted depends on a variety of factors such as the affinity and duration of the interaction, and notably the presence or absence of costimulation deriving from interactions between the CD28 costimulatory receptor on T cells and the B7 molecules on APCs, as well as between the CD8 or CD4 costimulatory receptors on T cells and MHC Class I or Class II molecules, respectively, on the APCs. The absence of costimulation renders the naive T cells anergic. CD28 costimulation, in particular, is recognized as a major T cell costimulatory pathway. It has been implicated in a variety of T cell responses including T cell proliferation, IL-2 (T cell growth factor) production, prevention of anergy, and the induction of anti-apoptotic factors [116, 117]. It also plays an important role in B cell differentiation and antibody production by controlling T cell help provided to B cells and germinal center formation. Finally, CD28 also directs T cells to sites of infection and inflammation by the production of certain cytokines and the regulation of cytokine receptors. All T cell effector functions involve cell-cell interactions. When armed effector T cells recognise specific antigen on target cells, they release mediators that act directly on the target cell, altering its behaviour. The triggering of properly activated effector T cells by peptide:mhc complexes is independent of co-stimulation, so that any infected target cell can be activated or destroyed by an armed effector T cell. CD8 + cytotoxic T cells kill target cells infected with cytosolic pathogens, thereby neutralizing pathogen replication. CD4 + T helper 1 cells (T h 1) activate macrophages to kill intracellular parasites. CD4 + T h 2 cells are essential in the activation of B cells to secrete the antibodies that mediate humoral immune responses directed against extracellular pathogens. Thus, effector T cells control virtually all known effector mechanisms of the adaptive immune response. 20 I n t r o d u c t i o n

34 While costimulation is essential to T cell activation and function, the signalling mechanisms critical for costimulation remain unclear. Current understanding of the process falls short of describing exactly how signals delivered by the TCR integrate with signals from coreceptors, such as CD28. The possibility exists then, for the discovery of missing signalling pathways comprised of unique components, such as FXa, and highlight the potential contribution of this thesis to the overall understanding of the process. RELEVANCE OF WORK FXa plays a pivotal role in blood coagulation as it serves to amplify the production of thrombin, which in turn facilitates the formation of a fibrin mesh that contributes to clot formation. In addition to its procoagulant activity, FXa has also recently been implicated in a variety of immunomodulatory roles such as inducing inflammatory cytokine expression by and proliferation of a variety of cells involved in inflammation and the immune response. Consistent with these observations, recent work done in our lab demonstrated that FXa enhances the proliferation and cytokine release (IL-2, IL-4 and IFN-γ) of antigen-independently sub-maximally activated enriched human T cells in vitro, and does so in the absence of added anti-cd28 costimulation, alluding to a potential costimulatory role. These effects were reportedly not due to thrombin, FX, or FIXa. In addition, through desensitization studies, PAR1 and PAR2 were implicated as potential FXa-signal mediators. Several questions remained unanswered however. The individual contributions of CD4 + and CD8 + T cells to the overall effect of FXa on enriched human T cells remained unclear. Furthermore, as discussed before, in normal physiology, T cells are activated by APCs in an antigen-dependent fashion. Hence, whether FXa enhanced the 21 I n t r o d u c t i o n

35 proliferation of T cells activated in a more physiologically relevant scenario was yet to be determined. Finally, although receptor-agonist desensitization assays were suggestive of PAR1 and PAR2 involvement in the mediation of the FXa signal, the individual contributions of PAR1 and PAR2 were not clear. A better understanding of the aforementioned will help define a clearer immunomodulatory role for FXa, and hence, serves as a rationale for the work done in this thesis. 22 I n t r o d u c t i o n

36 MATERIALS AND METHODS CELL CULTURE Jurkat E6.1 (Jurkat T lymphoblast, Clone E6.1, ATCC Cat. No. TIB-152) cultures were maintained in complete RPMI 1640 [RPMI 1640 (Sigma-Aldrich, R8758) supplemented with sodium pyruvate (GIBCO, ), minimal essential amino acids (GIBCO, 11140), L-glutamine (Wisent Biocenter, EL), HEPES buffer (Wisent Biocenter, # EL), 2-betamercaptoethanol (Sigma-Aldrich,3M-3158), penicillin/streptomycin (Wisent Biocenter, EL), and amphotericin B (Sigma-Aldrich, A2942)] and 10% fetal bovine serum (Wisent Biocenter, ). Medium was changed every 2-4 days depending on cell density (guidelines followed were as described by ATCC). Peripheral Blood Mononuclear Cell (PBMC) purification Blood was drawn from suitable donors into vacuum collection tubes containing sodium-heparin (Vacutainer, BD ). The blood was mixed 1:1 with phosphate buffered saline (PBS; Sigma-Aldrich, D8537) and underwent ficoll density centrifugation as per manufacturer s protocol (Ficoll-Paque, GE Amersham, ). Following centrifugation at 400xg for 30min at 18 C, the buffy coat layer residing between the plasma/pbs and the ficoll interface was removed and washed extensively with PBS. This population of cells represents the PBMC population. For T cell purification, the PBMCs were further subjected to magnetic purification to yield enriched total T cells, or enriched CD4 + or CD8 + T cell populations (Human T Cell Enrichment Kit (19051), Human CD4 + T Cell Enrichment Kit (19052), and Human CD8 + T Cell Enrichment Kit (19053), StemCell Technologies). 23 M a t e r i a l s a n d M e t h o d s

37 Primary T cell culture for ERK-Phosphorylation assays Enriched T cells were cultured in complete RPMI 1640 supplemented with 10% fetal bovine serum. In addition, between 10-50IU/mL Interleukin-2 (IL-2, Peprotech Inc., ) and 2.5ug/mL phytohemagglutinin (PHA-P, Sigma, L8902) were added. Cells were allowed to grow for 2 days, following another 2 days in culture without PHA-P, prior to the ERK phosphorylation assay. FLOW CYTOMETRY T cell purity Enriched primary T cells were either stained with antibodies against the CD3, CD4, and CD8 epitopes [Phycoerythrin (PE)-conjugated anti-cd3 mouse IgG1 monoclonal antibody (mab, ebioscience, Clone UCHT1, ); Fluorescein Isothiocyanate (FITC)-conjugated anti-cd4 mouse mab (BD Bioscience Pharmingen, ); and PE-conjugated anti-cd8 mouse IgG1 mab (ebioscience, Clone HIT8a, ). Briefly, cells were resuspended in ice-cold FACS buffer [PBS + 2mM EDTA (Bioshop Canada Inc, EDT ) + 5% FBS) at a concentration of 1x10 6 cells/ml. Anti-human CD3-PE, anti-human CD4-FITC or anti-human CD8-PE antibodies were added to 100uL volumes at 1/50 (v/v) dilution followed by incubation on ice for 30 min and further washing before analysis. HLA-A2 typing PBMCs from suitable donors, purified according to the Ficoll-Paque protocol (as described above), were resuspended in ice-cold FACS buffer at 1x10 6 cells/ml. A FITCconjugated anti-human HLA-A2 Ab (a kind gift of Dr. David Spaner, University of Toronto) was added to 100uL sample volumes at a dilution of 1/50 (v/v). Following incubation on ice for 30 minutes, the cells were washed with ice cold FACS buffer twice before analysis. 24 M a t e r i a l s a n d M e t h o d s

38 Influenza A MP58-66 response Cultured PBMCs were either stained with antibodies against CD8 epitopes (described above) and/or against the V β 17 segment of the T Cell Receptor (FITCconjugated anti-v β 17 mouse mab, Clone E17-5F3, BioDesign International, P91234F), in 100uL volumes. Briefly, anti-human CD8-PE mab or anti-human V β 17-FITC mab was added at 1/50 (v/v) concentration followed by incubation on ice for 30 minutes, followed by two washes with FACS buffer. In the case of dual staining, the procedure was repeated with the second antibody. PAR1 and PAR2 expression Enriched primary T cells were resuspended in ice-cold FACS buffer at concentration of 1x10 6 cells/ml. Anti-human PAR1 mabs, ATAP2-PE (PE-conjugated antihuman-par1 mouse IgG1 mab, Clone ATAP2, Santa Cruz Biotechnology, SC13503-PE) or WEDE15 (anti-human-par1 mouse IgG1 mab, Clone WEDE15, Immunotech, Beckman Coulter, PNIM2085), or an anti-human PAR2 mab PE (mouse IgG2a mab, R&D Systems, FAB3949P) were added to 100uL samples at a concentration of 1/50 (v/v). Following incubation on ice for 30 minutes, cells were washed twice with FACS buffer prior to analysis. In the case of staining with WEDE15 mabs, cells were stained further with a secondary goat-anti-mouse Ab (PE-conjugated goat-anti-mouse mab, BD Biosciences Pharmingen, ) for 30 min on ice, and washed twice with FACS buffer prior to analysis. Appropriate isotype controls pertaining to all Abs listed above were used as controls (mouse IgG1; Santa Cruz Biotechnology SC3877, mouse IgG1-PE; Santa Cruz Biotechnology SC2866, mouse IgG2a-PE; R&D Systems 1C003P). Flow cytometry was performed on a FACSCalibur (BD Biosciences, San Jose, CA) and results were analyzed using flow cytometry analysis software (FlowJo, Treestar Inc.). 25 M a t e r i a l s a n d M e t h o d s

39 ANTIGEN-DEPENDENT STIMULATION Tetanus Toxoid Assays Post-phlebotomy, PBMCs from suitable donors were purified by Ficoll-Paque separation as described previously. A portion of the purified PBMCs were kept aside to be used as APCs. These cells were resuspended in RPMI + 10% FBS, at room temperature, at 400,000 cells/ml. 50uL (20,000 cells) and were added to wells of a 96-well plate. They were then gammairradiated at 1500 RAD (Nordion Gamma Cell Irradiator, A234). Non-irradiated PBMCs were added to wells post-gamma-irradiation as a control. The rest of the PBMCs that were not to be used as APCs, underwent CD4 + T cell enrichment via magnetic separation (as described above). Enriched CD4 + T cells were then adjusted to 1,000,000 cells/ml in RPMI + 10% FBS. 100uL of this solution (corresponding to 100,000 CD4 + T cells) were added to the gamma-irradiated and non-gamma irradiated PBMCs. 25uL of a tetanus toxoid and diphtheria toxoid cocktail (DTT; at final dilution corresponding to 1/100 in 200uL total volume, Sanofi-Pasteur, DIN ) or PHA-P at 5ug/mL final concentration were added to wells. FXa (Enzyme Research Laboratories, HFXa 3460) at concentrations corresponding to 15nM, 25nm, 75nM and 150nM was added to certain wells. In all cases, total volume was adjusted to 200uL. Cells were seeded in replicates of 4 and grown for 3 days at 37 C, 5% CO2. Cells were then pulsed with 1.0uCi of 3 H-Thymidine (Perkin Elmer, NET027X001MC) for 6 hours at 37 C, and were subsequently harvested on filter plates ( Unifilter-96, GF/C, Perkin Elmer, ) using and allowed to dry overnight. 25uL of liquid scintillation cocktail (Microscint, Packard Bioscience Company, ) were added to 26 M a t e r i a l s a n d M e t h o d s

40 each well before assessing counts per minute (CPM) using a liquid scintillation counter (Canberra Packard Top Count NXT, ). Influenza A Matrix Peptide Assay HLA-A2 + donors were tested further for a response to the Influenza A Matrix Peptide MP58-66 (residues of the matrix protein, synthesized by JPT Peptide Technologies GmBH, Germany). PBMCs from donors were cultured in AIM-V medium (GIBCO, 12055) supplemented with sodium pyruvate, minimal essential amino acids, L-glutamine, HEPES buffer, 2-betamercaptoethanol, penicillin/streptomycin, and amphotericin B, for two weeks in the presence of the MP58-66 peptide, or alternately, in the presence of the EBV peptide, BMLF1 (a kind gift of Dr. Spaner, U of T), as a control, in 6-well plates, at a concentration of 1x10 6 cells/ml. IL-2 at various concentrations, between 10IU/ml and 50IU/mL, was added every 3-4 days to certain samples. After two weeks, cells were counted via a trypan-blue exclusion assay using a hemacytometer, washed with PBS, and resuspended in ice-cold FACS buffer at a concentration of 1x10 6 cells/ml before undergoing cell-surface analysis via flow cytometry. Once positive responders were identified, the assay was repeated to determine the effect of FXa. Briefly, FXa was added at various concentrations, between 25nm and 300nM, to certain wells at the start of the assay, and once again after a week. ANTIGEN-INDEPENDENT STIMULATION Post-phlebotomy, PBMCs were purified from donors using ficoll density centrifugation (as previously described). Half of the purified PBMCs underwent CD8 + T cell enrichment, and half underwent CD4 + T cell enrichment, using magnetic separation (previously described). Cells were 27 M a t e r i a l s a n d M e t h o d s

41 then adjusted to 400,000 cells/ml in AIMV medium. Wells of a 96-well plate were coated with an anti-human CD3 mab (OKT3 hybridoma, a kind gift of Dr. Tania Watts, University of Toronto) and an anti-human CD28 mab (9.3 hybridoma, a kind gift of Dr. Tania Watts, U of T) for 2 hours prior to cell plating, and were washed once with PBS. FXa, at various concentrations, between 25nM and 300nM, was added to certain wells. 50uL of the CD4 + or CD8 + cell solution were then added to wells in replicates of 4. Total volumes were adjusted to 100uL. Cells were then grown for 2 days at 37 C, 5% CO 2, following which cells were pulsed with 1.0uCi of 3 H-Thymidine for 6 hours at 37 C. Cells were then harvested on filter plates, as described above, and were allowed to dry overnight. 25uL of liquid scintillation cocktail were added to wells before assessing CPMs using a liquid scintillation counter. REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION (RT-PCR) Total RNA was extracted from Jurkat E6.1 and primary T cells at 0h or 2h, 6h, 12h, 24h, and 48h post-stimulation with anti-human CD3 mab (1ug/mL), and anti-cd3 mab (1ug/ml) and antihuman CD28 mab (5ug/mL) using a total RNA isolation reagent (TRIZOL Reagent, Life Technologies, Invitrogen, ) according to manufacturer s instructions. 2ug of RNA were pre-treated with RNase-free DNase (Fermentas Life Sciences) prior to cdna synthesis via RT-PCR. First-strand synthesis was primed using random hexanucleotides (Fermentas Life Sciences) with a 10 min annealing incubation at 70 C. 1ug total RNA was then reverse transcribed in 20uL volumes. 1uL of cdna from each sample was amplified by PCR using primer pairs specific for human PAR1 (PAR1 forward, 5 -CGCAGAGCCCGGGACAA-3 ; PAR1 reverse 5 - GATGAACACAACGATGG-3 ), and human PAR2 (PAR2 forward 5 -TGCAGTGGCACCATCCAAGG-3 ; 28 M a t e r i a l s a n d M e t h o d s

42 PAR2 reverse 5 -GCAAACCCACCACAAACA CA-3 ).The amount of cdna synthesized from each sample was calibrated according to the relative expression of ABL or 18s rrna, as determined by agarose gel electrophoresis of RT-PCR products generated using the oligonucleotide primers ABL forward (5 - CCCAACCTTTTCGTTGCACTGT-3 ), ABL reverse (5 - CGGCTCTCGGAGGAGACGTAGA-3 ), 18s rrna forward (5 -GTAACCCGTTGAACCCCATT-3 ) and 18s rrna reverse (5 -CCATCCAATCGGTAGTAGCG-3, all oligonucleotide primers were synthesized by IDT Inc). Amplification by PCR was performed using 2.5 U Taq DNA polymerase (Choice Taq, Denville Scientific) in a buffer containing MgCl 2 (Denville Scientific CB ) and the deoxynucleotide phosphates datp, dttp, dgtp and dctp ( Fermentas Life Sciences), using a thermal cycler (Brinkmann Eppendorf, Model 5345). An initial denaturation step of 4 min at 94 C, followed by 40 cycles of 30 sec at 94 C, 30 sec at 58 C, and 45 sec at 72 C, with a final extension step of 10min at 72 C was used. PCR products were separated by electrophoresis on a 1.8 % agarose (Denville Scientific, CA3510-8) gel in TAE buffer and visualized using 0.5ug/mL ethidium bromide on a gel imager (Bio-Rad UVP Gel Doc Sys 7500). ERK PHOSPHORYLATION ASSAYS Prior to assays, Jurkat E6.1 cells were serum-starved for 18 hours by growing them in medium without the presence of FBS. For both JE6.1 and primary T cells, ~3x10 6 cells were used per sample. Cells were pelleted from culture and resuspended in complete RPMI 1640 at room temperature. In certain cases, cells were blocked with anti-human PAR1 Abs (mouse mab clone ATAP2; Santa Cruz SC13503; and mouse mab clone WEDE15) and anti-human PAR2 Abs (mouse mab clone SAM11; Santa Cruz SC13504) prior to stimulation. For blocking, cells that were 29 M a t e r i a l s a n d M e t h o d s

43 pelleted from culture were resuspended at 12x10 7 cells/ml in PBS in 25uL volumes. Blocking antibodies were added at various concentrations, followed by incubation on ice for 30min. For antigen-independent sub-maximal stimulation of T cells, wells of a 12-well plate were precoated overnight with anti-human CD3 Abs (OKT3 mab) at various concentrations, between 0.05ug/mL and 10ug/mL, and washed once with PBS prior to assay. For antigen-dependent submaximal stimulation of T cells, PHA-P was added to certain wells prior to addition of cells at various concentrations between 0.1ug/mL and 10ug/mL. FXa was added at various concentrations, between 25nM and 150nM, to certain wells. Cells were added to wells in total volumes of 1mL complete RPMI 1640 per well. Following a 10 min incubation at room temperature, cells were pelleted and lysates were prepared for SDS-PAGE and western blotting (see below). SDS-PAGE Lysates were prepared from cell pellets by agitation on ice for 30 minutes in NP40 lysis buffer [50mM Tris-HCl (Bioshop Canada, TRS001.1), 150mM NaCl (Bioshop Canada, SOD ), 1% Nonidet P40 detergent (Bioshop Canada, NON )] supplemented with protease inhibitor cocktail (1ug/mL each of AEBSF.HCL, aprotonin, leupeptin/pepstatin, all purchased from Bioshop Canada), and phosphatase inhibitors sodium orthovanadate (25mM, Sigma, S6508) and sodium pyrophosphate (500mM, Sigma Chemical Company, S-9515). Following centrifugation at 10,000xg for 15 minutes, the supernatants were collected and quantified for protein content using a Lowry based protein quantification assay (DC Protein Assay Kit, Bio-Rad Laboratories, ). Equal amounts of total protein (10ug) were normalized to the same volume 30 M a t e r i a l s a n d M e t h o d s

44 (adjusted with lysis buffer) and mixed 3:1 with sample buffer [1% SDS (Bioshop Canada, SDS ), 10% glycerol (Sigma), 10mM Tris-Cl, 2mM EDTA, 2-ME, Bromphenol Blue, ph 8.0]and boiled for 5 minutes. These were then size-separated electrophoretically on 10% SDS- PAGE gels run at 90V in SDS running buffer [25mM Tris, 192mM Glycine (Bioshop Canada, GLN001.5), 0.1% SDS] for 2 3 hr. The gel was then equilibrated in transfer buffer [25mM Tris, 192 nm Glycine, 20% Methanol (Caledon Laboratories Ltd, ), ph7.8] by washing for 5 minutes. Typically gels were electrotransferred overnight to PVDF membranes (Immobilon-P, Millipore Corporation, IPVH00010) by a wet transfer at 24V. Upon completion of transfer, PVDF membranes were washed with TBST [Tris buffered saline (50mM Tris-Cl, 150mM NaCl) with 1% v/v Tween-20 detergent (Bioshop Canada, TWN ), 2x5min]. WESTERN BLOTTING PVDF membranes were blocked non-specifically for 1hr at RT with blocking buffer [TBST + 5% BSA (Protease-free grade, Bioshop Canada, ALB ), ph5.0]. Following washes with TBST (2x5min), the membrane was incubated overnight, with gentle agitation, in 10mL of a rabbitanti-phospho-erk antibody (Cell Signaling Technologies, 9101S) solution (1:10,000 v/v dilution in TBST + 1%BSA). Following washes with TBST (2x5min), the membrane was incubated for 1hr, with gentle agitation, in 10mL of a goat-anti-rabbit IgG-HRP (horseradish peroxidase; Bio-Rad Labs, ) antibody solution (1:20,000 v/v dilution in TBST + 1%BSA). Following further washes, phospho-erk1/2 signals were visualized using an enhanced chemiluminescence detection system (ECL Plus Western Blotting Detection System, GE Healthcare/Amersham, RPN2132). Prestained standard markers (PageRuler Prestained Protein Ladder, Fermentas Life 31 M a t e r i a l s a n d M e t h o d s

45 Sciences, SM0671) were used to verify the approximate molecular weights. All membranes were stripped for 30 minutes at RT with stripping solution (25mM Glycine-HCl, 1% SDS, ph 2.0) and washed 2x10min with TBST. Following blocking of non-specific binding with a 1 hr incubation in blocking buffer, membranes were re-probed with polyclonal anti-total-erk1/2 Ab (Cell Signaling Technologies, 4696; 1:1000 v/v dilution) as a loading control. DATA ANALYSIS Data are presented as mean +/- SE. Statistical analysis of experimental data was performed using the two-tailed Student s t test and the level of significance was set at a probability of < M a t e r i a l s a n d M e t h o d s

46 RESULTS FXA ENHANCES THE ANTIGEN-INDEPENDENT PROLIFERATION OF BOTH HUMAN PRIMARY CD4 + AND CD8 + T CELLS Previous studies in our laboratory have demonstrated that FXa enhances the proliferation of enriched human T cells that are activated submaximally in an antigen-independent fashion, i.e. with anti-human CD3 antibodies that bind to and crosslink the CD3 epitopes that are associated with the T cell receptor, or in conjunction with anti-human CD28 antibodies that stimulate the costimulatory molecule, CD28, on the surface of T cells. Importantly, these early results were not due to thrombin, the direct downstream product of FXa in the coagulation cascade, as no prothrombin was detected in culture supernatants, no prothrombin transcripts were detected in T cells, and the effects were not inhibited by lepirudin, a hirudin analog that inhibits thrombin. It was unclear, however, what the individual contributions of subsets of T cells, CD4 + and CD8 + T cells, were to the overall effect. We wanted to determine if there was cellular specificity in the response to FXa. Hence, primary human T cells were purified into CD4 + and CD8 + T cell subpopulations and cultured for 48 hrs in the presence of plate-coated anti-human CD3 and/or anti-human CD28 antibodies and various concentrations of FXa. Following this, the cells were pulsed with 3 H-Thymidine, a nucleotide analog that is radioactively labelled and incorporates into sister DNA strands during DNA replication events, and were assessed for incorporated radioactivity using a liquid scintillation counter. In both CD4 + and CD8 + T cells, mean counts per minute (CPM) from three donors, based on 4 replicates each, demonstrate that FXa enhances the proliferation induced by submaximal activation, and that this effect is 33 R e s u l t s

47 dose-dependent (Figures 5 and 6). Importantly, the FXa effect occurs in the absence of added anti-human CD28 antibodies. This, together with the observation that FXa does not induce proliferation of T cells without prior submaximal activation (Figure 5a) suggests that FXa likely delivers a costimulatory signal to T cells. Having showed that the proliferation of both primary human CD4 + and CD8 + T cells is enhanced in the presence of FXa, we next wanted to determine if the same occurred in response to a specific antigen. This would represent a more physiologically relevant scenario of T cell activation as it requires antigen presentation to T cells via APCs, which is how native activation of T cells typically ensues in vivo. FXA ENHANCES THE ANTIGEN-DEPENDENT PROLIFERATION OF HUMAN PRIMARY CD8 + T CELLS The Influenza A Matrix Peptide, residues of the core matrix protein of the virus (MP58-66), was chosen as an antigen for primary human CD8 + T cells due to several convenient properties. First, the antigen is presented to T cells exclusively via MHC Class I molecules on APCs, ensuring that only CD8 + T cells are engaged, thereby circumventing the need to purify CD8 + T cells. There exists allelic restriction, however, as the antigen binds only to derivatives of the HLA-A*0201 allele [118] (for a review of MHC class of genes, see Introduction). For this reason, HLA-A2 + donors were first identified by flow cytometry. The typical frequency of HLA- A2 + donors is around 45% in a Caucasian population, and within an HLA-A2+ population, the frequency of HLA-A* individuals is almost 95%. To determine if the donors carried the HLA-A*0201 allele, they were tested to see if they could mount a response to MP58-66 in vitro. PBMCs from HLA-A2 + donors were cultured for two weeks in the presence of MP58-66 as well 34 R e s u l t s

48 Mean CPM Mean CPM A B Donor 3 Donor nM Fxa 0 0.5ug/mL OKT3 150nM Fxa Unstim. 2.5ug/mL OKT3 Unstim. 0.5ug/mL 9.3

49 Figure 5: FXa enhances the proliferation of CD4+ primary human T cells, in vitro. Primary human CD4 + T cells were stimulated with either 2.5 5ug/mL immobilized anti-cd3 (OKT3) mabs, or with ug/ml immobilized anti-cd3 mabs and ug/ml immobilized anti-cd28 (9.3) mabs, in the presence or absence of 75, 150, or 300 nm FXa. Proliferation after 48 hours was determined by 3 H-thymidine incorporation. A. The addition of FXa in the absence of anti-cd3 did not induce T cell proliferation. Mean counts (+SE) from three donors are shown (SE is based on 4 replicates). 35 R es u l t s

50 Mean CPM Donor ug/mL OKT3 Unstim. 0.5ug/mL nM Fxa

51 Figure 6: FXa enhances the proliferation of CD8+ primary human T cells, in vitro. Primary human CD8 + T cells were stimulated with either 3.5 5ug/mL immobilized anti-cd3 (OKT3) mabs, or with ug/ml immobilized anti-cd3 mabs and ug/ml immobilized anti-cd28 (9.3) mabs, in the presence or absence of 75, 150, or 300 nm FXa. Proliferation after 48 hours was determined by 3 H-thymidine incorporation. Mean counts (+SE) from three donors are shown (SE is based on 4 replicates). 36 R e s u l t s

52 as various concentrations of IL-2, which was used to amplify any response. To determine whether there was a response, I took advantage of another convenient property of the MP58-66 peptide. Studies have shown that the population of CD8 + T cells that proliferate in response to MP58-66 is skewed heavily (85%) towards those that bear the V β 17 rearrangement of the variable region of the TCR [119]. As such, dual staining of PBMCs with an anti-human CD8 antibody and an anti-human V β 17 antibody, at the start and at the end of the assay, as assessed by flow cytometry, revealed whether there was skewing within the CD8 + T cell population towards CD8 + / V β 17 + T cells, which would indicate that a response to MP58-66 had occurred. Donors that responded to MP58-66 were then tested further to see whether FXa enhanced the MP58-66 response. The initial percentage of CD8 + / V β 17 + within the total CD8 + population, determined by flow cytometry, typically ranged from between 2-5%. Flow cytometric analysis shows that FXa significantly enhanced the proliferative response of CD8 + T cells activated by MP58-66 (Figure 7). In certain donors, FXa enhanced the MP58-66 response between 8-10 fold more (Figure 7b). Interestingly, it appears that IL-2 signalling might be synergistic with FXa signalling to some degree, as the response to Xa appears to be masked by the addition of IL-2 in certain cases (Figure 7b, c). Similar to the results from antigen-independent assays, FXa appears to be able to stimulate T cells only when they have already been activated submaximally by antigen, as it has no effect on T cells that have not been exposed to antigen (Figure 7b), an observation consistent with the notion that FXa provides a co-stimulatory signal to T cells. 37 R e s u l t s

53 A B Donor 6 C

54 Figure 7: FXa enhances a primary human CD8+ T cell response to Flu MP58-66 antigen, in vitro. PBMCs were grown for two weeks in the presence or absence of 10ug/mL Flu peptide, 10ug/mL EBV BMLF1 peptide, 10 IU/mL IL-2, and 75 or 300nM FXa. A. Following this, two-colour flow cytometry was performed on cell cultures by staining them with anti-human V β 17 mabs and anti-human CD8 mabs to determine V β 17 positivity within CD8 + T cell population. Percentage of double positive (V β 17 + /CD8 + ), top right quadrant, shows a robust increase. B. Effects of 75nM FXa and 300nM FXa are shown on cells from Donor 6. Black bars indicate Flu peptide, grey bars indicate Epstein-Barr virus (EBV) peptide BMLF1, and clear bars indicate no antigen. No response is seen to FXa in the absence of antigenic stimulation. A robust response is seen even in the absence of added IL-2 stimulation. C. Results from another donor, Donor 13, show a robust increase in the response of CD8 + / V β 17 + T cells to Flu MP58-66 peptide in the absence of added IL-2 stimulation. The response reaches saturation with added IL-2 stimulation indicating potential synergism between FXa and IL-2 signalling. Grey bars indicate 25nM FXa, black bars indicate 300nM FXa, and white bars indicate no FXa. Mean counts (+SE) are shown (SE is based on 3 replicates). 38 R e s u l t s

55 FXA ENHANCES THE ANTIGEN-DEPENDENT PROLIFERATION OF HUMAN PRIMARY CD4 + T CELLS Next, I wanted to identify whether CD4 + T cells respond to FXa similarly. A cocktail of tetanus toxoid and diphtheria toxoid (DTT) was chosen as antigen as their presentation to T cells is restricted via MHC Class II molecules on the surface of APCs [120], which ensures that only CD4 + T cells are engaged. Since 3 H-Thymidine incorporation assays were used to measure proliferation, autologous primary PBMCs that were used as APCs were gamma-irradiated first to ensure that they did not undergo mitosis, and that any measured proliferation could be attributed solely to responsive CD4 + T cells (Figure 8b). Suitable human donors were chosen according to whether they mounted a response to the DTT cocktail, a reflection of their immunization record. Primary CD4+ T cells were mixed with autologous gamma-irradiated PBMCs at a ratio of 100,000:20,000 cells and were incubated for three days in the presence of the various concentrations of the DTT toxoid cocktail. After pulsing the cells with 3 H-Thymidine, cells were assessed for incorporated radioactivity. Once responding donors were identified, the effect of FXa on submaximally activated CD4 + T cells was evaluated. Data from several donors demonstrate that FXa enhances the proliferation of primary CD4 + T cells that are activated by the DTT cocktail (Figure 8). This effect is seen with concentrations of FXa as low as 25nM for all donors. Similar to previous results, the necessity for submaximal activation of T cells was a requirement for the effect, as cells that were exposed to FXa without simultaneous antigendependent stimulation showed no proliferative response, again implicating FXa in a costimulatory role. 39 R e s u l t s

56 Mean CPM A B Donor 1 APC PHA-P response Gamma-irradiated Non-irradiated

57 Figure 8: FXa enhances the proliferation of primary human CD4+ T cells in response to DTT. A. Enriched primary human CD4 + T cells were grown with autologous gamma-irradiated (1500 RAD) PBMCs in the presence or absence of the DTT cocktail and 15, 25, 75, or 150 nm FXa. Proliferation after 72 hours was determined by 3 H-thymidine incorporation. No proliferation is seen in response to FXa in the absence of antigen. Responses to FXa are seen as low as 25nM in all donors tested. B. As a control, autologous gamma-irradiated PBMCs were shown to be unable to undergo mitosis in response to PHA (phytohemagglutinin) compared to nonirradiated autologous PBMCs, demonstrating that any measured proliferation is attributable solely to CD4 + T cell populations. Mean counts (+SE) from three donors are shown (SE is based on 4 replicates). 40 R e s u l t s

58 The results so far demonstrated that FXa enhanced the proliferation of T cells that were activated submaximally in both antigen-independent and antigen-dependent scenarios. However, as the readouts for the assays above were taken between two days to two weeks post-stimulation, autocrine and paracrine activation feedback loops within T cell populations and between T cells and APCs are likely responsible, at least in part, for the amplification of phenotypic changes, and therefore leave ambiguous the direct consequences of FXa mediated signalling. A clearer understanding of how FXa interacts with T cells and the nature of the signal transduction was required. Since the identity of a previously determined putative FXa receptor on lymphocytes known as EPR-1 was brought under suspicion recently, the obvious next step was to determine a novel mechanism by which FXa could interact with T cells, thereby possibly shedding light as to how FXa interacts with T cells and the nature of the signal transduction. As mentioned before, the PARs have been shown to mediate signals by a variety of serineproteases, including FXa, in several non-lymphoid cells. PAR1 and PAR2 have been notably implicated in FXa mediated signalling. As such, I sought to determine the role of these receptors as a potential point of interaction between FXa and T cells. PAR1 AND PAR2 ARE EXPRESSED BY T CELLS As a preliminary step in evaluating the role that the PARs might play in mediating the FXa signal in T cells, we wanted to identify whether primary T cells and Jurkat E6.1 cells express PAR1 and PAR2 on the cell surface. In the ensuing sections of the results, experiments using the Jurkat E6.1 T lymphoblastic cell-line (JE6.1) were performed in parallel with experiments using primary human T cells, as results that were obtained using JE6.1 cells were more consistent compared 41 R e s u l t s

59 to those using primary human T cells. Enriched primary human T cells and JE6.1 cells were stained with fluorochrome conjugated antibodies directed against the extracellular N-termini epitopes of either PAR1 or PAR2 and were evaluated for fluorescence via flow cytometry. The results indicated that both PAR1 and PAR2 were expressed on both primary human T cells and JE6.1 cells (Figure 9). Interestingly, the expression of both PAR1 and PAR2 appeared to be elevated on JE6.1 cells (Figure 9c, f). As these cells lack PTEN (phosphatase and tensin homologue) leading to constitutive activation of the phosphatidylinositol 3-kinase (PI3K)- signalling pathway [as reviewed in 121], an important mitogenic pathway, I wanted to determine whether activated T cells had a distinct PAR1/PAR2 expression profile. As a preliminary observation, JE6.1 cells, when serum-starved, did not show any increase in the level of surface PAR2 expression depending on the magnitude and duration of activation (Figure 10). Thus, the slightly higher expression of PAR2 by JE6.1 cells is likely independent of activation. PAR1 AND PAR2 MRNA EXPRESSION IS ACTIVATION-INDEPENDENT To determine further whether PAR expression varied in T cells depending on the state of activation of the cells, mrna expression of PAR1 and PAR2 were evaluated in primary human T cells and Jurkat E6.1 cells at various time-points post-stimulation with anti-human CD3 and antihuman CD28 antibodies. The time-points were chosen to coincide with possible cellular events such as any immediate signal transduction that occurs in response to activation stimuli, downstream responses such as the initiation of protein synthesis, and further downstream responses such as any phenotypic changes in the cells. Total RNA was harvested from cells, depleted of genomic DNA, and converted to complementary DNA via reverse transcription 42 R e s u l t s

60

61 Figure 9: PAR1 and PAR2 are expressed on the surface of human primary T cells and JE6.1 cells. PAR1 and PAR2 surface expression (indicated by green lines) was analyzed by flow cytometry using the anti-human PAR1 mabs, ATAP2 (B. and C.) or WEDE15 (A.), and an anti-human PAR2 mab, (D., E., and F.), respectively. Unstained controls are indicated by red lines, and appropriate isotype controls are indicated by blue lines. 43 R e s u l t s

62

63 Figure 10: Activation of JE6.1 cells does not modify PAR2 surface expression. Jurkat E6.1 cells that were activated by other 1ug/mL immobilized anti-cd3 (OKT3) for 10 minutes (B.) or 6 hours (C.) show no increase in cell surface PAR2 expression (indicated by green lines), as measured by the anti-human PAR2 mab , compared to unstimulated cells (A.). Appropriate isotype controls are indicated by blue lines, and unstained controls are indicated by red lines. 44 R e s u l t s

64 using random hexamer priming. The presence of PAR1 and PAR2 mrna was confirmed via PCR amplification of segments of the PAR1 and PAR2 genes. Primers that gave rise to products spanning both exons were used in order to ensure that there was no genomic DNA contamination, the presence of which would give rise to a larger amplified product due to the inclusion of the intron. Subsequent electrophoretic separation and visualization revealed that PAR1 and PAR2 mrna expression was visible and roughly constant at all time-points (Figures 11 and 12) indicating that the expression of these proteins, at least at the mrna level, did not change with the type and duration of antigen-independent activation. mrna expression was normalized by RT-PCR analysis of 18s rrna and ABL transcripts (Figure 13). FXA ENHANCES ERK1/2 PHOSPHORYLATION IN T CELLS One of the downstream signalling events during PAR1/PAR2 receptor cleavage and activation is the phosphorylation of the MAP kinases, ERK1 and ERK2, which are also downstream signal transduction components of T cell activation. By demonstrating that ERK1/2 are phosphorylated in T cells upon exposure to FXa, we could demonstrate a direct and immediate consequence of FXa mediated signalling, and could further speculate that PAR1/PAR2 may be involved somehow in the mediation of the FXa signal. A western blotting technique was used to determine whether, and to what degree, ERK1/2 were phosphorylated upon exposure of primary human T cells and JE6.1 cells to FXa. Typically, JE6.1 cells were serum-starved prior to assays to downregulate constitutive activation of the MAPK pathway, thereby allowing any subtle changes in FXa-induced ERK1/2 phosphorylation to be revealed. Enriched primary human T cells were first cultured in medium for 4 days to allow background activation of the MAPK 45 R e s u l t s

65 .

66 Figure 11: PAR1 mrna expression in primary human T cells and JE6.1 cells is not activationdependent, in vitro. Primary human T cells and JE6.1 cells were grown in the presence of 1ug/mL anti-cd3 mabs with or without 5ug/mL anti-cd28. Total RNA from samples at O hr (10 minutes stimulation), +2hr, +6hr, +12hr, +24hr and +48hr was harvested and analyzed by RT-PCR using human PAR1- specific oligonucleotide primers. PAR1 expression by JE6.1 cells and two representative donors is shown. RT lanes indicate no genomic DNA contamination. 46 R e s u l t s

67

68 Figure 12: PAR2 mrna expression in primary human T cells and JE6.1 cells is not activationdependent, in vitro. Primary human T cells and JE6.1 cells were grown in the presence of 1ug/mL anti-cd3 mabs with or without 5ug/mL anti-cd28. Total RNA from samples at O hr (10 minutes stimulation), +6hr, +24hr and +48hr was harvested and analyzed by RT-PCR using human PAR2-specific oligonucleotide primers. PAR2 expression by JE6.1 cells and two representative donors is shown. RT lanes indicate no genomic DNA contamination. 47 R e s u l t s

69 A. B C.

70 Figure 13: 18s rrna and ABL mrna expression in primary human T cells and JE6.1, in vitro. The expression of the housekeeping genes, 18s rrna and ABL, was assessed in samples used in Figure 11., and Figure 12, as a control. RT-PCR analysis was performed using 18s rrna-specific and ABL-specific oligonucleotide primers. A. 18s rrna expression in Donors 1 and 3, B. 18s rrna expression corresponding to PAR1 expression in JE6.1 cells, and C. ABL expression corresponding to PAR2 expression in JE6.1 cells, all indicate similar levels across activation scenarios. RT lanes indicate no genomic DNA contamination. 48 R e s u l t s

71 pathway (a consequence of the purification method) to subside. Cells were activated submaximally by either plate-bound anti-human CD3 antibodies or by soluble PHA antigen, and were simultaneously exposed to FXa at various concentrations. Protein lysates from samples were evaluated for the presence and degree of ERK1/2 phosphorylation using anti-phospho- ERK1/2 antibodies. An anti-total-erk1/2 antibody was used as a loading control to normalize results prior to densitometry analysis. In both human primary T cells and JE6.1 cells that were submaximally activated, there was a robust increase in the level of ERK1/2 phosphorylation upon exposure to FXa (Figure 14). Similar to previous findings, there was a requirement for submaximal activation of T cells in order to see any FXa induced ERK1/2 phosphorylation (Figure 14a, b). As well, this effect was observed regardless of whether cells were activated submaximally by plate-bound anti-human CD3 antibody (Figure 14) or by soluble PHA antigen (Figure 15a), demonstrating that FXa enhances both antigen-independent and antigendependent activation induced ERK1/2 phosphorylation. PAR2 LIKELY MEDIATES FXA SIGNALLING IN T CELLS The results thus far were consistent with a model where PAR1 and/or PAR2 mediated the FXa signal in T cells, but fell short as far as establishing PAR1 or PAR2 as FXa receptors on T cells, and determining what their individual contributions were. I therefore performed receptor blocking studies to determine whether the FXa-induced enhancement of ERK1/2 phosphorylation was abrogated when either PAR1 or PAR2 were blocked. ERK1/2 phosphorylation assays were performed as described above using human primary T cells and JE6.1 cells. However, prior to stimulation, cells were blocked with the PAR1 blocking 49 R e s u l t s

72 Donor 5 Donor 4 Donor 1 Jurkat E6.1 cells

73 Figure 14: FXa induces ERK1/2 phosphorylation in human primary T cells and JE6.1 cells, in vitro. Primary human T cells or JE6.1 cells were stimulated for 10 minutes in the presence or absence of 0.1 1ug/mL immobilized anti-cd3 (OKT3) mab, with or without 5ug/mL immobilized anti- CD28 mab, with or without 25nM, 75nM, 150nM or 300nM FXa. Lysates were analyzed for ERK1/2 phosphorylation via western blotting using an anti-phospho-erk1/2 mab. Results were normalized using an anti-total-erk1/2 mab and analyzed via densitometry in certain cases. Results from three donors and JE6.1 cells are shown. 50 R e s u l t s

74 A. Donor 1 B. Jurkat E6.1 cells

75 Figure 15: PAR2 mediates FXa signal in T cells and JE6.1 cells. Primary human T cells or JE6.1 cells were blocked with an anti-human PAR2 mab (SAM11) prior to being stimulated for 10 minutes in the presence or absence of 0.25ug/mL immobilized anti- CD3 (OKT3) mab or 0.05ug/mL PHA, with or without 75nM or 150nM FXa. Lysates were analyzed for ERK1/2 phosphorylation via western blotting using an anti-phospho-erk1/2 mab. Results were normalized using an anti-total-erk1/2 mab and analyzed via densitometry. Blocking of PAR2 results in abrogation of FXa induced ERK1/2 phosphorylation. Results from one of two representative donors and JE6.1 cells are shown. 51 R e s u l t s

76 monoclonal antibodies (mabs), ATAP2 and WEDE15, or the PAR2 blocking mab, SAM11. The ATAP2 and WEDE15 mouse mabs target the PAR1 tethered ligand domain and the hirudin-like sequence that promotes thrombin binding and receptor cleavage, respectively. The mouse antihuman PAR2 mab SAM11 is raised against amino acid residues of PAR2 corresponding to the extracellular tethered ligand epitope, and has been shown to inhibit Trypsin-induced IL-6 release in human conjunctival epithelial cells (HCECs) by blocking protease activation of PAR2 [122]. Densitometry analysis on western blots revealed that when both primary human T cells and JE6.1 cells were blocked with anti-human PAR2 (SAM11) antibodies, the FXa-induced enhancement of ERK1/2 phosphorylation above submaximal activation-induced levels was abrogated almost completely (Figure 15). These results seemed to suggest that PAR2 plays a key role in mediating the FXa signal. Studies using PAR1 blocking antibodies were inconclusive, however, and therefore any role that PAR1 may play in mediating the FXa interaction with T cells remains unclear (see Discussion). In summary, it is my hope that the results obtained will help establish a clearer understanding of the nature of the interaction between FXa and T cells. I have shown that both CD4 + and CD8 + T cells receive a signal from FXa, likely a co-stimulatory signal, which induces an enhanced proliferative response when the cells are activated submaximally in an antigen-independent fashion (Figures 5 and 6). Furthermore, I have shown that this effect is observed when T cells are natively activated through interaction with APCs that present antigen, which represents a more physiologically relevant scenario. Specifically, FXa enhances the proliferative response of both CD8 + T cells and CD4 + T cells to Flu MP58-66 antigen (Figure 7) and to diphtheria/tetanus toxoids (Figure 8), respectively. I have also demonstrated that ERK1/2 phosphorylation in T cells 52 R e s u l t s

77 is a direct consequence of their interaction with FXa (Figure 14), thus establishing a more concrete causal role for FXa in T cell activation. Finally, I have shown that the addition of antihuman PAR2 blocking antibodies abrogates FXa-induced ERK1/2 phosphorylation (Figure 15), thereby implicating PAR2 as a key player in the mediation of the FXa signal in T cells. In the following section, I will discuss the implications of these results and any unresolved issues. As well, I will discuss possible models of how and where T cells may interact with FXa that are consistent with the results, and will outline any future studies that will help further determine the specific nature of the interaction. 53 R e s u l t s

78 DISCUSSION Although a crucial role for FXa in hemostasis is well-established, increasing evidence suggests that FXa elicits various and complex signalling events on a wide range of cell types by activating protease-activated receptor (PAR)-1 and PAR-2, which act as receptors for multiple coagulation factors (see Introduction). Of relevance to this thesis, many of the effects induced by FXa are proinflammatory in nature [78, 79, 90, 123], and have established FXa as an important link between coagulation and immune responses. However, further investigation is required in order to gain a definitive understanding of the role that FXa plays in inflammatory and immune responses. The work presented in this thesis demonstrates a clear immunomodulatory role for FXa in T cell function, further highlighting the proinflammatory nature of FXa, and overall, contributing to the growing body of work that describes the intimate linkage between coagulation and inflammation. FXA ENHANCES THE PROLIFERATION OF BOTH CD4 + AND CD8 + T CELLS From the antigen-independent stimulation studies that I have done, it is clear that FXa can directly enhance the proliferation of submaximally activated primary human T cells without the need for antigen presentation through interactions with antigen presenting cells such as dendritic cells, B cells and macrophages. Importantly, both subsets of T cells, CD4 + and CD8 +, contribute to this effect. Interestingly, this FXa-induced proliferative response is seen in the absence of added anti-cd28 mabs, suggesting a costimulatory role for FXa. This notion will be discussed further in subsequent sections. Physiologically, however, the activation of naive T cells occurs in response to specific antigenic stimuli mediated via interactions with accessory 54 D i s c u s s i o n

79 antigen presenting cells. To address this issue, I have assessed the effect that FXa has on the proliferation of T cells when the cells undergo antigen-dependent activation. Specifically, I have shown that FXa enhances the proliferation of both CD4 + and CD8 + T cells in response to the specific antigens, diphtheria/tetanus toxoid and the influenza A Matrix peptide MP58-66, respectively. Furthermore, one can speculate from the antigen-independent studies, that FXa is likely directly affecting the T cells responding to the antigens. However, due to the involvement of APCs in these assays, we must also consider any effect or interaction that FXa may have with these cells. Antigen-dependent activation of naive T cells proceeds both directly, via contact between the T cell receptor (TCR) and the MHC molecule/ antigen complex on the APC, and the various costimulatory receptors and their respective ligands, as well as more indirectly, through paracrine and autocrine cytokine feedback loops between the T cells and the APCs. Previous studies have demonstrated that FXa has a multitude of proinflammatory effects on a variety of cell types (see Introduction). Of relevance to the T cell-apc interaction, is the fact that FXa induces IL-1 release from macrophages, which among other roles, function as antigen presenting cells. IL-1 is one of three cytokines (IL-1, IL-6, and TNF-α) that are crucial during the acute phase response of inflammation. During the adaptive phase of an immune response, however, IL-1 is also crucial to macrophage activation and T helper cell costimulation. Therefore, FXa that is present during the T cell-apc interaction may enhance the activation of not only the T cell, but also the macrophage, which in turn could ensue in a positive feedback loop in activation leading to a stronger response. Indeed, there may be other such FXa induced proinflammatory effects that are as yet unknown and may serve to enhance, modulate or 55 D i s c u s s i o n

80 regulate the interaction between T cells and accessory cells. In the flu-peptide study I have presented in this thesis, the magnitude of the response may therefore be attributable not only to the effect that FXa has on responsive CD8 + T cells, but also to the effect that FXa might have on accessory cells such as B cells, dendritic cells and macrophages, potentially enhancing their respective responses. Similarly, in the diphtheria/tetanus toxoid assay I have performed, though APCs cannot undergo proliferation due to gamma irradiation, their ability to release cytokines, which is preserved at the gamma irradiation level used, may be enhanced in the presence of FXa thereby, potentially further contributing to the FXa-induced proliferative effect on CD4 + T cells. As newer studies evaluate the effects of FXa on accessory cells such as dendritic cells, B cells, and macrophages, a clearer understanding of the role that FXa plays in the T cell activation milieu will be achieved. FXA PROVIDES A COSTIMULATORY SIGNAL TO T CELLS The data presented in this thesis are consistent with a costimulatory role for FXa in T cell signalling. The strongest argument for this proposal comes from the observation that T cells must be activated submaximally, either in an antigen-independent fashion via CD3 crosslinking, or in an antigen-dependent fashion using specific antigenic stimuli, in order for any FXa enhancement of proliferation to occur. In antigen-independent studies, primary T cells must at least be activated via CD3 crosslinking to see any FXa induced proliferation. This is an interesting result, as unlike the antigen-dependent studies where T cells are activated by accessory cells, here, due to the high purity of the CD4 + and CD8 + T cell populations, the lack of accessory cells, and the absence of added anti-human CD28 mabs, one can speculate that it is 56 D i s c u s s i o n

81 unlikely that the T cells receive adequate CD28 stimulation. This suggests that FXa signalling can, to a certain degree, compensate for the lack of CD28 stimulation, in vitro, at least with regard to proliferation. However, the magnitude of the proliferative response is much greater when cells undergo both CD3 crosslinking and CD28 stimulation, via added anti-human CD28 antibodies, simultaneously with FXa stimulation, suggesting that while FXa may provide a costimulatory signal, CD28 signalling represents a more crucial activation signal. Also, physiologically, it is unlikely that T cell activation proceeds without CD28 stimulation from accessory cells, as this would render the T cell anergic or deactivated. This is consistent with literature, as to date, no surrogate costimulatory signalling pathway has been demonstrated to completely replace CD28 signalling. Further evidence for a costimulatory role for FXa comes from the antigen-dependent proliferation studies presented in this thesis. FXa robustly enhances the proliferation of CD8 + /V β 17 + in response to the Flu MP58-66 peptide antigen, but no proliferation is observed in the absence of antigenic stimulation. These results were similar to the finding that added IL-2, the major T cell growth factor, could only enhance the proliferation of CD8 + /V β 17 + T cells in the presence of antigenic stimulation, consistent with a pre-established costimulatory role for IL-2, and hence, suggestive of the same for FXa. Furthermore, what is interesting is that in the absence of added IL-2, a dose-dependent response to FXa was much more evident in comparison to when IL-2 was added, in which case, although a robust response was seen with the lowest concentration of FXa, a higher dose of FXa did not significantly increase the response. This is suggestive of potential synergism between the signalling pathways of IL-2 and FXa as the induced proliferative responses appear to be additive until a threshold is reached, 57 D i s c u s s i o n

82 and therefore, further suggestive of a costimulatory role for FXa. This same restriction on FXainduced enhancement of proliferation is observed with CD4 + T cells that are activated by the diphtheria/tetanus toxoids. No CD4 + T cell proliferation was observed in response to FXa without antigenic stimulation. Taken together, these results are suggestive of a general costimulatory role for FXa that is not T cell subset-specific. Lastly, evaluation of ERK1/2 phosphorylation, which is a more immediate and direct indicator of signal transduction, provides another line of evidence suggesting a costimulatory role for FXa. Although FXa has been shown to induce ERK1/2 phosphorylation and Ca 2+ oscillation on its own in a variety of non-lymphoid cells through PAR1 and PAR2, the lack of submaximal activation precludes any FXa-induced effect in T cells. No ERK1/2 phosphorylation is observed in T cells when they are stimulated with FXa in the absence of antigen-independent or antigendependent submaximal activation. Taking all of the above observations together, it is likely that FXa mediates a general immunostimulatory effect in T cells. The exact function of FXa, however, remains undefined. Several T cell costimulatory roles for FXa can be envisioned. The historical two signal hypothesis of T cell activation states that naive T cells require at least two signals for proper activation and immunological function one through the TCR and associated CD4 or CD8 molecule (depending on the subset of T cell), and another costimulatory signal through the CD28 molecule. It is now believed that the two signal model is too simplistic, as proper T cell activation, differentiation and function is dependent on a multitude of signalling events that occur over a long period in various physiological environments, and depending on the specific nature of the immune 58 D i s c u s s i o n

83 response [124]. A variety of molecules and ligand-receptor pairs, including lymphocyte function-associated antigen 1 (LFA-1)/ intercellular adhesion molecule (ICAM)-1 [ ], CD45 [128], CD2/CD48/CD59 [129], ICOS/B7RP-1 [130], CD40/CD40 ligand [131], 4-1BB/4-1BB ligand [132,133], and OX40/OX40L [ ], have thus far been implicated in T cell costimulation. Furthermore, within hours of activation, additional costimulatory molecules are upregulated and may serve to sustain or diversify the T cell response [ ]. However, none of the alternative costimulatory pathways can completely replace CD28 signalling in resting T cell activation, but compared to CD28, such alternative costimulatory molecules can act at different stages of T cell activation and differentiation, on different subsets of T cells, or can promote the development of different effector functions. FXa may be one such alternative costimulatory molecule, as I have shown that it can compensate for inadequate CD28 stimulation to activate T cells. It is likely, then, that FXa contributes some accessory interaction that is separate, but synergistic in some way, to the effect of CD28. What remains to be determined is when and where FXa signalling in T cells likely occurs, how FXa is presented to T cells, how the FXa-T cell signal is transduced, and lastly, the significance of such signalling. PAR2 IS INVOLVED IN FXA SIGNALLING IN T CELLS In this thesis, I have demonstrated that FXa provides a direct costimulatory signal to T cells which is reminiscent of earlier reports that suggested a direct effect of FXa on lymphocytes. In these earlier studies, FXa reportedly contributed to an alternative costimulatory pathway of T cell proliferation through binding to a novel cellular receptor deemed effector cell protease (EPR)-1. This putative FXa receptor was identified immunochemically via cross-reacting FV/Va 59 D i s c u s s i o n

84 monoclonal antibodies (mabs) [88], and shown to be expressed on monocytes and monocytoid cells, neutrophils, natural killer cells, and on a small subset of circulating CD3 + lymphocytes [88, 142]. Cloned as a 337aa protein, EPR-1 was characterized phenotypically as a T cell activationdependent antigen and was reported to bind FXa specifically [88, 89]. The inter-egf sequence of FXa was identified as the novel recognition site for EPR-1, and the EPR-1 mediated FXa effects in T lymphocytes and in other cell types were reportedly not dependent on protease activity and could be mimicked by mabs and peptides that functioned as surrogate EPR-1 ligands [78, 80, 83, 85, 90, 91, ]. Recent studies, however, have brought the very identity of EPR-1 into question. The EPR-1 gene could not be detected in the mouse or human genomes [94]. Furthermore, the cdna encoding Survivin, an inhibitor of apoptosis, was shown to be nearly identical to the reverse orientation of the reported EPR-1 cdna sequence [147]. Moreover, the various EPR-1 detecting mabs were shown to have non-epr-1 specificities [88, 89, 145, 146] bringing into question the reported expression patterns of FXa receptors on lymphocytes and other cells. As well, as the majority of the reported effects were produced using surrogate EPR-1 ligands (now of unclear specificity) rather than FXa itself, the relationship of these observations to FXa became unclear. Furthermore, several studies since have demonstrated that the serine protease activity of FXa is necessary for signalling in endothelial, vascular and smooth muscle cells [79, 92, 93]. Consistent with these observations, previous work done in our laboratory demonstrated that the serine protease activity of FXa was necessary for signalling in human primary T cells. Taking these observations together, it is likely that EPR-1 does not exist and that FXa signalling is mediated by another mechanism. 60 D i s c u s s i o n

85 The PARs are attractive FXa receptor candidates as they have been shown to mediate signalling by various other coagulation factors, the most well described being thrombin. Furthermore, the PARs are expressed on a variety of immune cells and several immunological roles for the PARs have been described, thus establishing them as an important link between blood coagulation and immune responses. The role of PAR1, the main receptor for thrombin, has been studied extensively in pathophysiology over recent decades. FXa and thrombin share PAR1 as a cellular receptor, and this led to the assumption that FXa signalling would be highly reminiscent of thrombin signalling. Therefore, the significance of FXa-induced cellular responses remained largely underexplored, and their contribution to pathophysiological processes was not recognized. Interest in FXa signalling was ignited after the realization that PAR2, rather than PAR1, acts as a key player in the progression of a wide pattern of pathologies at the fibro-proliferative interface (among others). The fibro-proliferative process, the formation of fibrous tissue in a repair or reactive process, as opposed to formation of fibrous tissue as a normal part of the tissue or organ, is intimately linked to tissue remodelling, fibrosis and cancer. Because coagulation cascade activation in general appears to be closely linked to these processes, investigators started to challenge the concept that the functional consequences of FXa-induced signal transduction are simply redundant of thrombin, dramatically boosting research linking coagulation cascade activation to the progression of various diseases. The results of these efforts have uncovered FXa signalling as a cardinal player in fibro-proliferative pathology and now have led to the realization that FXa is situated on the crossroads between coagulation and inflammation and that this is of significance in pathophysiology [as reviewed in 148]. 61 D i s c u s s i o n

86 An overwhelming amount of data suggests that FXa mediates intracellular signalling via activation of PAR1 and/or PAR2 (dependent on cell type and concentration used). Several studies have determined the preferential receptor that mediates FXa-dependent signalling. A picture emerges that FXa activates PAR2 when engaged in the ternary TF FVIIa FXa complex, whereas soluble FXa activates both PAR1 and PAR2. However, the cell type and the receptor repertoire on individual cell types is also of major importance for receptor selection by FXa. Specifically, PAR activation is also tightly regulated by heterologous receptor crosstalk. For instance, during angiogenesis, TF exerts a negative regulatory control on PAR2 signalling that is released by the phosphorylation of the cytoplasmic domain of TF. Moreover, alternative receptors, for instance those of the sphingosine 1-phosphate (S1P) receptor family, might also modify FXa-dependent PAR activation [as reviewed in 148]. The results presented in this thesis have established a role for PAR2 in mediating FXa signalling in T cells. An anti-human PAR2 blocking antibody, SAM11, that binds to the tethered ligand motif in the extracellular portion of PAR2, corresponding to amino acids 37-50, was used to determine the contribution of PAR2 to FXa signalling in T cells. This antibody has been shown to inhibit Trypsin-induced IL-6 release in human conjunctival epithelial cells (HCECs) by blocking protease activation of PAR2 [122]. The results I have presented indicate that when PAR2 is blocked in primary human T cells and JE6.1 cells, the ERK1/2 phosphorylation that is induced by FXa signalling is almost entirely inhibited. This is indicative of the preferential use of PAR2 for FXa signalling in T cells, and is consistent with certain literature. Since studies that I performed 62 D i s c u s s i o n

87 using blocking antibodies to PAR1 were inconclusive, largely due to an unexpected mitogenic effect attributed to the blocking antibodies used, a role for PAR1 in FXa signalling in T cells can neither be established nor ruled out. Notwithstanding, recent studies have shown that both PAR1 and PAR2 are involved in soluble FXa signalling. Furthermore, as certain studies have shown in other cell types, this likely proceeds via intermolecular PAR signalling whereby the activation of PAR1 transactivates uncleaved PAR2 via the tethered ligand domain of PAR1 (Figure 16). Peptides corresponding to the tethered ligand of PAR1 (SFLLRN) can also activate PAR2, but not vice versa. It is possible then, that soluble FXa signalling in T cells may proceed via a similar mechanism where PAR1 is first cleaved and then transactivates PAR2 through the action of the newly exposed tethered ligand. Thus, depending on whether T cells encounter soluble FXa or FXa in the context of ternary complexes, there may be differential usage of the PAR1 and PAR2 receptors. I will speculate as to how FXa in both these contexts may be presented to T cells, physiologically, in the following section. It remains to be determined what the consequences of signalling through one or both receptors are. ROLE OF FXA IN INNATE/ ADAPTIVE IMMUNITY In order to understand the potential role of FXa-T cell signalling in innate and adaptive immune responses, it is necessary first to understand the progression of the adaptive immune response - the events leading to the recruitment of armed effector T cells to the site of local pathogenic infection. T cells that are recruited to such sites have already been activated into effector cells by antigen presenting cells that are predominantly comprised of dendritic cells, and to a lesser degree, macrophages and memory B cells. These antigen presenting cells capture antigen at the 63 D i s c u s s i o n

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