Phospholipid signaling pathways triggered by the antigen receptors in t lymphocytes

CHAPTER INTRODUCTION 1.1 T cells in the immune system T cells are of prime importance to the immune system, having a central role in the immunosurveillance and in adaptive immune responses to antigens. T cells orchestrate functions as diverse as cytokine and antibody production, the priming of cytotoxic T lymphocytes (CTLs), and tolerance to self-antigens. The high antigen specificity, potent effector functions and long lasting immunological memory makes T cells an integral component of host defense system. Usually, T cells are restricted to recognize antigens which are presented in the context of appropriate self major histocompatibility complex (MHC). Proteins introduced into the cell cytoplasm are presented on MHC class I, whereas proteins that remain in the endosomal/lysosomal system are presented by MHC class II molecules. Antigen presenting cells (APCs) present peptides, associated with MHC, to naive T cells as well as to the memory T cells. In addition, these APCs also provide “second signals” required for optimal T cells responses. The activation of T cells by APCs results in T cell proliferation, differentiation and acquisition of effector functions as well as the stimulation of other cells of the adaptive immune system, such as B cells, to mount an effective defense against the pathogens. 1.1.1 Types of T cells T cells undergo development in the thymus, where a diverse population of T cells is produced by random recombination of T cell receptor (TCR) gene segments; V, D and J. Thus, various populations of T cells express different TCR. T cells further undergo positive and negative selection in the thymus. During positive selection, developing T cells that fail to interact with endogenous peptide-MHC complex (pMHC) not receive a survival signal and die by neglect and only those cells whose TCR binds to pMHC with low affinity undergo further development. During the process of negative selection, T cells whose TCR binds to pMHC complex in a robust manner, are eliminated by apoptosis as these cells will be prone to interact with normal self antigens (Werlen et al., 2003). Those cells that pass the selection process emigrate from the thymus and based on their TCR are called T cells, γT cells and NK T cells. Based on the expression of CD4 and CD8 coreceptors,  T cells are further subdivided into CD4+ and CD8+ T cells. CD4+ T cells are restricted to recognize peptides presented by class П MHC molecules while CD8+ cells recognize class І MHC associated peptides. Upon activation, naive CD4+ and CD8+ T cells undergo clonal expansion and acquire effector functions (Abbas and Litchman, 2003). Following activation, most of T cells undergo apoptosis and only a small fraction differentiates into memory cells. Memory T cells can further be central or effector memory T cells. Central memory T cells reside in the lymphoid organs and undergo proliferation upon stimulation by the same antigen. Effector memory T cells reside in the non-lymphoid organs and upon encountering the suitable antigen perform immediate effector functions without undergoing further proliferation. 1.1.2 T cell receptors T cells are required for maintaining tolerance to self-antigens, but they also posses the ability to recognize and eliminate the body cells that have undergone malignant transformation. Thus, T cells are able to discriminate between the fragments of “self” and “non-self” antigens as well as recognize endogenous proteins with altered expression. T cells therefore, need to exhibit high sensitivity but also high selectivity in their antigen recognition. These strenuous demands are mainly met by the T cell Antigen receptor complex (TCR) (Clements et al., 2006). 1.1.2.a T cell antigen receptor complex (TCR) T cells express highly polymorphic and clone specific TCR that recognizes the peptides presented by APCs. TCR comprises of the antigen receptor, noncovalently linked to a signaling subunit, constituted by the CD3 and δ chain (Weiss, 1993) (Fig. 1). The antigen receptor is a heterodimer of two polypeptide chains covalently linked to each other by disulfide bond. Most commonly these chains are the  and  chains constituting  TCR. Rarely are they the γandchains forming γ TCR. Each of these polypeptide chains has N terminal followed by a short hinge region containing cystine residues, a hydrophobic transmembrane region and a short cytoplasmic region. Transmembrane region has positively charged amino acid residues, lysine in  chain and lysine and arginine in β chain. The N terminal has a variable (V) region and a constant (C) region. The V region of each chain has three complementarity determining regions (CDRs) which are short stretches of amino acids where variability between different TCRs is concentrated. CDRs are the antigen binding sites of the TCR. The interaction between TCR and pMHC is highly specific as a difference of even one amino acid between MHC allotypes can profoundly effect TCR recognition (Clements et al., 2006). CD3 molecule consists of γ,  and chains which are arranged into dimers of γ and . All of the three CD3 chains contain negatively charged aspartic acid residue in their membrane-spanning segment, which binds to the positively charged residues in the transmembrane region of TCR  and  chains, thus keeping the complex intact. The cytoplasmic tail of CD3 and  chain also contain immunoreceptor tyrosine based activation motif (ITAM), one in each CD3 chain and three in  chain. These ITAMs are crucial for coupling of TCR with intracellular tyrosine kinases and hence, for the activation of TCR (Qian and Weiss, 1997). Reprinted with kind permission from Elesevier; Abbas and Lichtman (2003); Cellular and molecular Immunology; Fifth edition; W.B. Saunders Company; page 111 (Antigen receptors and accessory molecules of T lymphocytes). Figure 1: Structure of TCR. The TCR complex consists of  chains non-covalently linked to CD3 and  proteins. Association of these proteins is mediated by charged residues in their transmembrane regions. 1.1.2.b Other receptors on T cells In addition to TCR, T cells also express a number of accessory molecules/cosignaling receptors that are generally members of immunoglobulin (Ig), integrin and selectin protein families. These coreceptors bind to the ligands present on APCs or other cells such as vascular endothelial cells and play an important role in modulating T cell functions. Mainly, these receptors help to increase the strength and duration of interactions of T cells with other cells. Adhesion molecules on T cells, primarily the integrins, stabilize the attachment of the T cells to APCs, thus ensuring that T cells are engaged for long enough to trigger functional responses (Burbach et al., 2007). Many of these accessory molecules also transduce signals, such as CD4, CD8 and CD28 receptors (van der Merwe and Davis, 2003; Schmitz and Krappmann, 2006). CD4 receptor recognizes peptides associated with class II MHC, while CD8 receptor recognizes peptides presented by class I MHC molecules. The CD4 and CD8 receptors also transduce activating signals to T cells, thus modulating the response threshold of T cells. In addition, some of these accessory molecules, such as L-selectin, are needed for homing of T cells to tissues and the sites of inflammation (van der Merwe and Davis, 2003). 1.1.3 Activation of T cells Naive T cells, after leaving the thymus, continue to circulate between blood and lymph nodes looking for a suitable antigen. When a foreign antigen enters the body it is taken up by dendritic cells which, upon binding the antigen in peripheral tissue, migrate to the T cell area of regional lymph nodes via lymphatic vessels and present antigens to naive and memory T cells. The interaction between dendritic cell and T cells results in the activation of T cells and the differentiation of naive cells into effector and memory cells as well as expansion of the antigen-specific T cell pool (Mempel et al., 2004). Activated CD4+ T cells, also called helper T cells, stimulate the production of antibodies by B cells while activated CD8+ T cells, also called cytotoxic T cells, mediate lysis of the cells infected by intracellular pathogens. Under different activation conditions, naive CD4+ T cells may differentiate into subsets that secret distinct sets of cytokines and perform different effector functions. The best defined of these subsets are the T helper (TH1) and T helper (TH2) populations of CD4+ helper T cells. Under the influence of IL-12, TH0 cells differentiate into TH1 cells while in the presence of IL-4, TH0 cells differentiate into TH2 cells. TH1 cells, primarily associated with cellular immunity, secrete interferon-γ (IFN- γ), tumor necrosis factor- (TNF-) and TNF- (also called lymphotoxin) and are important for the eradication of intracellular pathogens. TH2 cells, mainly associated with humoral immunity, produce interleukin (IL) 4, 5, and 13 which are essential for optimal antibody production and elimination of extracellular microorganisms (Kidd, 2003). In addition, there is another type of CD4+ T cells called regulatory T cells (Tregs), that play important role in immune suppression by controlling the activation and expansion of aberrant overactive T cells (Valencia and Lipsky, 2007). 1.1.4 Signal transduction by the TCR complex Interaction of TCR with suitable peptide-MHC complex or cross-linking by antiTCR or anti-CD3 antibodies initiates the TCR signal transduction cascade consisting of intricate signaling networks that contain multi-protein complexes which assemble at various intracellular compartments and integrate and transmit signals that will lead to the activation of various transcription factors and elicitation of T cell functional responses. 1.1.4.a Activation of tyrosine kinases The polypeptide chains of TCR lack any intrinsic tyrosine kinase activity but are associated with other proteins that recruit adaptor molecules and enzymes to form a scaffold for the assembly of signaling molecules. Binding of TCR with a suitable pMHC results in initiation of a series of intracellular protein tyrosine phosphorylation events that include kinase recruitment and activation leading to substrate phosphorylation, subsequent mobilization of adaptor proteins and activation of several second messenger cascades (Sedwick and Altman, 2004). These phosphorylation events are initiated within seconds of TCR engagement and are sequentially mediated by three families of non-receptor protein tyrosine kinases; Src, Syk and Tec (Van leeuwen and Samelson, 1999; Nel, 2002). Src kinases p56Lck and possibly p59fyn are one of the first tyrosine kinases that were identified as crucial part of TCR signaling cascade. These Src kinases are recruited to TCR following its activation where they phosphorylate ITAMs bound to CD3 and  chain. Phosphorylated ITAMS recruit SH2 domain of ZAP-70, a 70 kDa chain associated protein tyrosine kinase of the Syk family, to the TCR where the activation loop of ZAP-70 is phosphorylated by Lck. Activated ZAP-70 in turn recruits and phosphorylates several substrates, including transmembrane adaptor molecule called linker for activated T cells (LAT) and the cytosolic adaptor protein called leukocyte protein of 76 kDa (SLP-76) (Di Bartolo et al., 1999). LAT contains tyrosine based motifs, which when phosphorylated by ZAP-70, recruit SH2 proteins including Grb2, phospholipase C-γ (PLCγ) and PI3 kinase. SLP-76 is also an adaptor protein that contains proline rich motifs and an SH2 domain that associates with other molecules such as Vav, Itk and LAT. Thus, these proteins serve as docking sites for other signaling proteins and adaptor molecules, recruiting them to the site of TCR activation (Nel, 2002). The activation of these cytoplasmic tyrosine kinases leads to downstream signaling events critical for T cells function such as flux of cytosolic calcium, activation of protein kinase C (PKC) and mitogen activated protein kinase (MAPK) pathway. The stimulation of these signaling molecules eventually results in the transcriptional activation of various genes that control T cell responses. 1.1.4.b Formation of immunological synapse Engagement of TCR induces the formation of a highly ordered, membraneassociated junction at the interface of T cell and APC termed the T-cell immunological synapse (IS) (Grakoui et al., 1999). Immunological synapse consists of a central signaling zone that surrounds clustered TCRs known as the supramolecular activation cluster (SMAC) and is further subdivided into the central SMAC (cSMAC) and peripheral SMAC (pSMAC). Different sets of signaling proteins appear to be relegated to one or the other of these distinct regions, where they can generate unique signals. TCR, the CD4, CD8 and CD28 receptors, and associated signaling molecules such as Lck and PKC are generally concentrated in cSMAC while larger and heavily glycosylated molecules such as CD44, CD45 and CD43 preferentially occupy pSMAC (Jury et al., 2007). However, it has been observed that early tyrosine phosphorylation events precede the formation of immunological synapse. Therefore, it is now beleived that initial TCR activation occurs in TCR containing microclusters which are formed with in seconds of receptor activation and contain TCR as well as cytoplasmic proteins and adaptor molecules such as Lck, ZAP-70 and LAT (Seminario and Bunnell, 2008). Later these microclusters move to the site of receptor engagement and thus, they help to shuttle the essential signaling components to immunological synapse. 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J Cell Physiol 208(1): 109-15. 204 [...]... calmodulin Activated calmodulin binds to the calmodulin-binding domain of a serine phosphatase, calcineurin and removes the autoinhibitory domain from the active site of the enzyme, allowing it to act on its substrates which include NFAT proteins Activated calcineurin binds to its docking site on NFAT where it dephosphorylates the serine residues thereby 14 causing a conformational change that uncovers the. .. repressors via their C-terminal ankyrin repeats that retain the Rel proteins in the cytoplasm (Ghosh and Karin, 2002) Prior to cell activation, NF-B proteins reside as homo or hetero dimers in the cytosol in the inactive state repressed by their association with members of inhibitor of  B (IB) family Activation of signaling pathways upon receptor stimulation results in the activation of multi-subunit complex... another important regulator of PKC activity (Newton, 1995) In addition, the enzyme is also regulated by an autophosphorylation mechanism whereby a psuedosubstrate regulatory domain occupies the active site of the enzyme Normally, inactive PKC resides in the cytosol of the cells with its active site masked by the pseudosubstrate domain and upon activation translocates to the 12 membrane in the vicinity... T cells in the quiescent state (class 1) and those that are upregulated in response to TCR activation and are involved in the feedback inhibition of TCR activation (class 2) (Liu, 2005) One of the first biochemical events, following TCR activation, is the activation of Lck which is regulated by other enzymes Prior to TCR activation, Lck is kept in inactive state by the action of a tyrosine kinase, Csk,... phosphorylate ITAMs Thus, TCR activation is regulated by an equilibrium between phosphorylation and dephosphorylation events that will determine the strength of the response (Acuto and Cantrell, 2000) The initial TCR activation is followed by attenuation and eventual termination of signal by multiple mechanisms that act in consort to regulate the signaling cascade These signaling repressors include MAPK... regulatory mechanisms operate at the transcriptional, translational and post-translational levels, including regulation by other kinases, phospholipases, scaffolds and serine/threonine protein phosphatases (Baier, 2003) These regulating factors either interact with the regulatory domain of PKC or modulate enzyme function by transphosphorylation The subcellular distribution of both the enzyme and substrate... Activated NFAT then moves to the nucleus where it occupies the NFAT binding sites on proximal and distal regions of various cytokine genes and thus, regulates transcription NFAT proteins bind their DNA target sequences with relatively weak affinity and, therefore, participate in gene trnascription in synergy with other transcription factors such as AP-1 (Savignac et al., 2007) Activation of NFAT is turned... and stimulation of receptor associated JAKs which in turn phosphorylate tyrosine residues in the receptor‟s cytoplasmic domain This results in the recruitment of several proteins with SH2 domains, including members of a family of DNA binding proteins called signal transducers and activators of transcription (STAT) STAT family of transcription factors includes seven members: STAT1, STAT2, STAT3, STAT4,... receptor complex The membrane translocation of the enzyme seems to be crucial for its activation, as that is where the accumulation of DAG occurs following agonistic stimulation When the enzyme is inactive, its autoinhibitory pseudosubstrate domain is protected from proteolysis Binding of PKC to DAG results in a significant change in the conformation of PKC, leading to the degradation of the pseudosubstrate... higher affinity for its ligands than CD28 CTLA4 is 22 upregulated in activated T cells where it competes with CD28 for its ligands and transduces negative signals to antigen stimulated T cells Thus, CTLA4 pathway acts as a negative regulator of T cell activation and responses by helping to limit and terminate them CTLA4 pathway, therefore, plays an important role in induction of tolerance and maintenance . associates with other molecules such as Vav, Itk and LAT. Thus, these proteins serve as docking sites for other signaling proteins and adaptor molecules, recruiting them to the site of TCR activation. conformation of PKC, leading to the degradation of the pseudosubstrate resulting in release of autoinhibitory interactions between the regulatory and kinase domains of the enzyme. The activation. calmodulin. Activated calmodulin binds to the calmodulin-binding domain of a serine phosphatase, calcineurin and removes the autoinhibitory domain from the active site of the enzyme, allowing it to