Phospholipid signaling pathways triggered by the antigen receptors in t lymphocytes

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

CHAPTER 1 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 failto interact with endogenous peptide-MHC complex (pMHC) do 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 tointeract 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, non-covalently 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)

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

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)

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 1 (TH1) and T helper

2 (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, 6 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 TCR 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

anti-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 9 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, associated 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 signalingzone 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

membrane-regions, where they cangenerate 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 Subsequent immunological synapse formation not only further potentiates TCR signaling by providing a sustained signal for gene transcription but also helps in the eventual downregulation and cessation of signal

1.1.5 Downstream effects of TCR activation

1.1.5.a Increase in cytosolic calcium

Calcium (Ca2+) is utilized as a second messanger by essentially all cells in multicellular organisms and acts as a universal regulator of intracellular signaling Cells contain numerous intracellular Ca2+ sensors, such as calmodulin, that are activated following increase in cellular Ca2+ levels When activated, these Ca2+ sensing proteins in turn stimulate downstream targets

Ca2+ homeostasis is tightly regulated in the cells by four different channels These include voltage-gated Ca2+ channels; channels gated by physical parameters (such

as temperature, mechanical forces, etc.); channels gated by ligand/receptor interaction; and store-operated Ca2+ channels (SOCs) gated by depletion of intracellular Ca2+ stores SOCs channel open upon stimulation of receptors coupled to phospholipase C (PLC) G protein coupled receptors (GPCRs) activate phospholipase C- (PLC) while immune receptors such as Fc receptors and tyrosine kinase receptors activate phosholipase C-γ (PLCγ) (Gwack et al., 2007)

In tyrosine kinase receptors such as TCR, PLCγ is recruited by the binding of its SH2 domain to the phosphorylated tyrosines on the receptor This is followed by the tyrosine phosphorylation of PLCγ resulting in its catalytic activation Activated PLCγ cleaves membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2), to produce two second messengers, inositol-1,4,5-triphosphate (IP3) and diacylglycerol (DAG) IP3 that is produced binds to specialized IP3 receptors on the membrane of endoplasmic reticulum (ER) resulting in the release of Ca2+.The depletion of ER Ca2+ stores results in opening

of SOCs in the plasma membrane resulting in the influx of extracellular Ca2+, thereby causing the stabilization of Ca2+ signal This sequential release of Ca2+ from internal and external stores results in sustained elevation of intracellular

Ca2+ which is critical during the initial phases of T cell activation, especially for induction of cytokine gene expression and for controlling T cell cytolytic functions Sustain elevation also acts a trigger for activation of various transcription factors such as NFAT (Acuto and Cantrell, 2000; Nel, 2002)

1.1.5.b Activation of protein kinase C (PKC)

PKC is a family of serine/threonine protein kinases that has nine functionally distinct isoforms divided into three subgroups; the conventional or classical PKCs (, 1, 2, and γ), which are regulated by Ca2+ and DAG; novel PKCs (, , ν, and ), which are Ca2+ insensitive but DAG sensitive and atypical PKCs (δ and ), which lack Ca2+ or DAG binding domains (Mellor and Parker, 1998) Some of these isoforms are ubiquitously expressed while some show tissue specific distribution It is believed that the various isotypes act, in part, in distinct

signaling pathways, though, little specificity has been observed in vitro It has

been suggested that intracellular location of various isotypes, regulated by

specific scafoflds and adaptors, mainly contributes to the specificity in vivo

(Bauer and Baier, 2002)

Complex PKC 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 is also 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

membrane in the vicinity of 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 resulting in release of autoinhibitory interactions between the regulatory and kinase domains of the enzyme The activation of PKC is terminated by either the metabolism of DAG or by proteolytic cleavage of the enzyme by Calpain The degradation of PKC is particularly enhanced following prolonged stimulation (Newton, 1995; Sedwick and Altman, 2004)

PKC isoenzymes are involved in a wide range of physiological processes such as cell growth, differentiation and transformation, and play critical roles in transducing signals from a plethora of extracellular receptors, including those for hormones, neurotransmitters, growth factors and antigen receptors The enzyme can also be directly activated by phorbol esters such as PMA and other tumor promoters that can substitute for DAG PKCs can phosphorylate a number of downstream substrates such as receptors and membrane proteins, cytoskeletal proteins and enzymes PKC pathway also cross-talks with a variety of other signaling pathways in cells, such as mitogen activated protein kinase (MAPK) pathway and protein kinase D (PKD) pathway (Nishizuka, 1995)

1.1.5.c Activation of nuclear factor of activated T cells (NFAT)

NFAT, initially thought to be an inducible nuclear factor found only in activated

T cells, has now shown to be expressed by almost all the cells of the body NFAT not only plays an important role in in immune responses, but is also involved in cell proliferation, differentiation, migration, angiogenesis and musculoskeletal development (Horsley and Pavlath, 2002) The NFAT family consists of five members; NFAT1 (also called NFATc2), NFAT2 (also called NFATc1), NFAT3 (also called NFATc4), NFAT4 (also called NFATc3) and NFAT5 All family members share a conserved DNA binding domain called Rel Homology Region (RHR) In addition, NFAT contains a potent transactivation domain as well as a moderately conserved regulatory domain, called NFAT-homology region (NHR) which contains docking sites for NFAT kinases and calcineurin (Klee et al., 1998; Macian, 2005)

NFAT is normally present in the cytosol as inactive serine phosphorylated protein with its nuclear localization signal (NLS) masked NFAT proetins are activated following the engagement of receptors that are coupled to calcium signaling pathway such as antigen receptors on B and T cells, G protein coupled receptors and Fc receptors Ca2+ that is released following receptor activation binds to a cytosolic protein called 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

causing a conformational change that uncovers the NLS 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 off by the action of various protein kinases, such as casein kinase1 (CK1), glycogen synthase kinase3 (GSK3) and dual-specificity tyrosine-phosphorylation regulated kinase (DYRK) Phosphorylated NFAT exposes a nuclear export sequence (NES), which binds the exportin, Crm1, and relocates back to cytosol (Gwack et al., 2007)

Calcineurin plays an important role in calmodulin-calcineurin-NFAT module and

is a direct target of two immunosuppressants, cyclosporin A (CsA) and FK506, which complex with low molecular weight proteins called immunophilins CsA binds to cyclophilin A while FK506 binds to FK506 binding protein 12 (FKBP12) Heterodimers of drug and immunophilin form stable trimolecular complex with calcineurin and inhibit its enzymatic activity Without active calcineurin, the activity of NFAT is suppressed and the cell activation is blocked (Serfling et al., 2006)

1.1.5.d Activation of nuclear factor-B (NF-B)

NF-B is the generic name for a family of transcription factors that regulate genes involved in immune and inflammatory responses as well as in cell growth and differentiation The NF-B family comprises of five sub-units each encoded by a

distinct locus; RelA (p65), RelB, c-Rel, NF-B1 (p50 and its precursor p105) and NF-B2 (p52 and its precursor p100) NF-B proteins are characterized by the presence of a conserved N terminal domain called Rel homology domain (RHD)

Of these isoforms, RelA, RelB and c-Rel contain a C-terminal transcription activation domain (TAD) which is absent in NF-B1 and NF-B2 Therefore, NF-B1 and NF-B2 can not drive transcription rather they act as transcriptional 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 called IB kinase (IB) composed of two catalytic subunits (IB and IB) and a regulatory subunit (NEMO or IB γ) Activation of IB catalytic subunits results in phosphorylation of IB at two serine residues This results in ubiquitination of IB proteins by E3 ubiquitin ligase followed by their degradation by 26S proteasome NF-B dimers that are released then translocate to the nucleus where they bind to the promoter or enhancer regions and thus, participate in the transcriptional activation of various cytokines and cytokine receptors (Li and Verma, 2002)

Activation of a number of receptor systems such as GPCRs, tyrosine kinase receptors, cytokine and chemokine receptors, leads to stimulation of NF-B

among various receptors and seems to be specific to each receptor, though the downstream events follwing IB activation are shared regardless of the initial stimulus

Activated NF-B is downregulated through multiple mechanisms including the negative feedback pathway whereby newly synthesized IB protein binds to nuclear NF-B and exports itout to the cytosol.Thus, IB and IB act as key regulators of NF-B signaling pathway (Hayden and Ghosh, 2004)

1.1.5.e Activation of mitogen activated protein kinase (MAPK) pathway

MAPK pathway is a signal transduction pathway that is activated in response to diverse stimuli and plays in important role in both physiological as well as pathological responses such as proliferation, differentiation, stress responses, inflammation, growth arrest and apoptosis MAPK are family of serine and threonine protein kinases with well conserved evolutionary functions The pathway consists of three sequentially activated kinases which are themselves regulated by phosphorylation First are the MAPK kinase kinases (MKKKs) which phosphorylate and activate specific MAPK kinases (MKKs) MKKs in turn phosphorylate and activate MAPK through dual phosphorylation on threonine and tyrosine residues Activated MAPK in turn phosphorylate and activate various substrates (Katz et al., 2007) Various phosphatases that include phospho-tyrosine, phospho-serine/threonine, or both types of phosphoresidue phosphatases (dual-specificity phosphatase (DSP)), regulate MAPK cascade both positively and negatively (Raman et al., 2007)

Three well-characterized subfamilies of MAPK have been identified These include the extracellularsignal-regulated kinases (ERK), ERK1 and ERK2; the c-JunNH2-terminal kinases, JNK 1, JNK 2, and JNK 3 ; andp38 enzymes, p38, p38, p38 γ, and p38 The ERK pathway is mainly activated by growth factor-stimulated cell surface receptors while the latter two are mainly activated under conditions of cellular stress but also in response to growth factors (Owens and Keyse, 2007)

The most widely studied MAPK pathway is ERK 1 and 2 pathway which is activated by various stimuli such as by growth factors, cytokines, ligands for GPCRs and tyrosine kinase receptors, transforming agents and carcinogens ERK

1 and 2 are widely expressed and are involved in the regulation of meiosis, mitosis, and postmitotic functions in differentiated cells ERK 1 and 2 are both components of the three-kinase phosphorelay module thatincludes the Raf-1, B-Raf, or A-Raf as MAKKK, MEK1 and MEK2 as MAPKK and ERK1 and ERK2

as MAPK Activated ERK 1 and 2 phosphorylate and regulate the activities of a large number of substrates including various nuclear and cytoplasmic proteins and transcription factors As ERK pathway promotes cell proliferation and survival, it has been strongly implicated in the development and progression of cancer and inhibitors of the ERK pathway are enteringclinical trials as potential anticancer agents (Roberts and Der, 2007)

The JNKs were isolated and initially characterized as stress-activated protein kinases on the basis of their activation in response to inhibition of protein synthesis Later on it was found that JNKs are also involved in the activation and

phosphorylation of genes which are involved in cell differentiation and apoptosis One of the major consequences of activation of JNK pathway is the phosphorylation of DNA binding protein called c-Jun c-Jun is normally present

in the cytoplasm and translocates to the nucleus upon phosphorylation where it binds with Fos to form AP-1 complex, which then binds DNA and promotes transcription Activator protein-1 (AP-1) complex contributes to the control of many cytokine genes and is activated in response to environmental stress, radiation, and inflammatory cytokines Regulation of the JNK pathway is extremely complex as the pathway is influenced by more than 12 MAPKKKs This diversity of MAPKKKs allows a wide range of stimuli to activate this MAPK cascade As JNKs areimportant in controlling programmed cell death or apoptosis, therefore they are also an important target for the treatment of cancer (Johnson and Lapadat, 2002)

The p38 enzyme is expressed in most cell types, including the immune cells where it plays a significant role in the activation of the immune response and in the regulation of cytokine gene expression p38 MAPK are activated by various stimuli such as by hormones, ligands for GPCRs and particularly in response to stresses such as UV radiation, osmotic shock and heat shock MKK3 and MKK6 kinases phosphorylate p38 and are themselves phosphorylated by several MAPKKKs, some of which also stimulate JNK cascade (Katz et al., 2007)

1.1.5.f Production of cytokines

Cytokines include a large group of molecules including interferons (IFNs), interleukins (ILs), various colony-stimulating factors (CSFs), tumor necrosis factors (TNFs) and transforming growth factors (TGFs)

Cytokines are small proteins that bind to high affinity receptors on target cells resulting in induction of biochemical signals in the cells that profoundly affects their behavior (Paul and Seder, 1994) Cytokine receptors lack any intrinsic enzymatic activity but are constitutively associated with cytoplasmic kinases that consist of four members of the Janus Kinase (JAK) family: JAK1, JAK2, JAK3 and TYK2 Activation of cytokine receptors results in receptor dimerization 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, STAT5a, STAT5b and STAT6 Different cytokines activate different combinations of JAKs and STATs Activation of JAK/STAT pathway results in stimulation of transcription of cytokine-responsive genes and thus provides a rapid membrane to nucleus mechanism for the regulation of gene

expression (Shuai and Liu, 2003)

The role of cytokines in immunoregulation and inflammation is well established Multiple genome-wide association studies have associated the polymorphisms and mutations of cytokine receptors and their signaling components to autoimmune

disorders such as diabetes and multiple sclerosis In addition, anti-cytokine therapies are now being used for the treatment of chronic inflammatory diseases such as rheumatoid arthritis (O'Shea and Murray, 2008)

1.1.5.g Cell proliferation

One of the principle responses of T cells to TCR activation is the cell proliferation and clonal expansion The cell proliferation involves the rapid and transient expression of transcription units required for cell-cycle progression (Ballard, 2001)

Normally the non-proliferating cells stay in a resting state called G0 phase Cell proliferation requires DNA replication that occurs during a specific phase of cell cycle called S phase S phase is preceded by a gap called G1 phase during which the cell prepares for DNA synthesis S phase is followed by another gap called G2phase during which cells prepare for mitosis

The transition of proliferating cell from one phase to another occurs in an orderly fashion and is tightly regulated by a large number of proteins Key regulators of cell cycle progression are cyclin-dependent kinases (CDK), a family of serine/threonine protein kinases that are activated at specific points of the cell cycle as well as tumor suppressors such as p53 and Rb (Vermeulen et al., 2003)

1.1.6 Costimulation of T cells

Optimal activation of T cells requires two distinct but synergistic signals from APCs Firstly, an antigen specific signal mediated by the binding of the TCR to the pMHC complex on APC, and secondly, antigen nonspecific signals triggered

by cosignaling receptors expressed on surface of APCs as well as by soluble

factors such as cytokines (Bretscher, 1999) These second signals are essential for complete T cell activation, proliferation, and differentiation Activation of TCR alone, in the absence of costimulatory signal results in T cell anergy or apoptosis (Noel et al., 1996)

A number of T cell costimulatory pathways have been studied and described The best characterized interactions are the ones between CD28 receptors on T cell and CD80 (B7-1) and CD86 (B7-2) on APC; and between CD154 (CD40 Ligand) on

T cells and CD40 that is expressed by APCs, vascular endothelial cells and smooth muscle cells

CD28 is constitutively expressed by T cells, during all stages of activation, while expression of CD80 and CD86 is upregulated on APCs following antigenic stimulation Prolonged TCR activation is needed to upregulate CD86 expression which is very low in inactivated APCs Thus, it seems that CD80 helps in the initiation while CD86 helps in the potentiation of T cell responses Activation of CD28 pathway helps to lower the threshold for T cell activation, enhances the differentiation of TH0 cells, augments proliferation of antigen specific T cells and elicits T cell effector functions such as cytokine production It is still not clear if activation of CD28 only helps to augment the TCR mediated intracellular signaling cascade or if it also activates some distinct signaling pathways (Alegre

et al., 2001)

Another receptor, cytotoxic T lymphocyte antigen-4 (CTLA4), structurally homologous to CD28 is also expressed by T cells CTLA4 also binds to CD80 and CD86, and has a higher affinity for its ligands than CD28 CTLA4 is

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 of homeostasis (Rudd and Schneider, 2003)

CD40 is a transmemebrane protein whose extracellular domain is homologous to TNF receptor (TNFR) family CD40 is expressed by APCs such as dendritic cells and B cells as well as by several other cells such as epithelial and endothelial cells CD40 binds to its ligand (CD40L), a member of TNF family, expressed by activated T cells, eosinophils and platelets Engagement of CD40 by CD40L results in an enhanced expression of B7 molecules by APCs and increase in their production of cytokines such as IL-12 Therefore, CD40:CD40L ligation indirectly helps to augment T cell cytokine production, T cell differentiation and the development of T cell helper functions (Sebille et al., 2001)

Several other costimulatory molecules and pathways, such as inducible costimulator (ICOS): ICOS-L and programmed death-1 (PD-1): PD-L1 and PD-1:PDL2, have also been identified PD-1 and ICOS are induced on T cells after activation while their ligands are expressed on APCs as well as on a variety of non-lymphoid tissues suggesting a role for these molecules in regulation of T cell activation in the peripheral tissues (Greenwald et al., 2002)

1.1.7 Regulation of TCR

The stimulation of TCR signaling pathway leads to cytokine production, T cell proliferation and activation of other components of adaptive immune system Any irregularities in this pathway may have serious consequences for the host defense system Therefore, TCR signaling pathway is subject to stringent and extensive regulation by a complex network of signaling molecules, which can be broadly classified into two classes Those that maintain 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, which phosphorylates the negative regulatory C-terminal tyrosine residue of Lck Following TCR activation, Lck gets dephosphorylated by a tyrosine phosphatase CD45 (Weil and Veillette, 1994) Similarly, the phosphorylation of ITAMs by Src kinases is continuously regulated by cytoplasmic phosphatases During the formation of immunological synapse, these tyrosine phosphatases are removed from TCR, allowing Src kinases to 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 phosphatases (MAKPs) that

dephosphorylate and inactivate MAPK and Calcipressins (Csps) that inhibit the activated calcineurin, amongst many others (Liu, 2005)

1.1.8 Role of T cell signaling in diseases

The potent responses of the T cells to pathogens are integral for human health However, these responses, when misdirected or dysregulated contribute to various pathologies

T cells are crucial for maintaining tolerance to self-antigens Normally, T cells specific for self-antigen undergo apoptosis in the thymus However, T cells specific for organ-specific antigens that emigrate from the thymus have thepotential for causing organ-specific autoimmunity In most autoimmune disorders, autoantigen-specific responses are induced by the activation of T cells by self peptides displayed on activated APCs These T cells may then activate and drive

B cell responses that either initiate or contribute to disease pathogenesis

Advances in the understanding of TCR signaling pathways have led to the development of novel drugs that can provide immunotherapy for various allergic diseases, autoimmunity, malignancy, and transplant rejection These immunomodulators help to block the engagement and activation of cells, such as blocking TCR signaling (using non-mitogenic anti-CD3 monoclonal antibody), blocking CD28 or CD4 costimulation (anti-B7 or anti-CD4 monoclonal antibody blockade), blocking CD40 engagement on APCs (anti-CD154 monoclonal antibody blockade) or blocking individual signaling molecules such as PKC (inhibited by Rottlerin), ERK1/2 (inhibited by PD98059) and IB (Aspirin) (Nel and Slaughter, 2002)

1.2 Phospholipid signaling

Although the role of phospholipids as membrane constituents and their requirement for energy metabolism has been long acknolwdged, only over the past few decades phospholipid mediators began to be recognized as important endogenous regulators of cell activation, signaling, cell migration, proliferation and apoptosis Most of these bioactive phospholipids are produced by the cleavage of constituents of cellular membranes by the action of various phispholipd modifying enzymes Of note are the phospholipad modifying

enzymes, phospholipase D (PLD) and sphingosine kinase (SPHK)

1.2.1 Sphingosine Kinase (SPHK)

One of the earliest sphingolipids to be identified was sphingosine, which constitutes the backbone of all sphingolipids Sphingolipids have now been recognized as potent second messengers modulating biochemical intracellular events and acting as ligands to mediate extracellular systems Of key interest are ceramide and sphingosine-1-phosphate (S1P) While ceramide has been implicated in cell-stress responses such as apoptosis and growth arrest, S1P has been shown to play important roles in cell proliferation and survival

1.2.1.a Sphingolipid metabolism

The sphingolipid metabolic pathway is highly complex with several points of modulation and regulation (Fig 2)

Sphingomyelin (SM) is a major membrane sphingolipid Cellular SM is hydrolyzed by sphingomyelinases (SMase), resulting in production of ceramide Ceramide, which is produced, is then hydrolyzed by ceramidases to yield sphingosine Alternatively, ceramide can be utilised by sphingomyelin synthase to generate sphingomyelin or it can also be phosphorylated by ceramide kinase (CerK) to yield ceramide-phosphate Ceramide phosphate, in the cells can be dephosphorylated by the general lipid phosphate phosphatase (LPP) to reverse back to ceramide

Sphingosine, produced by the action of ceramidase, is phosphorylated by sphingosine kinase (SPHK) to yield sphingosine-1-phosphate (S1P) or is converted back to ceramide by ceramide synthase S1P, produced by the action of SPHK, is cleared by either desphosphorylation by either of the two specific S1P

phosphatases (SPP-1 and SPP-2) or by general lipid phosphate phosphatase (LPP)

to yield sphingosine S1P can also undergo irreversible cleavage by S1P lyase to hexadecanal and phosphoethanolamine (Melendez, 2008) (Fig 2) As ceramide, sphingosine and S1P can be interconverted by the sequential actions of the various sphingolipid modifying enzymes, the intracellular levels of these metabolites are tightly regulated by the balance between their synthesis and degradation It has also been proposed that the relative balance of these sphingolipid metabolites play an important role in cell-fate decisions Increased amounts of ceramide in the cells triggers apoptosis while an excess of S1P results

in cellular proliferation (Cuvillier et al., 1996) SPHK thus, plays a crucial role in

determining the cell fate by modulating the relative levels of S1P in the cell

Figure 2: The sphingolipid metabolic pathway

Sphinogmeylin, a membrane phosphoplipid is hydrolyzed to produce ceramide which is further converted to other bioactive molecules Sphingosine kinase (SPHK) predominantly phosphorylates sphingosine to produce sphingosine-1- phosphate (S1P), which is either cleaved by S1P lyase or converted back to sphingosine by S1P phosphatase or lipid phosphate phosphatase (LPP)

Reprinted with kind permission from Elesevier;

Sphingosine kinase signalling in immune cells: potential as novel

therapeutic targets; Melendez, AJ (2008) Biochim Biophys Acta

1784 (1), 66-75

1.2.1.b Properties of SPHK

Sphingosine kinases are members of a newly discovered class of the class of lipid kinases Other members of this class include ceramide kinases (CerK), diacylglycerol kinases (DAGK) and phosphatidylinositol 3-kinases (PI3K) Sphingosine kinases are highly conserved from protozoa to mammals and their expression is ubiquitous in almost all tissues

To date, two mammalian forms of SPHK have been cloned and characterized: SPHK1 (Kohama et al., 1998; Melendez et al., 2000) and SPHK2 (Liu et al., 2000) with molecular weight of 49 KD and 68 KD respectively Both forms have

a unique catalytic domain, which includes an ATP-binding site, as well as five conserved domains called C1 to C5 SPHK1 and SPHK2 have been found to share

a similar phosphate donor-binding site and phosphorylation mechanism as other kinases, such as the DAGK Both SPHK1 and SPHK2 are widely but differentially expressed SPHK1 is most highly expressed in brain, heart, thymus, spleen, kidney and lung (Melendez et al., 2000) while expression of SPHK2 is highest in kidney and liver (Liu et al., 2000) Also, SPHK1 has higher substrate specificity and enzymatic activity than SPHK2

SPHK is primarily a cytosolic enzyme and its activation is often accompanied by its translocation to the plasma membrane where its substrate is located Sphingosine kinases appear to be highly specific in their substrate preference Both SPHK1 and SPHK2 are capable of phosphorylating erythrosphingosine, dihydrosphingosine and phytosphingosine However, no other phospholipids appear to be significantly phosphorylated by these enzymes Although the activity

of SPHK could be inhibited by a number of compounds, the best known inhibitors

are analogues of sphingosine, such as DL-threo-dihydrosphingosine (DHS) and

N, N-dimethylsphingosine (DMS) (Melendez, 2008)

1.2.1.c Activation and regulation of SPHK

SPHK is stimulated in response to a number of biological effectors especially following activation by growth and survival factors These include growth factors such as ligands for G protein coupled receptors (GPCRs) e.g acetylcholine and lysophosphatidic acid (LPA) Activation of tyrosine kinase receptors such as platelet derived growth factor (PDGF) and nerve growth factor (NGF) receptors

as well as immunoglobulin receptors such as FcγR1 and FcR1 results in SPHK activation SPHK is also stimulated following activation of cell by various cytokines and chemotactic peptides such as by TNF-and C5a (Maceyka et al., 2002; Kee et al., 2005)

The possible mechanisms of SPHK regulation are translocation, phosphorylation, protein-protein interaction and calcium Translocation of SPHK from cytosol to plasma membrane in response to agonist stimulation is probably the most important regulatory mechanism as its substrate, sphingosine, is located in the plasma membrane (Wattenberg et al., 2006) Several factors such as PKC and ERK1/2 seem to play a role in the membrane translocation It has been shown that phosphorylation of SPHK1 on Ser225 by ERK1/2 is crucial for its translocation (Pitson et al., 2003) Similarly, activation of PKC also results in SPHK1 phosphorylation and translocation Moreover, tyrosine kinases Lyn and Syk have

been found to increase the activity of SPHK1 and 2 in a kinase independent

manner

1.2.1.d Role of sphingosine-1-phosphate (S1P) in cellular responses

Sphingosine-1-phosphate (S-1-P), produced by the action of SPHK, is a bioactive sphingolipid, levels of which are low in cells and are tightly regulated by the balance between its synthesis and degradation S1P has dual messenger functions;

it acts in an autocrine as well as paracrine manner Thus, it can cause direct activation of signaling proteins by acting an intracellular second messenger (Payne et al., 2002) In addition, intracellularly produced S1P can also bind to S1P specific cell surface GPCRs (“inside-out” signaling) Five type of cell surface S1P receptors have been identified; S1P1 (EDG-1), S1P2 (EDG-5), S1P3 (EDG-3), S1P4 (EDG-6) and S1P5 (EDG-8) These receptors are ubiquitously expressed and are coupled to multiple G proteins S1P via S1P receptors activates several downstream signaling molecules (Spiegel and Milstien, 2003)

As a second messenger, S1P has been implicated in cellular proliferation, cell survival and suppression of apoptosis (Cuvillier et al., 1996) It has also been suggested that SPHK and S1P mediate intracellular calcium release in an IP3 independent manner (Meyer zu Heringdorf et al., 1998; Melendez and Khaw, 2002), though specific receptors for S1P in ER have not yet been identified S1P also plays a central role in cell motility and migration by regulating factors critical for cytoskeletal rearrangement and cell locomotion (Donati and Bruni, 2006) Thus, it is not surprising that S1P is required for embryonic development, organogenesis, angiogenesis and wound healing (Spiegel and Milstien, 2003)

S1P also promotes cell proliferation by acting as a mitogen and regulating DNA synthesis (Olivera et al., 1999) In contrast, S1P precursors, sphingosine and ceramide have been demonstrated to play a role in growth arrest and apoptosis (Hannun and Obeid, 2002) As these various metabolites are interconvertible, therefore, it has been suggested that the cell fate is determined by the relative amount of these sphingolipids in the cells Higher amount of S1P in the cell promotes cell proliferation while an excess of ceramide in the cells triggers

apoptosis This balance of sphingolipid metabolites is called “sphingolipid rheostat” and has been proposed to play important role in regulating cell fate

(Cuvillier et al., 1996)

1.2.1.e Role of SPHK and sphingosine-1-phosphate (S1P) in immune cells

In immune cells, S1P has been shown to mediate the signals for cell activation and differentiation In T cells SPHK has been shown to promote cell survival in response to ceramide or Fas induced apoptosis Numerous studies have shown the role of SPHK and S1P in T cell migration An analogue of sphingosine, FTY720, blocks S1P receptors and prevents the egress of T cells from secondary lymphoid organs, thus preventing their migration to sites of inflammatory lesions (Mansoor and Melendez, 2008) S1P also enhances the proliferation of Th17 cells and increases their ability to produce IL-17 (Liao et al., 2007) Furthermore, it has been suggested that SPHK2 associates with the cytoplasmic region of the IL-12 receptor-chain and it is likely to positively modulate the effect of IL-12 on the T cell response, which leads to the generation of interferon-γ (INF- γ) Thus,

although not very well characterized, the role of SPHK on lymphocytes appears to

be significant

Studies have demonstrated that SPHK regulates neutrophil priming and activation, in order to provide critical defense against infections (Ibrahim et al., 2004) SPHK is also important for TNF- induced superoxide production in neutrophils suggesting that SPHK plays important role in the functional responses

of neutrophils (Niwa et al., 2000)

Monocytes, activated via FcγRΙ receptor, stimulate SPHK activity and this stimulation is essential for calcium release from internal stores and activation of oxidative burst (Melendez et al., 1998) Similarly, activation of mast cells via the cross-linking of FcRΙ induces SPHK activity which is important for rise in cytosolic calcium and mast cell functions such as degranulation (Melendez and Khaw, 2002)

1.2.2 Phospholipase D (PLD)

Phospholipase D (PLD) is a ubiquitous phosphodiestrase that catalyzes hydrolysis

of the terminal diester bond of phosphatidylcholine (PC) to liberate phosphatidic acid (PA) and choline (Billah and Anthes, 1990). Over the years, there is increasing evidence to support a role for PLD activation as an important component of the signaling cascades activated following receptor aggregation in various cells.

1.2.2.a Structure and properties of PLD

PLD genes from various organisms belong to an extended superfamily The defining feature of PLD enzymes is the HKD catalytic motif in the relatively conserved catalytic domain HKD motifs are essential for the enzyme‟s catalytic

activity, both in vitro and in vivo, as the point mutation in HDK motif disrupts

PLD activity (Sung et al., 1997) The catalytic domain of PLD is flanked by much less conserved regulatory N- and C- terminal regions Other highly conserved regions of the PLD genes are the phox consensus sequence (PX), the plekstrin homology domain (PH) and the PI4,5P2 binding site (Fig 3) The PX domain mediates protein-protein interactions and interactions between PLD and phosphatidylinositol phosphates (PIP) (Xu et al., 2001) while the PH domain plays an important role in the localization of the protein (Sugars et al., 2002)

In mammalian cells, two isoforms of PLD (PLD1 and PLD2) have been cloned, and characterized (Hammond et al., 1995; Lopez et al., 1998) Mammalian PLD1

is a 1071-amino acid protein, while PLD2 is a 933- amino acid protein The two isoforms share about 50% sequence identity, differing mainly in the N- and C-

terminal regions PLD1 is expressed as two splice variants, namely, PLD1a and PLD1b PLD1b, though shorter than PLD1a by 38 amino acids, has the same catalytic activity and regulation as PLD1a (Hammond et al., 1997)

PLD1 has a conserved loop region, which probably acts as a negative regulator of the enzyme as the deletion of this region from PLD1 increases the basal enzymatic activity by three folds This auto-inhibitory loop is absent in PLD2, thus, its not surprising that the basal activity of PLD2 is higher than PLD1 (Sung

et al., 1999)

of catalytic activity by protein kinase C and Rho are indicated and the PIP2interacting site is also denoted

-With kind permission from Springer Science + Business media;

Signalling roles of mammalian phospholipase D1 and D2 (figure 1);

Cockcroft (2001) Cell Mol Life Sci 58(11), 1674-1687

Most mammalian cells express both PLD1 and PLD2 although the relative expression in cell lines varies greatly Thus, Jurkat and Molt-4 cells express mainly PLD2, HL60 cells express mainly PLD1 and U937 cells express both isoforms (Meier et al., 1999) Due to the absence of specific inhibitors for each isozyme type, the exact function of PLD1 and PLD2 has been difficult to delineate However, it is believed that PLD1 and PLD2 play different roles within the cell PLD1 has a peri-nuclear localization, suggesting a Golgi, endoplasmic reticulum or late endosomal distribution (Freyberg et al., 2001; Lucocq et al., 2001), and upon stimulation it translocates to plasma membrane (Lucocq et al., 2001) PLD2 is mostly localized to cell periphery, although localization to sub-membranous vesicular compartments has also been reported (Divecha et al., 2000), and upon activation it becomes associated with membrane ruffles and endocytic vesicles (Colley et al., 1997; Du et al., 2004)

1.2.2.b Transphosphatidylation reaction

PLD cleaves the terminal diester bond of phosphatidylcholine with water acting

as a nucleophile in the reaction This hydrolysis of PC by PLD is a two-step reaction that requires both HKD motifs of PLD that form an active pocket The N-terminal HKD motif of PLD is normally protonated on the histidine residue Once the PC enters the active pocket of PLD, formed by two HKD motifs, free choline

is released by the release of N terminal proton resulting in the formation of PLD intermediate with histidine of carboxy terminal HKD domain Finally, N-terminal histidine re-acquires the proton from a water molecule, leaving the

PA-hydroxyl group to attack PA-PLD intermediate resulting in the release of PA (Jenkins and Frohman, 2005)

In normal circumstances, water acts as the phosphatidyl group acceptor in this hydrolytic reaction However, due to strong nucleophilic nature of short chain primary aliphatic alcohols such as ethan-1-ol or butan-1-ol, primary alcohols are preferentially used over water by at least 1000 fold Thus, in the presence of primary alcohols, PLD does not form PA but instead forms rare phospholipids such as phosphatidylethanol (PtdEtOH) or phosphatidylbutanol (PtdBut) (Fig 4) This reaction is a unique property of PLDs and the activation of PLD in the presence of primary alcohol is called transphosphatidylation reaction

Owing to their unique origin, low basal activity and metabolic stability, the naturally occurring phosphatidylalcohols produced in transphosphatidylation reaction are used as quantitative marker of PLD activity Moreover, in the presence of primary alcohol, PLD metabolic pathway is shunted to phosphatidylalcohols and the production of second messenger PA is abrogated resulting in the inhibition of downstream events normally triggered by PLD activation Thus, transphohatidylation reaction is commonly used as the gold standard method for measuring PLD activity in intact cells as well as to study the events down stream of PLD activation

non-Figure 4 PLD-catalyzed hydrolysis and transphosphatidylation reaction

Following the cleavage of choline, the first part of the reaction involves the formation of a PA-PLD intermediate by covalent linkage of PA to the histidine, present in the catalytic pocket of PLD Either water or a primary alcohol can act

as a nucleophile in the second stage of the reaction In the presence of water, the reaction product is phosphatidic acid (PA), and in the presence of primary alcohol, the product is a phosphatidylalcohol

With kind permission from Springer Science + Business media;

Signalling roles of mammalian phospholipase D1 and D2 (figure 2); Cockcroft (2001) Cell Mol Life Sci 58 (11), 1674-1687