DESCRIPCIÓN E INTENCIÓN DE ALGUNAS DE LAS ACTIVIDADES DE

The downstream effector pathways in the membrane domains contain the intracellular protein tyrosine kinases and a series of adaptor proteins that interpret signals in the context of „danger‟ and adopt an appropriate response (78). It is important that cells do not become activated in the absence of further danger signals. Activation requires two or more signals from the environment that are particularly important for immune cells to recognise foreign antigens and pathogens (79). The biochemical events in the membrane domains sense the environment and modulate cellular responses to ensure activations are appropriate for that environment (79). Membrane domains rich in signalling molecules are transported to the centre of the forming synapse. This allows sustained TCR signalling (Figure 1.2).

The first biochemical event that can be detected upon TCR/CD3-ligation is the lymphocyte-specific protein-tyrosine kinase (Lck)-mediated phosphorylation of tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAMs) (80). Protein kinase Lck is critical for initiation of the TCR signalling process through

37 the CD3 (81). Lck is a cytosolic protein that becomes membrane associated as a result of fatty acid modifications to its amino terminus, after which it can bind to the cytosolic tails of the transmembrane CD4 and CD8 co-receptors and thus initiate TCR signalling (82), (83). Other components of the TCR signalling complex, such as CD4 or CD8, are also likely to influence T cell signalling (55). Class I and class II MHCs are recognised by their respective CD8 and CD4 co-receptors (55). TCR signalling can be either co- receptor dependent or co-receptor independent. Co-receptor dependent TCR signalling is thought to be more efficient than co-receptor independent TCR signalling, requiring lower antigen doses for equivalent levels of stimulation (84). The CD4 co-receptor interaction is achieved in the MHC class II system (85). While both domains of CD8 cooperate to bind class I MHCs, only one domain (the N-terminal variable-like region) of CD4 makes contact with the class II MHCs, with the second tandem CD4 domain being distal to the interface (55). The co-receptor-Lck associations of CD4/Lck or CD8/Lck to the TCR/CD3 complex exist to enhance antigen-specific TCR signalling (86), (87), (88).

The cytoplasmic domains of the CD3 and δ polypeptides contain ITAMs (58). The ITAMs can appear in tandem thrice in the TCR zeta chain and once in each of the CD3 subunits (γ, δ, and ε) (58). Different ITAMs of the TCR/CD3 complex can interact with different adaptors that transmit antigen receptor signals, indicating that variations in the phosphorylation of individual ITAMs during T cell activation will generate a signalling diversity (89), (90). One of the main adaptors is the membrane-anchored adaptor molecule linker for the activation of T cells (LAT) which contains many ITAMs (91). The phosphorylation of ITAMs is determined by the avidity of ligation and the

38 resulting pattern of partial or complete ITAM phosphorylation and the differential recruitment of signalling molecules (79). Therefore, the phosphorylation of ITAMs leads to cell activation, depending on the intensity of the receptor stimulation and the presence or absence of co-stimulatory signals (79). ITAMs and the associated receptors mediate activating signals that are opposed by a distinct set of receptors that signal through cytoplasmic-domain Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs) (77). ITIMs recruit phosphatases, including the tyrosine phosphatases; Src homology 1-domain containing tyrosine phosphatase (SHP-1) and Src homology 2- domain containing tyrosine phosphatase (SHP-2) that attenuate ITAM-induced signalling by dephosphorylating and thereby inactivating signalling intermediates (77). ITAMs can also generate inhibitory signals and attenuate signalling by heterologous receptors (92), (93), (94). Although specific ITAM-associated receptors may be predominantly inhibitory, several experimental systems show that the same ITAM- coupled receptors can generate both positive and negative signals (77).

Next, ITAMs with both tyrosines phosphorylated are required to recruit the non- receptor tyrosine kinase, zeta-chain-associated protein kinase of 70 kDa (ZAP-70), through its paired Src homology 2 (SH2) domains (95). The ZAP-70 kinase was originally identified by virtue of its association with the TCR zeta homodimer, an association mediated by the binding of the two Src homology (SH2) domains of ZAP- 70 to tyrosine-phosphorylated motifs in the zeta protein (95), (96). The recruitment and activation of ZAP-70 then phosphorylates a series of adaptor proteins that lead to the recruitment of phospholipase C-γ (PLC-γ) and phosphatidylinositol-3 kinase (PI3-K) to the activated TCR complex (97). The ZAP-70 phosphorylate sites that lead to the

39 recruitment of adaptor proteins; SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), LAT, Gads (Grb2-related adaptor downstream of Shc) and Grb 2 (growth factor receptor bound protein 2) (97). The SLP-76 recruits the guanine nucleotide exchange factor (Vav) and the non-catalytic region of tyrosine kinase (Nck), which then recruits the Wiscott-Aldrich syndrome protein (WASP). The activated WASP orchestrates the formation of complex actin-based structures which induce cytoskeletal rearrangements (98). These rearrangements are important for integrin signalling and the formation of the immunological synapse. The immunological synapse is triggered by tyrosine phosphorylation by ITAMs and Ca2+ mobilization by PLC-γ (58). The predominant effector systems involved in the intracellular transmission of TCR signals resemble those defined in other systems, such as calcium- dependent kinases and phosphatases (77). Also, the guanine triphosphate (GTP)- binding proteins control common signalling effectors such as serine / threonine kinase cascades of the mitogen-activated protein kinase (MAPKs) type. Signalling effectors act to regulate the function of transcription factors, such as the nuclear factor of activated T cells (NF-AT) (77). NF-AT and the nuclear factor kappa-light-chain- enhancer of activated B cells (NF- B) promote the transcription of a number of genes, most importantly interleukin-2 (IL-2), which is a cytokine which promotes the long term proliferation of activated T cells (77).