Antigenic stimulation which leads to an internal T-cell signalling cascade is termed TCR triggering. However, the precise details of how antigen binding mediates this process is still unclear. Several models have been suggested, which are categorised as aggregation,
70 segregation, and conformational alteration, all of which are summarised by figure 1.4 (van der Merwe and Dushek, 2011).
When considering TCR triggering models it is important to remember that TCR recognition of ligand differs somewhat to other ligand-receptor interactions. One of the most fundamental of these is that a TCR must be able to bind a wide range of ligands including those which do not originate from humans. This function can be performed by TCRs due to the non-conserved binding residue profile of TCRs which provides a great range of binding diversity. Specifically, while the interface on the MHC has the ability to form many contacts, only a few residues are required to bind in order to activate a TCR and the interaction of peptide with TCR is therefore by no means an exact fit (Tynan et al., 2005).
The TCR also has to be highly sensitive to its corresponding ligand as a non-self-peptide is often only present at very low concentrations, bound to a subset of MHC molecules on APCs. The chance of TCR-antigen binding is therefore low and requires high affinity binding to ensure peptide detection. This low abundance of non-self-antigen presents another problem, discrimination between this and the much more abundantly presented self-peptides which are often referred to as “noise”. To account for this, it is thought that there are co-operative effects that aid this distinction. This may involve the interaction of numerous TCR-CD3 complexes which allow for internal signal amplification from self- peptides, or alternatively, a process known as signal spreading. Signal spreading is based upon the fact that non-self-peptide TCR affinity is greater than that of self-peptides. The high affinity binding overcomes a threshold whereby the signalling cascade is initiated. Extracellular signal-regulated kinase (ERK) is activated to phosphorylate, and therefore activates lymphocyte-specific protein tyrosine kinase (Lck), while self-peptides provide a suboptimal response only great enough to trigger a negative feedback loop whereby the
71 tyrosine phosphatase SHP-1 is recruited to inactivate Lck (Schamel et al., 2006; Štefanová et al., 2003; van der Merwe and Dushek, 2011).
1.7.2.1 Aggregation model.
Two models of TCR triggering are grouped into a category based on aggregation (Figure 1.4a). The first is the coreceptor heterodimerisation model in which binding of the MHC- TCR is accompanied by binding of either the CD4 or CD8 coreceptor to the MHC. If both interactions occur then Lck located on the intracellular portion of the coreceptor is brought into close proximity with the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR CD3 chains and stimulates a phosphorylation cascade that leads to T- cell activation. However, the majority of studies have found that MHC monomers bound to peptide are unable to fully activate the TCR which lead to the proposal of multiple MHC-TCR interactions being required for T-cell activation. This adapted aggregation model is termed the pseudodimer model, as it relies on not only binding of non-self- peptide with TCR and MHC, but also a secondary interaction between an adjacent TCR and MHC molecules which may contain self-peptide. The coreceptor in this instance is bound to the self-peptide-MHC complex but due to the dimer formation of the TCR, the associated Lck is also able to phosphorylate the non-self-peptide TCR. While both monomeric and polymeric TCR complexes have been identified, it is thought that antigenic signals promote the dimerisation and clustering of the TCR complexes. In support of this polymeric model, it has been shown that self-peptide MHC complex formation allows for better recognition of non-self-peptide (van der Merwe and Dushek, 2011). It appears however that neither aggregation model can account for all cases of T- cell activation. Whilst for CD8+ T-cell activation, binding of the coreceptor appears crucial
72 for TCR activity, for CD4+ T-cells, those exposed to higher densities of an antigen can be
activated in the absence of coreceptor binding (Irvine et al., 2002; Purbhoo et al., 2004).
1.7.2.2 Conformational change model.
An alternative model of TCR triggering is based upon the observation that the TCR undergoes a conformational change during T-cell activation (Figure 1.4b). It has been shown that the presence of lipid vesicles, mimicking the lipid structure of the plasma membrane, was able to promote the folding of the CD3ζ chain into a state where it was unable to be phosphorylated by Lck, therefore blocking zeta chain-associated protein kinase (Zap70) recruitment and T-cell activation (Aivazian and Stern, 2000). It is well established that the TCR undergoes conformational change upon binding to an antigen but this is primarily in and around the antigen binding region and not within the T-cell. Indeed, studies have reported altered TCR constant domain structures, but are generally reliant on the use of low resolution crystallographic techniques (Beddoe et al., 2009). It is now thought that these extracellular conformational changes are translated through the plasma membrane to the intracellular portion of the TCR where the CD3 chains themselves then undergo a conformational change. Using a molecular dynamics model, Martinez-Martin et al were able to identify a structural alteration in CD3ε subsequent to ligand binding resulting in a more rigid structure (Martínez-Martín et al., 2009) thought to expose ITAMs which can then be phosphorylated. More thorough analysis of this alteration showed that by introducing mutations TCR activation could be prevented. Furthermore, it was concluded that TCRs may form clusters within which TCRs are able to signal to one another as signalling in this model could not be restored by addition of non-mutated CD3ε.
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1.7.2.3 Segregation model.
The final TCR triggering model is termed the segregation model, often referred to as the redistribution model (Figure 1.4c). This refers to the separation of the TCR complex from inhibitory proteins such as CD45, a receptor tyrosine phosphatase known to prevent T- cell activation. Indeed it is activation of cell surface proteins such as CD45 which prevent Lck from constitutively proceeding with the TCR signalling cascade. Although no more Lck is activated after TCR ligation, the constitutively active Lck is then free to phosphorylate downstream proteins (Nika et al., 2010). The kinetic segregation model excludes TCR inhibitory phosphatases based on their longer length and maintains an area of close contact between T-cells and APCs using short adhesion molecules (Davis and van der Merwe, 2006). Both lengthening of the MHC complex, and the use of shortened CD45, can prevent TCR activation thus supporting this theory (Choudhuri et al., 2005; Irles et al., 2003). A slight variation to this model, states that TCR activation requires the presence of lipid rafts which are associated with the absence of inhibitory proteins such as CD45 but express others such as the T-cell activation cascade initiating kinase, Lck. While the presence of these rafts during T-cell activation is questionable due to the lack of markers identifying the co-localisation with TCR clusters, the theory is quite plausible as lipid rafts are known to be vital for translocating important proteins to the membrane (Hashimoto-Tane et al., 2010).
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Figure 1.4 T-cell receptor triggering models. Aggregation model – (A) the co-receptor heterodimerisation model states that the TCR-MHC interaction is accompanied by binding of the CD4/8 coreceptor. Lck, a tyrosine kinase bound to the coreceptor is brought into close proximity with the ITAMs on the TCR CD3 chains to begin a phosphorylation cascade. (B) is similar and termed the pseudodimer model, but requires the additional presence of a second TCR-MHC interaction presenting self-peptide to boost downstream signalling. Conformational change model – (C) The piston-like model states that interaction of the TCR with a peptide-MHC complex initiates an extracellular conformational change that translates to the intracellular portion of the TCR to ‘open’ the folded conformation of the CD3 chains thus exposing ITAMs which can subsequently be phosphorylated/activated. (D) The piston-like clustering model is an adaption of this whereby piston-like movement occurs and results in TCR clustering and amplification of downstream activation pathways. Segregation/redistribution models – (E) The kinetic model identifies that cell surface proteins such as the tyrosine phosphatase CD45 prevent T-cell activation. Exclusion of these long inhibitory molecules from sites of TCR-MHC interaction would allow T-cell activation to proceed and thus may occur in areas of close contact between APCs and T-cells. (F) The lipid-raft model states that lipid rafts containing T-cell activation molecules such as Lck, but excluding the presence of inhibitory proteins (CD45) may associate with TCRs and allow for T-cell activation. Figure adapted from (van der Merwe and Dushek, 2011).
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