Consolidación de Son Batá: las notas que los llevaron a las raíces

Immunological Synapse

T cells are activated in response to binding of the T cell antigen receptor to peptide presented by major histocompatibility (MHC) molecules on the surface of antigen-presenting cells (APCs) (Tseng and Dustin, 2002).

T cell-APC adhesion is essential in the formation of the immunological synapse (IS) (Bromley et al., 2001; Dustin, 2002a; Sims and Dustin, 2002). The IS can be broadly defined as any T helper cell-APC or cytotoxic- or NK T cell-target cell interface that is involved in ‘information transfer’. The T cell-APC immunological synapse is a long-lived structure that can be stable over many hours and incorporates a migratory stop signal that ‘arrests’ the T cell upon encounter with the antigen-presenting cell to allow signalling and T cell activation to occur (Dustin, 2002b; Sims and Dustin, 2002).

IS formation is a multi-step process with two major morphologically defined stages: the nascent or immature IS and the mature IS. In the nascent IS the integrins are initially engaged in the centre of the contact area, and TCRs are engaged in the periphery of the contact. This TCR ring of the nascent IS is the site of robust Lck and ZAP-70

activation. The mature IS has two major features. First, the pattern of the LFA-1 and TCR are inverted compared to the nascent IS, such that the TCR is clustered in the centre. Second, this central cluster of TCR-MHC interactions is stabilized. The cytoskeletal protein talin remains associated with the active form of LFA-1; talin is a defining component of the LFA-1 ring in the IS (Bromley et al., 2001 ; Sims and Dustin, 2002).

The first evidence that an active cytoskeletal mechanism is involved in driving receptor accumulation at the T cell-APC interface during T cell activation stems from studies by Wulfing and Davis; they showed that this mechanism requires the engagement of the major costimulatory receptor pairs B7-CD28 and ICAM-1-LFA-1 and is dependent on myosin motor proteins (Wulfing and Davis, 1998). Myosin motor proteins move molecules that are linked to the actin cytoskeleton toward the T cell-APC interface. This study suggested that this active cytoskeletal movement is a central part of costimulation, as it would effectively amplify TCR-mediated signals as a result of the increased receptor densities.

While stable IS formation may not be essential for T cell activation, this stable structure achieves the important goal of stopping the T cell by coordinating the antigen (- MHC-APC) recognition process with migration, and thereby facilitating signalling to occur (Dustin, 2002b). The antigen-dependent stop signal, inherent to the stable IS, may be the primary mechanism by which APCs retain antigen-specific T cells in the secondary lymphoid tissues that have been exposed to antigen; antigen-specific T cells stop re­ circulation but subsequently re-initiate migration.

Interestingly, T cells are able to interact with the APC also through antigen- independent mechanisms prior to TCR interaction with MHC-peptide complexes; this interaction can be seen as scanning, in which it is likely that TCR will interact with peptide-MHC complexes; LFA-1 may be involved in this scanning interaction (Sims and Dustin, 2002). Scanning is thought to be an important first step in antigen recognition. If agonist MHC-peptide complexes are detected in the interface between the T cell and APC, then LFA-1 may be activated. This combination of signals from LFA-1 and the TCR leads to polarization of the T cell towards the APC (Ardouin et al., 2003). The response of T cells to MHC-peptide complexes is extremely rapid (minutes) (Hailman et al., 2 0 0 2).

Very recently, for the first time, the formation of an active signalling complex in thymocytes undergoing positive selection as a result of a TCR-MHC-dependent interaction with thymic epithelial cells has been described (Hare et ah, 2003). The observation that thym ocyte-epithelial cell interactions that increase thymocyte intracellular calcium levels, induce phosphotyrosine accumulation and polarization of CDS, CD4, CDS, CD45, and the key signalling molecules p56Lck and LAT, to the point of thymocyte-epithelial cell contact, also take place during the initial stages of positive selection, provided evidence for similarities between thymocyte selection and T cell activation during mature T cell-APC interactions.

1.4.5.1 LFA-1

LFA-1 plays a key role in the structure of the IS; LFA-1 adhesion to surfaces coated with ICAM-1 is low on resting T cells, but is induced in response to activation by a large number of signalling pathways that include the TCR and chemokine receptors (Ardouin et ah, 2003; Hogg et ah, 2003; Krawczyk et ah, 2002; Sims and Dustin, 2002).

This inside-out signalling enhances both interaction with ICAM-1 and with cytoplasic proteins like talin that are associated with integrin cytoplasmic domains.

The P2 subunit of LFA-1 has been implicated in signalling events thought to be

associated with adhesion. Recent work has shown that during T cell activation, the p2

integrin chain CD 18 becomes phosphorylated on serine and functionally important threonine residues located in the intracellular C-terminal tail (Fagerholm et ah, 2002).

Using a peptide corresponding to the cytoplasmic tail of the integrin P2 (CD 18)

chain, the sites phosphorylated in vitro were identified as Ser-745 and Thr-758. Catalytic domain fragments of protein kinase C (PKC)ô and PKCpI/II have been identified as the major protein kinases in leukocyte extracts that are responsible for this phosphorylation. 14-3-3 proteins present in the cytoplasm of T cells bound to the C-terminal peptide of CD18 in vitro when the peptide was phosphorylated at Thr-758. Thus, PKC-mediated phosphorylation of CD 18 at Thr-758 after cell stimulation could lead to the recruitment of 14-3-3 proteins to the activated integrin. 14-3-3 proteins are adaptor proteins that bind to phospho-serine- and phospho-threonine-containing-motifs and are known to be involved in regulating a number of signalling molecules. 14-3-3 proteins could therefore be

involved in the initiation of signalling complex formation (recruitment of Vav or other secondary messengers) ‘downstream’ of the phosphorylated integrin molecules which may play a role in regulating its adhesive state and/or ability to signal. Truncation or mutation of the p2 cytoplasmic tail inactivates adhesion and stimulation through phorbol

ester (PMA). The requirement or contribution of this particular phosphorylation sites, Thr-758 and Ser-745, to integrin activation/adhesion has not been answered yet.

The modifications of the tail that take place in response to integrin activation may be very important for cytoskeletal remodelling.

LFA-1-deficient mice have a severe defect in the ability of their T cells to arrest on high endothelial venules (Berlin-Rufenach et al., 1999). Studies using LFA-1-deficient mice have further demonstrated that LFA-1 facilitates T cell activation by lowering the amounts of antigen necessary for T cell activation (Bachmann et al., 1997).

In the absence of LFA-1, 100-fold more antigen was required for T cell/antigen- presenting cell (APC) interaction and all subsequent events of T cell activation, including TCR down-regulation, Ca^"’-flux, T cell proliferation and lytic effector cell induction. Another cell surface molecule that has been implicated in T cell-APC interactions and TCR signalling is the costimulatory molecule CD28. CD28 interacts with B7-1 and B7-2 on antigen-presenting cells and T cell responses are greatly impaired in the absence of B7-CD28 interaction, which may result in T cell anergy (Appleman and Boussiotis, 2003). Nevertheless, it has been shown that high affinity ligands could activate T cells in the absence of CD28.

In summary, the study proposed that LFA-1 facilitates the functional triggering of TCR by promoting adhesion of T cells to APCs. In contrast, CD28 has been shown to reduce the number of TCRs that had to be triggered for T cell activation, which allowed activation of T cells by low affinity ligands.