DYNAMIC INTERACTIONS AT THE IMMUNOLOGICAL SYNAPSE
As we have described above, successful TCR triggering involves a multitude of signal transduction events that culminate in T‐cell activation. But what is the probability of this occurring in an in vivo setting? T‐cells need to be highly efficient at finding their cognate antigen and discriminating between activating and non-activating peptide–MHC complexes for several reasons.
First, the numbers of T‐cells bearing the correct TCRs for productive engagement with peptide–MHC are typically small; 1 in 100 000 or fewer cells being capable of responding to a particular peptide–MHC combination is not unusual. Therefore, T‐cells need to be able to efficiently recognize the correct peptide–MHC combination in a veritable sea of self peptide–MHC and non‐activating peptide–MHC molecules. Because of the need to search for the correct peptide–MHC combination, naive T‐cells are in continual motion within a lymph node, scanning at speeds that enable them to visit up to 5000 DCs in 1 hour; quite the social networkers! Because of this ferocious rate of movement, TCR–peptide–MHC inter actions are very fleeting, as cells brush past each other at high speed. When an activating TCR–peptide–MHC interaction does occur, as few as 10 peptide–MHC complexes can persuade a T‐cell to stop and linger, forming a more stable interaction that leads to the productive assembly of the immunological synapse (described below). Of course, the reader will under stand that the T‐cell needs to be pretty certain that this is the correct peptide–MHC complex to respond to, for if an error is made, the consequences are potentially calamitous and can result in autoimmunity.
The behavior of a T‐cell within a lymph node as it searches for the correct peptide–MHC combination can be divided into several phases. Whereas T‐cell movement is rapid during the seeking phase (phase I), encounter with agonistic peptide–MHC leads to stable T‐cell–DC interactions lasting approximately 12 hours (phase II), during which cytokines such as IL‐2 are produced. This is followed by a return to rapid movement involving further transient DC interactions (phase III), during which the T‐cell divides a number of times and exits the lymph node.
A serial TCR engagement model for T‐cell activation
We have already commented that the major docking forces that conjugate the APC and its T‐lymphocyte counterpart must come from the complementary accessory molecules such as ICAM‐1/LFA‐1 and LFA‐3/CD2, rather than through the relatively low‐affinity TCR–MHC plus peptide links (Figure 7.3). Nonetheless, cognate antigen recognition by the TCR remains a sine qua non for T‐cell activation. Fine, but how can as few as 10 MHC–peptide complexes on a DC, through their low‐affinity complexing with TCRs, effect the Herculean task of sustaining a raised intracellular calcium flux for the 60 minutes required for full cell activation? Any fall in calcium flux, as may be occasioned by adding an antibody to the MHC, and NFATc dutifully returns from the nucleus to its cytoplasmic location, so aborting the activation process.
Figure 7.19 Serial triggering model of T‐cell receptor (TCR) activation. Intermediate‐affinity complexes between MHC–peptide and TCR survive long enough for a successful activation signal to be transduced by the TCR, and the MHC–peptide dissociates and fruitfully engages another TCR. A sustained high rate of formation of successful complexes is required for full T‐cell activation. Lowaffinity complexes have a short half‐life that either has no effect on the TCR or produces inactivation, perhaps through partial phosphorylation of ζ chains. Green dots denote successful TCR activation; red dots denote TCR inactivation; and a dash denotes no effect. The length of the horizontal bar indicates the lifetime of that complex. Being of low affinity, they recycle rapidly and engage and inactivate a large number of TCRs. High‐affinity complexes have such a long lifetime before dissociation that insufficient numbers of successful triggering events occur. Thus modified peptide ligands of either low or high affinity can act as antagonists by denying the agonist access to adequate numbers of vacant TCRs. Some modified peptides act as partial agonists in that they produce differential effects on the outcomes of T‐cell activation. For example, a single residue change in a hemoglobin peptide reduced IL‐4 secretion 10‐fold but completely knocked out T‐cell proliferation. The mechanism presumably involves incomplete or inadequately transduced phosphorylation events occurring through a truncated half‐life of TCR engagement, allosteric effects on the MHC–TCR partners, or orientational misalignment of the peptide within the complex.
Surprisingly, Salvatore Valitutti and Antonio Lanzavecchia have shown that as few as 100 MHC–peptide complexes on an APC can downregulate 18 000 TCRs on its cognate T‐lymphocyte partner. They suggest that each MHC–peptide complex can serially engage up to 200 TCRs. In their model, conjugation of a MHC–peptide dimer with two TCRs activates signal trans duction, phosphorylation of the CD3‐associated ζ chains with subsequent downstream events, and then downregulation of those TCRs. Intermediate affinity binding favors dissociation of the MHC–peptide, freeing it to engage and trigger another TCR, so sustaining the required intracellular activation events. The model for agonist action would also explain why peptides giving interactions of lower or higher affinity than the optimum could behave as antagonists (Figure 7.19). The important phenomenon of modified peptides behaving as partial agonists, with differential effects on the outcome of T‐cell activation, is addressed in the legend to Figure 7.19.
Figure 7.20 The immunological synapse. (a) The formation of the immunological synapse. T‐cells were brought into contact with planar lipid bilayers and the positions of engaged MHC–peptide (green) and engaged ICAM‐1 (red) at the indicated times after initial contact are shown. Reproduced with permission of AAAS.). (b) Diagrammatic representation of the resolved synapse in which the adhesion molecule pairs CD2/LFA‐3 and LFA‐1/ICAM‐1, which were originally in the center, have moved to the outside and now encircle the antigen recognition and signaling interaction between TCR and MHC–peptide and the co‐stimulatory interaction between CD28 and B7. The CD43 molecule has been reported to bind to ICAM‐1 and E‐selectin, and upon ligation is able to induce IL‐2 mRNA, CD69, and CD154 (CD40L) expression and activate the DNA‐binding activity of the AP‐1, NFkB, and NFAT transcription factors.
The immunological synapse
Experiments using peptide–MHC and ICAM‐1 molecules labeled with different fluorochromes and inserted into a planar lipid bilayer on a glass support have provided evidence for the idea that T‐cell activation occurs in the context of an immunological synapse. These and other imaging studies have revealed that the immunological synapse between the T‐cell and the DC has a “bull’s eye” pattern with a central cluster of TCR– peptide–MHC, known as the cSMAC (central supramolecular activation complex), surrounded by a ring of the integrin LFA‐1 interacting with its cognate ligand ICAM‐1 on the DC (Figure 7.20). Initially unstable TCR–MHC interactions occur outside of the integrin ring, followed by transit of the peptide–MHC molecules into the cSMAC, changing places with the adhesion molecules that now form the outer ring (Figure 7.20). It has been suggested that the generation of the immunological synapse only occurs after a certain initial threshold level of TCR triggering has been achieved, its formation being dependent upon cytoskeletal reorganization and leading to potentiation of the signal. LFA‐1 engagement with ICAM‐1 is essential for formation of immunological synapses for a number of reasons. In the early stages of synapse formation, these molecules serve in a predominantly adhesive capacity to tether the opposing cells to facilitate TCR and peptide–MHC interactions, thereby allowing the T‐cell to scan the peptide–MHC complex on offer.