Cytolytic T lymphocyte (CTL) interaction with an antigen-bearing cell results in generation of transmembrane signals which activate responses, including degranulation and delivery of the 'lethal hit' to kill the target cell. Recognition and signalling are governed by the antigen-specific T cell receptor (TCR), but additional 'accessory' proteins on the CTL also contribute as a result of interactions with ligands on the target cell. A number of transmembrane signalling events occur upon CTL-target interaction, including activation of tyrosine kinase (TK) activation of the polyphophoinositide (PI) pathway to result in a rise in [Ca++]int and translocation of protein kinase C (PKC). In addition, we have obtained considerable evidence that activation of PLA2 to release arachidonic acid occurs rapidly upon CTL stimulation, and is required for triggering responses. A continuing aim of this project is to further elucidate the role of PLA2 activation in CTL signalling, and determine if activation is directly linked to the TCR. The CD8 T cell accessory protein binds to class I protein, and previous work by ourselves and others had suggested that CD8 might have both adhesion and signalling roles. We have found that CD8 on CTL is normally inactive but can be 'activated' via the TCR to mediate CTL adhesion to class I protein on artificial membranes. We also found that activated CD8 binding to class I results in generation of a signal required to initiate degranulation. The PI pathway is not activated by a minimal TCR stimulus, but is activated if CD8 then binds to class I. Thus, CD8 can play a critical role in coupling TCR occupancy to PI pathway activation. Experiments will be done to determine the nature of the TCR-dependent signal which 'activates' CD8, and the mechanism by which CD8-class I binding activates the PI pathway. Experiments will also be done using purified ligands to examine the adhesion and signalling roles of LFA-1 and VLA receptors on CTL, and to quantitate the relative contributions of these, and CD8, to antigen recognition and CTL activation. It is anticipated that the proposed studies will provide a better understanding of the complex set of ligand binding/signalling interactions which mediate CTL recognition and lysis of foreign cells; an understanding which should contribute to more effective manipulation of these responses for protective and therapeutic purposes.
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