Although natural abundance of antigenic peptides in some virus-infected is very low, these cells are still lysed by virus-specific cytotoxic T lymphocytes (CTL). We have found that less then a dozen cognate peptide-MHC (pMHC) complexes on target cells are sufficient to render them susceptible for specific lysis by CTL. The molecular mechanism accounting for the striking sensitivity of target cell lysis by CTL is not understood. Our preliminary data suggest that release of a small amount of cytolytic granules would not induce target cell lysis unless the granules are concentrated in a limited space between CTL and target cell membranes. We hypothesized that immunological synapse concentrates the granules preserving their activity at the precise location and accounts for the sensitivity and specificity of target cell lysis by CTL. We have also found that LFA- 1 -1CAM- 1 interactions are sufficient for early stages of immunological synapse formation by CTL. This let us to propose that signaling through adhesion molecules results in the formation of transient synapse that has two typical domains or supramolecular activation clusters (SMACs). The transient synapse is likely to facilitate effective scanning of the surface of target cells by CTL. Most recently, we have found that MHC-I and ICAM-1 molecules are co-localized biochemically and by imaging within membrane rafts. We also showed that raft integrity facilitates viral peptide-MHC (pMHC) detection by CTL through mechanisms that require interaction of LFA-1 and ICAM-1. Based on this we hypothesized that ICAM-1-MHC-I co- clustering in rafts provides the linkage between early immunological synapse formation on CTL and the presentation of antigen on target cells. We further hypothesize that MHC-I and ICAM-1 are recruited to the rafts through adapter proteins to form complex molecular assemblies, which might have distinct structures on various cell types. Using precise manipulation of human CTL clones with known specificity, we will test this hypothesis by pursuing 3 specific aims: (i) To determine the significance of cytolytic molecule concentration by the immunological synapses by studying cytolytic granule release at the interface between the T cell and target cell membranes and the role of LFA-1-ICAM-1 interactions that lead to the synapse formation and effective target cell lysis by CTL; (ii) To determine the mechanism of immunological synapse formation by CTL and NK cells studying adhesion molecule patterns and functional correlates of these patterns; (iii) To determine the functional importance and mechanism of MHC-I and ICAM-1 interactions in membrane rafts.