Several lines of evidence, including our own data, suggest that a fraction of CD8, p56lck- associated protein that marks a population of cytotoxic T lymphocytes (CTL) and binds to the nonpolymorphic domain of MHC class I (MHC-I) molecules, form molecular assemblies with T-cell receptor (TCR) on the CTL surface. The formation of these assemblies facilitates the sensitivity and the specificity of CTL responses against target cells. We and others have shown that MHC proteins on the cell surface are also concentrated in clusters and that disruption of the clusters affects the ability to effectively present peptide antigens to T cells. We have recently simulated MHC clusters with a novel tool, semiconductor nanoparticles, quantum dots (QD), bearing controlled numbers of pMHC complexes, and found that the cognate and non-cognate pMHC complexes on QDs effectively cooperate in the binding of QD/pMHC to live T cells and in the induction of T cell responses. Our working hypothesis is that clustering of immune receptors on the cell surface leads to increased proximity between the receptor molecules that are essential for their ability to cooperate in the induction of downstream receptor-mediated signaling. The hypothesis will be tested by pursuing 2 specific aims: (i) To evaluate the proximity between TCR and CD8 on the surface of naive and antigen experienced CD8+ T cells and to relate changes in the extent of CD8-TCR clustering to the sensitivity and specificity of CTL responses;(ii) To estimate the separation distances between pMHC complexes attached to QDs and to compare them with corresponding distances between MHC proteins on the surface of living cells and to evaluate the impact of the proximity between pMHC with various biological activities bound to QDs on the efficiency of such QD/pMHC biosensors to elicit T cell responses. The proposed experiments are expected to generate sufficient amount of experimental parameters, which then can be utilized for building comprehensive models to explain mechanisms of triggering of the proximal signaling. Understanding the role of separating distances between immune receptors on T cells and target cells should enable us to make progress in understanding molecular mechanisms controlling T cell activation and will ultimately permit manipulation of various T cell activities to influence T cell-mediated immunity. Public Health Relevance: Inasmuch as CD8+ cytotoxic lymphocytes are indispensable component of anti- virus and anti-cancer defense of the host body, bettering understanding of the mechanisms of their actions, which is expected to emerge from the proposed research, will facilitate the development of new strategies to enhance the performance of these lymphocytes and to facilitate the ability of patients to fight viral infections and cancer.