The goal of this research effort is to understand how an important type of white blood cell, called a T lymphocyte, recognizes the presence of a microorganism or cancer cell in the body, or inappropriately recognizes a normal component of the body (an "auto-antigen"). This research tries to provide a detailed understanding of how the substances (antigens) making up these microorganisms, cancer cells, or normal self-components, are made visible to the defending T cells or the auto-reactive T cells. We have examined the structure and function of special cellular proteins called major histocompatibility complex (MHC) molecules that are essential for antigens to be recognized by T cells. Specific questions involve understanding how structural differences in the MHC molecules of different individuals affect the ability of these proteins to stimulate T cells and the exact nature of the antigens bound to the MHC molecules. Our work has described the events within a cell that bring the antigen and MHC molecule and the cellular distribution of antigenic complexes within the body (antigen processing and presentation). By understanding these events, we will be able to identify the most important components of microorganisms or cancer cells to use in protective or therapeutic vaccines and how best to deliver these substances to stimulate an effective immune response. We have made several advances in the past year. We have developed a novel method for the direct microscopic visualization in real time of the interactions of T cells and antigen presenting cells in intact lymphoid tissue. We have uncovered a key role for microbial signals in enhancing antigen processing by dendritic cells and increasing their contact with T cells in secondary lymphoid tissue. Our studies have for the first time visualized the sites of intracellular association of antigenic peptides with MHC class I molecules after exposure to heat shock proteins associated with antigen; these same studies reveled a key role of receptor -mediated uptake in Hsp-based antigen presentation and also demonstrated two distinct intracellular routes of MHC class I loading. Finally, using high efficient methods for retroviral transduction of dendritic cells grown ex vivo, we have been able to modify the function of these key antigen presenting cells and to develop a new model for understanding the molecular basis of CD40-dependent signaling that plays a central role in dendritic cell function.