T cells initiate the immune response to foreign antigens following direct contact with primed antigen presenting cells (APC) such as dendritic cells.  T cell receptor (TRC) engagement results in a cytosolic Ca (Ca) signal that leads to Ca dependent gene expression, cytokine secretion, proliferation, and differentiation.  Our experimental approach using live-cell imaging will parallel this sequence of events.  Two-photon microscopy has recently enabled visualization of T and B cells deep within the intact lymph node, and has revealed a dynamic pattern of motility and cell-cell interaction, offering a unique opportunity for in vivo imaging of immune system function.  Thus, our approach will aim to consolidate results from in vitro and in vivo experiments performed on antigen-specific primary T cells from transgenic mice and T cell lines.  A variety of introduced and genetically encoded fluorescent reports will monitor the TCR directly, (Ca), gene expression, and secretory responses in T cells before and after stimulation.  A further goal of this project is to compare the functional responses of quiescent, activated, and chronically activated auto reactive T cells in light of their differing ion channel phenotypes.