The long-term goal of this project is an understanding of how costimulatory signals provided by antigen-presenting cells (APC) contribute to the productive activation of CD4+ T-cells. In vitro approaches have shown that the maximal production of the growth factor IL-2 by CD4+ T-cells requires T-cell antigen receptor (TCR) recognition of antigenic peptide/MHC complexes, and a costimulatory signal transduced by CD28 following interaction with B7-1 and B7-2 molecules on the APC. Although this two signal model has provided an important framework for the study of T-cell activation, it does not account for the profound effects that inflammation and survival proteins have on T-cell immunity in vivo. Therefore, in the current application, T-cell costimulation will be studied with a novel system that allows in vivo tracking of the fate of a small population of adoptively transferred TCR transgenic T-cells in vivo. Results from this system have led to the hypothesis that at least three steps are required for the productive activation of naive antigen-specific T-cells in vivo: initial proliferation of the antigen-specific T-cells in response to antigen-presentation by dendritic cells; adjuvant-related sustenance of the expanded T-cell population; and differentiation of the T-cells into cells capable of producing effector cytokines such as IFN-gamma (IFN-g). This hypothesis will be tested by using confocal microscopy and in situ hybridization to visualize the interactions between adoptively transferred fluorescent dye-labeled antigen-specific T-cells, antigen- specific B-cells, and dendritic cells to determine whether CD28- and IL-2-dependent proliferation by naive T-cells is initiated by peptide/MHC complex-bearing dendritic cells. Flow cytometry will be used to determine whether this interaction causes CD40 ligand expression on the antigen- specific T-cells and whether CD40 ligand or lymphokines produced by the T-cells induce B7 molecules on antigen-specific B-cells or macrophages. These methods and RT-PCR will be used to determine whether adjuvants sustain the T-cell response by promoting T-cell proliferation and/or survival and whether these effects are mediated by CD28 or products of the TNF-a cascade through the action of IL-2 or bcl-2 family proteins. Finally, the spatial relationships between antigen-specific T-cells and IL-12 producing APC will be visualized by immunohistology and the in vivo mechanism by which IL-12 promotes T-cell differentiation in vivo will be studied by flow cytometric detection of cytoplasmic IFN-g within antigen-specific T-cells.
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