The T cell repertoire is composed of two distinct properties - the recognition specificity of the TCR heterodimer and the functional response of the cell after TCR stimulation. It is now clear that once a particular TCR heterodimer is expressed on the T cell surface, the antigen specificity is frozen for all the clonal progeny of that cell. The functional responses available, however, are quite extensive and range from programmed cell death to initiation of distinct modalities of active immune response. This proposal is designed to elucidate the precise molecular mechanisms which determine the selection among possible functional responses and the clonal stability of such selection events. The experimental approach will be to stimulate alpha beta-TCR transgenic T cells with molecularly defined """"""""artificial"""""""" APCs constructed by transfection, and to analyze these activated cells using sensitive in situ techniques. The broad long term objective of this study is to determine the molecular interactions that determine the functional activity of T cells so that these interactions can be altered to achieve more clinically useful immune responses (or lack of immune responses) in clinical medicine. The first specific aim is to generate a panel of """"""""artificial"""""""" APCs by transfecting a fibroblast line with MHC class II antigen and a series of putative costimulatory molecules, including B7/BB-1, ICAM-1, HSA, and various combinations of these molecules. These APC will then be used to dissect the relationship between costimulation and cytokine expression by both established T cell clones and by naive CD4+ T cells isolated directly from unimmunized TCR transgenic mice. The analysis of these responses will rely heavily on a sensitive in situ hybridization procedure to enumerate the frequency of individual T cells that express mRNA for different cytokines (IL-2, IFNgamma, LT, IL-4, IL-5, and IL-10). Production of cytokine protein will be determined in parallel by standard sandwich ELISA assays. The frequency of cells that undergo apoptosis will also be measured by a sensitive single cell technique utilizing TdT mediated end labeling of fragmented DNA. The focus of these investigations will be to determine the particular costimulator combinations that direct T cells into stable cytokine expression profiles, including the split between Th1- and Th2-like cells and the IL- 2 deficient state often termed anergy. The second specific aim is to examine the clonal heterogeneity of cytokine expression in both naive transgenic T cells and in established T cell clones. These studies will focus on a double label in situ hybridization procedure and physical sorting of cells based on CD45RB antigen expression. The focus will be to determine whether a restricted cytokine phenotype develops due to selective growth of committed individual cells or represents molding the activity of cells capable of multiple cytokine expression. Experiments are also proposed to examine the stability of cytokine phenotype in established clones. The third specific aim is to examine the role of anatomical localization and time after thymic emigration on cytokine expression phenotype development.
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