The main goals of this application are to further elucidate the function of CD8 in immune responses and to define the molecular mechanisms involved in the regulation of expression of this protein. A cell-cell binding assay will be used to analyze the interaction between human CD8 and class I major histocompatibility complex (MHC) proteins. The ability of CD8AlphaBeta heterodimers to bind to class I molecules will be compared to that of CD8Alpha homodimers. The binding site on class I MHC proteins for CD8 will be compared for CD8AlphaBeta heterodimers and CD8AlphaAlpha homodimers. The relative abilities of distinct forms of CD8 (AlphaAlpha, Alpha'Alpha, AlphaBeta, Alpha'Beta) to stimulate T cell activation will be assessed by measuring the release of interleukin-2 (IL-2) and the phosphorylation of the CD3 zeta chain in response to antigen stimulation, as well as the activation-induced binding to class I MHC proteins. These studies will be done both in a class I-specific T cell hybridoma, in which CD8 and the T cell receptor can bind to the same MHC protein, and in a class IIrestricted T cell hybridoma, in which CD8 and the T cell receptor can only bind to separate MHC proteins. Chimeric CD8 molecules in which the ligand binding portion consists of CD8Beta dimers will be constructed and assayed for their ability to interact functionally with class I MHC proteins. The mouse CD8Alpha and CD8Beta genes are known to be located 36 kb apart in the mouse genome. An attempt will be made to link the human CD8Alpha and CD8Beta genes by chromosomal walking. The sequences required for proper tissue-specific, subset specific and stage-specific expression of human CD8 will be identified using the native human CD8Alpha and CD8Beta genes in transgenic mice and a reporter gene system in tissue culture cells.
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