The overall objective of this project is to identify the molecular and cellular basis for the immunoregulatory effects of cyclosporine, a naturally occurring cyclic polypeptide that has had major impact on clinical organ transplantation. The hypothesis that normal human peripheral blood T-cells distinguished from one another on the basis of expression of cell-surface antigens, and with distinct functional potential, have intrinsic differential sensitivity to the inhibitory activity of CsA will be tested. The experimental approach to test the hypothesis includes: (1) Isolation of phenotypically distinct and functionally relevent subsets of normal human T-cells. The CD4+ cells, and subsets of CD4+ cells (CD4+CD45R+, CD4+CD45R-, D4+Leu8+(p80+), CD4+p8O-, CD4+CD45R-p80+ r CD4+CD45R-p80- cells), CD8+ cells and subsets of CD8+ cells (CD8+CD1lb+ or CD8+CD1lb- cells) will be isolated by panning and fluorescence activated cell sorting (FACS) of normal human T-cells. (2) Utilization of two novel models of T-cell activation. Transmembrane signaling of T-cell subsets with the synergistic combinations of: sn-1,2-dioctanoylglycerol (DAG, physiologic activator of protein kinase C [PKC]) and ionomycin (Calcium ionophore) or crosslinked anti-CD2 and anti-CD3 monoclonal antibodies. (3) Determination of effects of CsA on T-cell activation parameters initiated with the novel stimuli described in 2. A functionally linked cascade of activation events pertinent to signal transduction, cellular signaling and functional programming and cell growth will be examined for sensitivity to CsA. The parameters examined for inhibition by CsA will include: (1) calcium mobilization, (2) PKC activation, (3) induction of gene expression for Interleukin-2 (IL-2), lL-2 receptor (IL-2R, alpha and beta chains), IL-4, Interferon-gamma and the protooncogene, c-myc, (4) cell surface and functional expression of IL-2R, (5) production of IL-2, IL-4 and IFN-gamma (6) cell cycle progression and proliferation. CsA' 5 effects will also be examined in the presence of recombinant IL-I, lL-2, IL-4 and/or IL-6. A novel methodology for mRNA analysis, sequential reverse transcription and polymerase chain reaction as well as Southern and Northern hybridization will be utilized for the transcriptional phenotyping of functionally relevent and phenotypically distinct subpopulations of normal human T-cells.
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