204: mTOR knockdown inhibits anti-HLA antibody induced MHC class I signaling

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mediated rejection continues to be a significant source of morbidity and mortality in heart transplant patients past the first year. T cell antigen receptor (TCR) signaling is the essential element in these alloresponses against donor MHC and non-MHC antigens. Yet there is no therapy to specifically target this process. Dasatinib, an oral kinase inhibitor used for chronic myeloid leukemia, is exquisitely specific for Abl and Src family kinases (SFK), which are critical for the first step of T cell activation. The immunosuppressive effects of Dasatinib have not yet been examined, thus we conducted the following studies to determine the effects of this drug on in vitro T cell responses. Methods and Materials: PBMC were isolated from healthy donors and stimulated with PHA or anti-CD3 (1-100 ng/ml) plus/minus Dasatinib (0.25-1000 nM). IL-2 expanded T cells were analyzed in the same fashion. Flow cytometric analysis for CD69, CD38, CD25, and HLA-DR was performed on total CD3 and CD4 or CD8 gated T cells. Proliferation was analyzed via CSFE staining. Immunoblotting was performed for total phosphotyrosine, phospho (p)-SFK, p-ZAP-70, p-ERK, p-AKT, p-STAT3, and p-STAT5. Results: Dasatinib inhibits T cell proliferation without inducing apoptosis. T cells remain viable and capable of activation upon drug withdrawal. Low nanomolar concentrations of Dasatinib markedly inhibit TCR induced expression of activation markers CD69, CD25, CD38, and HLA-DR. IFN-gamma production is inhibited by Dasatinib Immunoblot and intracellular flow cytometric analysis reveals specific targeted inhibition of the TCR signaling complex Alternative activation via phorbol ester, bypassing the TCR, is not affected even by micromolar concentrations of Dasatinib, demonstrating specificity for SFK Conclusions: We demonstrate that targeted inhibition of the kinases acting at the earliest stage of lymphocyte activation is a novel immunosuppressive approach that may offer a distinct advantage for combating rejection.