Rapamycin does not induce anergy but inhibits expansion and differentiation of alloreactive human T cells

Studies in mice have shown that rapamycin inhibits cell cycle progression and promotes the development of clonal anergy. We here addressed the question if rapamycin can induce anergy of human T cells and studied the effects of rapamycin on activation, proliferation and expression of cytotoxic effector molecules of alloresponsive T cells in mixed lymphocyte cultures. Peripheral blood mononuclear cells from healthy individuals were labeled with CFSE to monitor subsequent cell divisions. Cells were cocultured with allogeneic irradiated cells in the presence or absence of rapamycin. Flowcytometric analysis was performed after staining for surface CD4, CD8, and CD25 and for intracellular perforin, granzyme B, active caspase-3, and TGF-beta. Bio-Plex cytokine assay was done to measure the secretion of IL-2, IL-4, IL-10, and IFN-gamma. Addition of rapamycin at a final concentration of 10 ng/ml strongly decreased precursor frequencies of alloreactive CD4+ and CD8+ T cells. However, when these cells were washed and subsequently specifically restimulated in the absence of rapamycin, the proliferative capacity appeared normal. Next to lowering precursor frequencies, rapamycin also inhibited T cell expansion by inducing apoptosis in divided alloreactive CD4+ and CD8+ T cells. Rapamycin did not interfere with the formation of CD25brightCD4+ T cells during allogeneic stimulation and did not inhibit their suppressive function. Furthermore, the drug decreased production of effector molecules perforin and granzyme B by alloreactive T cells and diminished alloreactive cytotoxicity. Our data show that rapamycin strongly inhibits proliferation and effector functions of alloreactive T cells in vitro, but does not induce alloantigen specific nonresponsiveness.