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The frequency of T cells that can respond to alloantigens is unusually high. It remains unclear how T cells would respond when stimulated by multiple major histocompatibility complex (MHC) disparate alloantigens in the same cultures. In this report, we examined potential interactions of T cell clones that were stimulated simultaneously by two sets of complete MHC disparate alloantigens using mixed lymphocyte reaction (MLR). In this assay, we observed that proliferation of B6 lymphocytes (H-2b) stimulated by both BALB/c (H-2d) and C3H (H-2k) allogeneic cells was not increased but rather reduced as compared to B6 cells stimulated with either BALB/c or C3H allogeneic cells. Interestingly, interleukin (IL)-10 expressions at both protein level and mRNA level was significantly increased in cultures stimulated with the two MHC alloantigens, while IL-2, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β1 production did not show any differences. In addition, Foxp3 mRNA expression was comparable amongst all groups. In conclusion, we observed an inhibitory effect in T cell proliferation in response to multiple MHC mismatched alloantigens in MLR, and this effect might be associated with the upregulation of IL-10 expression.