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Postthymic T cell receptor (TCR)-mediated cell death offers the potential for creating antigen-specific transplant tolerance analogous to thymic clonal deletion. Murine splenic CD4+T cells were rigorously purified by: (a) adherent cell depletion and (b) magnetic bead/monoclonal antibody (mAb) depletion of macrophages, Ia+cells, μ-chain+cells, NK cells, and CD8+T cells. CD4+s were typically >95% pure by flow cytometry. Resting CD4+s stimulated by plastic-immobilized anti-TCR/CD3 mAb were shown to die in the absence of exogenous interleukin (IL)-2. Blasting CD4+s showed dose-dependent cell death upon religation of TCR/CD3 in the presence of IL-2; however, withdrawal of IL-2 from blasting CD4+s also resulted in cell death. Cell death was shown to be apoptotic by flow cytometry DNA content analysis. Anti-CD28 mAb, co-immobilized with anti-TCR/CD3 mAb, inhibited cell death of resting CD4+s in the absence of exogenous IL-2; however, anti-CD28 mAb showed minimal cell death inhibition of CD4+blasts when TCR/CD3 was religated. In contrast, splenic adherent cells effectively inhibited cell death of blasting CD4+s induced by TCR/CD3 mAb religation.We conclude that TCR-mediated programmed cell death of highly purified splenic CD4+s is dependent upon activation state, availability of IL-2, and accessory cell or CD28 costimulator signals. Furthermore, IL-2 acts to protect against cell death in both resting and activated CD4+T cells. IL-2 protection could be overcome by high concentrations of anti-TCR/CD3 mAb, which results in cell death of CD4+blasts. In the effort to understand potential mechanisms of peripheral tolerance induction, these findings assist to distinguish and define conditions for antigen receptor-mediated programmed cell death of mature CD4+T cells.