Post-IL2 receptor blockade increases fas ligand expression and apoptosis in allospecifically-activated human helper T cells

Abstract

Introduction: Pre-interleukin-2 receptor (IL-2R) blockade with cyclosporine (CsA) abrogates apoptosis of activated allospecific lymphocytes. The mammalian target of rapamycin (mTOR) is a post-IL-2R signaling molecule with anti-apoptotic effects. We examined the effects of rapamycin (RPM) and CsA on apoptosis and surface expression of the apoptotic mediators fas and fasL in human mixed lymphocyte cultures (MLC). Methods: Peripheral blood lymphocytes were isolated by Ficoll-Paque gradient centrifugation. Responder and irradiated stimulator cells were co-cultured with logfold concentrations of RPM or CsA. Cells were stained with PE-labeled anti-CD4 antibody and annexin-V and 7-AAD, FITC-labeled anti-fas antibody, or FITC-labeled anti-fasL antibody and analyzed by flow cytometry. Cells staining for annexin-V, but excluding 7-AAD, were interpreted as apoptotic. Results: While CsA treatment decreased CD4+ T cell surface expression of fasL by up to 60% by day 7, RPM increased fasL express by up to 40%. Interestingly, fas surface expression was decreased by both RPM (up to 83%) and CsA (up to 89%). RPM increased CD4+ T cell apoptosis by up to twofold, while CsA decreased apoptosis by up to 50%. Conclusions: Post-IL-2R blockade with RPM increases apoptosis of allospecifically-activated helper T cells. This effect is consistent with the antiapoptotic function of mTOR, and may explain the dissimilar apoptotic effects when compared to pre-IL-2R blockade with CsA. The increase in fasL expression, an apoptotic effector molecule, on the cell surface in response to post-IL2R blockade suggests a potential mechanism for this effect.