INTRODUCTION: Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes following interleukin-2 (IL-2)-dependent T-lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal rabbit antithymocyte antibody thymoglobulin (rATG) and monoclonal anti-IL-2 receptor antibody basiliximab. METHODS: Human lymphocytes were isolated using Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (10(6) cells/mL) were cocultured with equal numbers of responder cells. Apoptosis was measured using annexin-V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed using Western blotting with densitometry. RESULTS: Apoptosis increased at days 3 and 7 in rATG MLC compared with control and basiliximab MLC. Fas was up-regulated in rATG MLC in a dose-dependent manner, whereas basiliximab did not alter fas. FasL was increased initially and at late time points in rATG MLC. CONCLUSIONS: Polyclonal rATG increased apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, monoclonal basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that a specific IL-2 pathway blockade may prevent allospecific tolerance and that a non-IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cells.
INTRODUCTION: Transplant tolerance is dependent on the apoptotic deletion of allospecific T lymphocytes following interleukin-2 (IL-2)-dependent T-lymphocyte activation. Current immunosuppressive strategies block IL-2 and may prevent T-cell activation. We examined apoptotic alterations in mixed lymphocyte culture (MLC), a model of allospecific lymphocyte activation, by polyclonal rabbit antithymocyte antibody thymoglobulin (rATG) and monoclonal anti-IL-2 receptor antibody basiliximab. METHODS:Human lymphocytes were isolated using Ficoll-Paque gradient. Cesium-irradiated (2500 rad) stimulator cells (10(6) cells/mL) were cocultured with equal numbers of responder cells. Apoptosis was measured using annexin-V staining and propidium iodide exclusion using flow cytometry. Isolated protein was analyzed using Western blotting with densitometry. RESULTS: Apoptosis increased at days 3 and 7 in rATG MLC compared with control and basiliximab MLC. Fas was up-regulated in rATG MLC in a dose-dependent manner, whereas basiliximab did not alter fas. FasL was increased initially and at late time points in rATG MLC. CONCLUSIONS: Polyclonal rATG increased apoptosis and production of the proapoptotic proteins fas and fasL. In contrast, monoclonal basiliximab did not change lymphocyte apoptosis or apoptotic protein production. These results suggest that a specific IL-2 pathway blockade may prevent allospecific tolerance and that a non-IL-2 pathway blockade may encourage apoptosis of allospecifically activated T cells.