Pan-Caspase Inhibitor Prevents Anti-CD3 Mediated T-Lymphocyte Proliferation

BACKGROUND. Host defense requires the ability for upregulation and downregulation of lymphocyte responses. It is well known that proliferative responses can be seen in conjunction with subsequent cell death suggesting that the two processes are linked.(1) Caspases are constitutively expressed zymogens which, upon activation, participate in receptor and nonreceptor mediated apoptosis.(2) The purpose of this study was to assess whether caspase activity was required for the proliferation of splenic-derived T lymphocytes by using the nonspecific, irreversible inhibitor z-VAD-fmk. METHODS. Splenocytes from C57Bl/6 mice were passed over an antibody coated column (R&D) to obtain a >90% CD3+ population. Cells were stimulated to proliferate with immobilized monoclonal anti-CD3 (BD Pharmingen, 100µg/ml) in 10% MLC media. Proliferation was assessed by 3 H thymidine incorporation at time points with and without zVAD-fmk (Alexis, 100µM). Western Blot analysis was performed by SDS-PAGE and probed with polyclonal Caspase 3 antibody H-277 (Santa Cruz, 1:1000). Caspase 3-like activity was assessed by 100µM Ac-DEVD-pNA (Alexis) substrate followed by quantitation by spectrophotometry at 405nm. RESULTS. TCR stimulation of purified T lymphocytes at 72 hours resulted in proliferation 500 fold greater than unstimulated controls. Treatment of lymphocytes with zVAD inhibited CD3 mediated proliferation by greater than 99%. Delay in the administration of inhibitor after stimulation decreased the effect on proliferation (see Figure 1).