IAP antagonist inhibits T cell proliferation in vitro. (A) A total of 3 × 105 CFSE-labeled wild-type (WT) splenocytes were stimulated with plate-bound anti-CD3 antibody. Splenocytes were exposed to LBW242 (red line) or solvent (green line). After 45 hours (left column) and 69 hours (right column), cultures were harvested completely and stained with anti-CD4 and anti-CD8 antibodies. Cells were resuspended in equal volumes of FACS buffer and counted in the flow cytometer for a defined time interval. The area under each curve indicates the total number of cells collected and the dilution steps of CFSE fluorescence represent the number of cell divisions. Red numbers represent a statistical analysis of the proportion of total T cells exposed to IAP antagonist relative to solvent control from 5 experiments. (B) Assessment of cell death in ConA/IL-2 stimulated CD8 T cells. Splenocytes were stimulated with 2 μg/ml ConA and 10 IU/ml IL-2 for 4 days in the presence of LBW242 and/or 10 µM necrostatin-1. CD8 T cells were analyzed for Annexin V exposure and Live/Dead FACS staining. Annexin V positive cells were classified as apoptotic and cells single positive for Live/Dead stain were classified as necrotic. Dot plots show a representative experiment and column graph shows the mean of 2 experiments.
