Effect of IAP antagonist is reduced in TNF-deficient mice. (A) CFSE-labeled WT and TNF^−/− splenocytes were stimulated with plate-bound anti-CD3 antibody and exposed to graded concentrations of LBW242 (red line) or solvent (green line). After 72 hours, the cultures were harvested and stained with anti-CD8 antibody. 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 represents the total number of cells collected. WT or TNF^−/− mice were infected intravenously with 200 PFU LCMV and treated daily with LBW242 or solvent intraperitoneally. At day 8 post-infection, (B) total counts of leukocytes, (C) CD8 T cells, and (D) GP33-specific CD8 T cells were determined in the spleen. (E) KLRG-1 upregulation was analyzed on gated CD8 T cells and total numbers are shown. (F) Expression of intracellular INFγ was quantified on CD8 T cells and total numbers are given. Numbers displayed in the panels indicate the ratio of the different cell populations from solvent and LBW242 treated animals for WT and TNF^−/− mouse groups, respectively. (G) Serum was taken from day 8 LCMV-infected mice and analyzed for TNF levels using enzyme-linked immunosorbent assay. Data are representative of 3 independent experiments.