Increased ex vivo lymphocyte proliferation in the absence of the intracellular FasL domain. (A) CFSE-labeled lymphocytes were either treated for 72 hours with 5 μg/mL coated anti-CD3 antibody (T cells), for 72 hours with 2.5 μg/mL soluble F(ab′)₂ anti-IgM antibody (B cells) or were left untreated (filled gray peaks, negative control). B cells (top panel), CD4⁺ CD25⁻ helper T cells without regulatory T cells (middle panel) and CD8⁺ cytotoxic T cells (bottom panel) were isolated from splenocytes. Activation-induced proliferation of living cells (propidium iodide negative) was measured by FACS analysis, as represented by the decrease in CFSE fluorescence intensity. The solid line represents wild-type cells while the dotted line represents cells from homozygous FasL ΔIntra mice. One representative experiment of 5 (T cells) or 1 of 4 (B cells) is shown. (B) The observed differences in proliferation are not due to differences in activation-induced cell death. Lymphocytes used for the CFSE proliferation assays were simultaneously seeded under identical conditions for annexinV/PI staining. The number of dead cells was quantified 48 and 72 hours after stimulation with anti-IgM (B cells) or plate-bound anti-CD3 (T cells) antibodies by FACS analysis. Dark gray columns represent wild-type and light gray columns FasL ΔIntra cells. The columns display the mean and bars the SE of 5 individual experiments.