Fig. 2.

RIPK3 deficiency restores proliferation and survival of FADDdd CD8⁺ T cells. (A) CFSE analysis of CD8⁺ T cells treated with α-CD3 (145-2C11; 1 μg/mL) plus αCD28 (200 ng/mL). Splenocytes were stained and analyzed by cytometry after 3 d. Shown are plots for CFSE vs. cell numbers of CD8⁺ splenocytes. (B) Rescue of enhanced death phenotype of FADDdd CD8⁺ T cells by RIPK3 deficiency. Plots for CFSE vs. 7-AAD of CD8 cells; upper and lower left quadrants represent populations that have divided and are dead or alive, respectively. (C) Recovery of live CD8⁺ T cells in FADDdd × RIPK3^−/− vs. WT cultures following 3 d stimulation. Activated splenocytes treated with or without 10 μM necrostatin-1 (Nec-1) added at start of culture. Graph represents percent viable CD8⁺ cells recovered ± SEM. (D) Fold change in cell death of CD4⁺ or CD8⁺ cells upon restimulation (restim) with or without Nec-1 relative to death observed in activated cells (1 μg 1 × 10⁵ cells αCD3, 200 ng/mL αCD28) not subjected to restimulation (dotted line). Error bars represent SEM (*P < 0.05, **P < 0.01, and ***P < 0.001 vs. WT restimulation). (E) Lymphoid tissue from aged (40 wk) mice of indicated genotypes. (F) FACS plots display percentage of double-negative population that are CD3⁺ B220⁺ in mice of indicated genotypes. (G) Graphs represent counts or percentage of total live cells (after RBC lysis) that are CD4⁻CD8⁻CD3⁺B220⁺. Error bars represent SEM; n = 3 of each genotype.