Figure 2. Regulation of perforin, granzyme B, and IFN-γ expression by T-bet and Eomes in differentiating CTLs. (A) IFN-γ expression by WT (Tbx21+/+) and T-bet–deficient (Tbx21−/−) T cells. Naive CD8+ T cells, or cells activated and cultured for 4 or 6 d, were restimulated with PMA and ionomycin for 6 h, and IFN-γ expression was assessed by intracellular staining. Numbers show the percentage of IFN-γ+ cells. (B) Northern blot analysis of Prf1 and GzmB mRNA expression in WT or T-bet–deficient CD8+ T cells activated and either left uninfected (uninf) or transduced with retroviruses expressing Eomes-VP16 (Eo-VP16) or an empty IRES-GFP cassette (GFP). Total cellular RNA was analyzed on day 6 of culture. The frequency of transduced cells in the cultures was equivalent for both constructs (∼65–70% GFP+ cells; not depicted). (C) Granzyme B and IFN-γ expression by Tbx21+/+ and Tbx21−/− T cells analyzed in restimulated cells that had been cultured for 5 d. (D) IFN-γ production by cells transduced with Eo-VP16 or control (GFP) retroviruses (RV) measured on day 4 after 6 h of restimulation with PMA and ionomycin. Numbers show the percentage of GFP+ IFN-γ+ cells. Results are representative of three (A and C) or two (B and D) independent experiments.