Perforin and granzyme B expression are not appreciably regulated by T-bet
To test the model outlined in the previous paragraph directly, we compared the expression of IFN-gamma, perforin, and granzyme B in CD8+ T cells from WT and Tbx21 (T-bet)-deficient mice. As expected (17, 21), naive Tbx21-/- CD8+ T cells produced IFN-gamma poorly upon activation (Fig. 2 A). Notably, this deleterious effect of T-bet deficiency was only observed in differentiating CD8+ T cells until day 4 of culture but was almost completely mitigated by day 6 (Fig. 2 A). This most likely reflected compensation by Eomes, which was strongly induced between days 4 and 6 (Fig. 1). In contrast, T-bet-deficient T cells cultured for 6 d showed no defect in perforin mRNA expression (Fig. 2 B, compare lanes 1 and 4). We consistently observed a modest reduction in GzmB mRNA in T-bet-deficient T cells (Fig. 2 B, compare lanes 1 and 4), which did not translate into a decrease in expression of granzyme B protein (Fig. 2 C). 
To examine the role of Eomes, we transduced naive CD8+ T cells from WT and Tbx21-/- mice with retroviruses containing internal ribosome entry site (IRES)-GFP that were either empty or encoded a strongly transactivating version of Eomes (Eo-VP16) (8), and expanded them for 6 d under our culture conditions. Eo-VP16, but not the empty GFP retrovirus, increased perforin expression in both WT and T-bet-deficient CD8+ T cells (Fig. 2 B, lanes 2, 3, 5, and 6). As expected, Eo-VP16 also rescued the early defect in IFN-gamma production observed in T-bet-deficient CD8+ T cells (Fig. 2 D). However, Eo-VP16 did not induce GzmB mRNA expression in either WT or T-bet-deficient cells; thus, the partial T-bet dependence of GzmB mRNA expression cannot be compensated for by Eo-VP16. 
