TLR2 stimulation on CD8 T cells increases the expression levels of effectors molecules and T bet. (A,F) Purified CD8 OT-1 T cells were activated with antigen-pulsed MyD88^−/− splenocytes in each of the absence (white bar) or presence of the TLR2 agonist, Pam₃Cysk₄ (10 μg/mL; black bar). Four days later, the intracellular level of granzyme-B, perforin, and IFN-γ were determined by flow cytometry. (B) The production of IFN-γ and granzyme B by OT-1 T cells, in response to varying concentrations of antigen, was determined by ELISA. (C) The IFN-γ, perforin, and granzyme-B transcript levels in OT-1 T cells were determined by RT-PCR. The fold changes in gene expression are shown. (D) The protein levels of T-bet and EOMES in nuclear extracts of OT-1 T cells were determined by Western blot analysis. (E) T-bet binding to the promoter of effectors molecules was examined by ChIP assays 3 days after T-cell activation. (F) Gene expression in OT-1 or T-bet^−/−OT-1 T cells was determined by RT-PCR. The data shown for panels A through D are representative of at least 4 independent experiments, each yielding similar results, whereas data shown in panels E and F are representative of 2 experiments. The fold changes in gene expression between TLR-stimulated and non–TLR-stimulated cells were analyzed by ANOVA; **P < .001, *P < .05