TLR2 stimulation on CD8 T cells in vivo augments the number of effectors gene transcripts and increases T-bet expression. (A) Purified OT-1 (CD90.1⁺ CD45.2⁺) and MyD88^−/−OT-1 (CD90.2⁺ CD45.2⁺) CD8 T cells were mixed at a 1:1 ratio, and 1 × 10⁶ T cells were injected intravenously into CD45.1⁺CD90.2⁺ mice. One day later, mice were injected intravenously with Ova_157-164–pulsed CD45.1 splenocytes plus TLR2 agonist (10 μg) or Ova_157-164–pulsed splenocytes alone. Five days after adoptive T-cell transfer, cells were sorted by flow cytometry, and the levels of effectors gene transcripts were determined by RT-PCR. The numbers of each transcript in OT-1 and MyD88^−/−OT-1 T cells were normalized to β-actin, and fold changes of gene transcripts are shown. Error bars represent the SD from the mean of 3 PCR reactions. (B) Alternatively, intracellular T-bet protein levels in transferred T cells were determined via intracellular staining and flow cytometry. The data shown are representative of 2 independent experiments, each yielding identical trends. The fold changes in gene expression between were analyzed by ANOVA; *P < .05, **P < .001.