The TLR2/MyD88 signaling pathway promotes adaptive immune response to VV in vivo (A) Maturation of splenic CD11c⁺ DCs. Wild-type C57BL/6 (WT), TLR2^−/−, or MyD88^−/− were administered intravenously with 1 × 10⁷ pfu VV and splenocytes were harvested 24 hours later, stained with anti-CD11c and anti-CD86 or an isotype control antibody (isotype), and analyzed for CD86 expression by FACS based on isotype control antibody staining on respective CD11c⁺ cells. The percentages of CD11c⁺ DCs expressing CD86 are indicated. (B) VV-specific T-cell activation. Seven days after infection, CD5⁺ T cells purified from splenocytes were restimulated with VV at an MOI of 0.5, 0.1, or 0.02 in the presence of corresponding naive irradiated splenocytes. Proliferation of virus-specific T cells was analyzed by ³H-thymidine incorporation. Data reflect the mean plus or minus SD of the stimulation index, calculated by dividing ³H counts in cpm in the presence of viral stimulation by those in the absence of stimulation, as a function of different virus doses. T cells harvested from uninfected C57BL/6 mice were used as control (control). (C) Activation of VV-specific effector CD8 T cells. Seven days after VV infection, splenocytes were stimulated with anti-CD3 and anti-CD28 for 4 hours and assayed for intracellular IFN-γ secretion by CD8 T cells. Splenocytes from uninfected C57BL/6 mice were used as control (control). In some TLR2^−/− mice, 30 μg LPS was co-injected at time of VV infection (TLR2^−/− plus LPS). Events were gated on total lymphocytes. The percentages of IFN-γ–producing CD8 T cells among total lymphocytes are indicated.