FV-specific CD4 +and CD8 +T cell responses in FV-infected TLR3 +/+and TLR3 −/−mice. TLR3+/+ and TLR3−/− mice were infected with 20,000 SFFU of FV. CD4+ and CD8+ T cells were analyzed at 10 dpi by flow cytometry. Percentages of activated (CD43+ CD62L−, A) CD4+ T cells were determined. For the analysis of virus-specific CD4+ T cells (B) splenocytes were stained with MHC class II-antibody tetramers specific for F-MuLV env fn20. To analyze the function of CD4+ T cells, the intracellular expression of IFN-γ (C), IL-2 (D) and TNF-α (E) was measured in activated effector CD4+ T cells. Percentages of activated (CD43+, F) CD8+ T cells and virus-specific effector CD8+ T cells (G), which are specific for the FV GagL epitope stained for GagL class I tetramers, were analyzed by flow cytometry. Uninfected TLR3+/+ and TLR3−/− mice were used as controls (white bars). At least five mice per group were analyzed and the mean value for each group is indicated by bars and dots. At least 2 independent experiments were performed. Statistical differences between the groups are indicated by * for p < 0.05, ** for p < 0.01 and *** for p < 0.001. (H) Splenocytes from naive mice were loaded with the FV-specific DbGagL peptide and labeled with CFSE. Target cells were injected intravenously into naive or FV-infected TLR3+/+ and TLR3−/− mice. Two hours after transfer, donor cells from spleen were analyzed. The figure shows the percentage of target cell killing in the spleen. Two independent experiments with three mice per group were performed and mean values are shown by bars. Differences between both groups are indicated by ** for p < 0.01.