Cytokine reduction of CD4
T cells with stimulation of IL-1R KO macrophages. (A) Isolated CD4+ T cells (1 × 105) from the spleen of OT-II mice were cultured with in vitro 10 MOI Theiler’s murine encephalitis virus (TMEV)-infected peritoneal macrophages (1 × 104) from either C57BL/6 or IL-1R knockout (KO) mice for 3 days in the presence of 2 μM OVA epitope peptides. T cell proliferative responses were analyzed using [3H]TdR uptake, and cytokine production (IFN-γ and IL-17) of the cultures were analyzed using specific ELISAs. (B) Isolated CD4+ T cells (1 × 105) from the spleen of B6 mice infected with TMEV at 8 days post-infection (dpi) were cultured with in vitro TMEV-infected (10 MOI for 24 h) peritoneal macrophages (1 × 104) from either naïve C57BL/6 or IL-1R KO mice for 3 days in the presence of viral epitope peptides. Message levels of the indicated genes were then analyzed by real-time PCR. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) level was used as an internal control. The values are expressed as the means ± SD of triplicates. Statistically significant differences are indicated with asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Data shown are the representation of three independent experiments. (C) Peritoneal CD11b+ cells from naïve B6 and IL-1R KO mice infected with TMEV in vitro for 24 h were analyzed for the expression of PDL-1 and TIM-3 T cell inhibitory molecules. A representative flow cytometry plot of three similar results is shown here.