(A) Adoptive transfer experiments. Spleen cells and draining lymph node cells isolated from immunized WT mice were used as donor cells and restimulated with 20 μg/ml of MOG peptides in vitro. 3 × 10⁷ cells were transferred into WT or TRIF^–/– naive recipient mice via tail-vein injection (5 mice per group). The mice were monitored daily for clinical signs of disease. (B) Spleen cells and draining lymph node cells isolated from immunized TRIF^–/– mice were restimulated with MOG peptide in vitro for 72 hours. 3 × 10⁷ cells were transferred into WT or TRIF-deficient naive recipient mice via tail-vein injection (5 mice per group). (C) Flow cytometry analysis of Th17 development in CD4⁺ T cells cocultured with BMMs. BMMs from WT mice, TRIF-deficient, or IFNAR-deficient mice were stimulated with LPS (100 ng/ml) for 24 hours, then were cultured with WT naive CD4⁺ T cells in the presence of anti-CD3 (1 μg/ml) for 72 hours. Cells were stained for surface CD4 and intracellular IL-17. Plots were gated on CD4⁺ T cells. Numbers indicate percentage of IL-17⁺CD4⁺ cells of total CD4⁺ cells. (D) IL-17 production in the coculture of WT CD4⁺ T cells and BMMs in experiments described in C. (E) IL-17 production from TRIF-deficient CD4⁺ T cells cocultured with BMMs.