Figure 3
Antigen specific proliferation is decreased in lymph node cells of STA-5326-treated mice, and IL-17 production and the proportion of Th17 cells from draining lymph nodes are significantly reduced in STA-5326-treated mice. (a) Antigen specific proliferation of draining lymph nodes in STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml human experimental autoimmune uveoretinitis (IRBP) peptide 1–20 for 72 hours and pulsed with bromodeoxyuridine for the last 24 hours. (b-d) Cytokine production of interferon (IFN) γ, interleukin (IL) 17 and IL-4 by draining lymph node cells from STA-5326-treated or vehicle-treated mice. Immunized mice were treated with 5 mg/kg or 20 mg/kg STA-5326 or vehicle from day 0 to day 14 after immunization. Draining lymph node cells collected on day 18 after immunization were pooled within each group. Cultures were stimulated with 10 μg/ml IRBP1–20 for 72 hours, and supernatants collected at 72 hours were assayed by ELISA. (a-d) Statistical analysis was performed using Student;s t-test. (e and f) Intracellular cytokine staining of draining lymph node cells in 20 mg/kg STA-5326 or vehicle-treated mice. Draining lymph node cells collected on day 18 were stimulated with IRBP1–20 for 72 hours, and the cultured cells were incubated with PMA plus ionomycin and brefeldin A and stained with CD4, CD8 and intracellular IFN-γ and IL-17. The percentage shown in the upper right quadrant is for IFN-γ or IL-17 positive cells in CD4+ T cells.