Fig. 4

Ex vivo analysis of the effect of FTY720 on the type II collagen (CII)-specific T-cell response. Mice were immunized with CII and complete Freund’s adjuvant and treated with FTY720 (1.0 mg/kg or 0.2 mg/kg) or vehicle, or they were not immunized and not treated (naive). Lymph node cells recovered from the mice treated with FTY720 or vehicle (a–c) or from naive mice (d) were stained with a DR1-CII tetramer and antibodies specific for CD19, CD3, CD4, CD8, CD25, and CD62L, and the cells were analyzed by flow cytometry. The mean percentage of CD4⁺/tetramer-positive, CII-specific T cells in the lymph nodes is shown in (e), and the number of CII-specific T cells present in the lymph nodes as determined by tetramer staining and cell counts is shown in (f). The expression of CD25 and CD62L by the tetramer-positive cells in the FTY720 and control groups as determined by flow cytometry is shown in (g) and (h). Data were generated by sequential gating on 4′,6-diamidino-2-phenylindole–negative cells, forward versus side scatter, CD19⁻ and CD8⁻ cells, and CD3⁺CD4⁺ cells. The tetramer-positive cells in (a–e) are shown as the percentage of CD3⁺CD4⁺ T cells, and this percentage value was used to calculate the total number of CII-specific T cells shown in (f). Open bars indicate mice treated with 1.0 mg/kg FTY720, gray bars represent mice treated with 0.2 mg/kg, and closed bars indicate the vehicle control group. Error bars indicate standard deviation, and p values indicate statistically significant differences between treatment groups and vehicle groups