Fig. 5

Proliferative response of type II collagen (CII)-specific T cells recovered from mice treated with FTY720 (FTY). Total lymphocytes or CD4⁺CD25⁻ T cells were recovered from draining lymph nodes of CII-immunized mice treated with 1 mg/kg of FTY720 or vehicle and cultured in the presence of the CII_257–274 peptide for 4 days. [³H]thymidine was added to the cultures on day 3, and cells were harvested on the following day. Controls were unstimulated cells. Error bars indicate standard deviation. a In vitro proliferative response of lymph node cells from DR1 transgenic mice immunized with CII and treated with 1.0 mg/kg FTY720 or vehicle three times per week for 3 weeks. b Lymph node cells from mice treated with 1.0 mg/kg FTY720 or vehicle were stimulated in vitro with anti-CD3/anti-CD28 antibodies (Ab). c Proliferative response of CD4⁺CD25⁻ lymph node cells from CII-immunized mice treated with 1.0 mg/kg FTY720 or vehicle. CD4⁺CD25⁻ T cells were purified using magnetic bead separation and mixed with antigen-presenting cells depleted of CD4⁺ cells recovered from the spleens of FTY720- or vehicle-treated mice. d Addition of interleukin (IL)-2 to cultures of T cells from mice treated with FTY720 restored the CII-stimulated proliferative response. e and f DR1-CII tetramer analyses of the proliferative response shown in (d). Lymph node cells from FTY720- or vehicle-treated mice immunized with CII were labeled with CellTrace Violet, placed in culture, and stimulated with the CII peptide. After 3 days, cells were recovered and stained with DR1-CII tetramer; antibodies to CD3, CD4, and CD8; and 4′,6-diamidino-2-phenylindole (DAPI). Data shown are the percentages of CD4⁺ DR1-CII tetramer-positive cells in cellular division as indicated by the loss of CellTrace Violet fluorescence. Data were sequentially gated on DAPI⁻, CD3⁺, and CD8⁻ cells. Gray histograms are the CD4⁺ DR1-CII tetramer-positive cells, and open histograms are derived from CellTrace Violet–labeled cells that were not stimulated to divide