Figure 5.

Combination treatment with FTY720 plus GPI_325‐339 induces GITR⁺ non‐Treg cell populations in inguinal lymph nodes. Cells were obtained from inguinal lymph nodes of treated mice at day 14 (completion of treatment). (A) The percentage of GITR⁺ cells in Foxp3⁻CD4⁺ cells was determined by flow‐cytometric analysis (four animals from one experiment). (B) Violet Fluorescence‐labeled GITR⁻CD25⁻CD4⁺ cells (5 × 10⁴ cells) were cultured with unlabeled‐GITR⁻CD25⁻CD4⁺ cells, GITR⁺CD25⁺CD4⁺ cells, or GITR⁺CD25⁻CD4⁺ cells (2.5 × 10⁴ cells) in the presence of IA/IE⁺ cells (5 × 10⁴ cells) and anti‐CD3 mAb (1.0 μg/mL) for 72 h. Proliferation index was analyzed by FlowJo software (five animals from one experiment). (C) GITR⁻CD25⁻CD4⁺, GITR⁺CD25⁺CD4⁺ cells, and GITR⁺CD25⁻CD4⁺ were stimulated with anti‐CD3/anti‐CD28 coated dynabeads and IL‐2 (50 U/mL) for 72 h. The percentage of Foxp3⁺ cells and IL‐10⁺ cells and mean fluorescence intensity of CTLA‐4 expression were determined by flow‐cytometric analysis (three determinations). The results are shown as mean + SD. The significance of differences was examined by Duncan's test (*denotes P < 0.05).