Anti-GITR mAb but not anti-OX40 mAb induces the proliferation of CD4⁺CD25⁺ T reg's. (A) CD4⁺CD25^- cells (1 × 10⁵) cultured with CD4⁺CD25⁺ (5 × 10⁴) cells were stimulated with 1 μg/mL of anti-CD3 and ACs (1 × 10⁵) and seeded in a 96-well plate in triplicates. After 66 hours, the triplicates were pooled and analyzed by flow cytometry. Where indicated, CD4⁺CD25⁺ cells were preincubated with anti-OX40 or anti-GITR mAbs (both at 30 μg/mL), washed twice, and added to the CD4⁺CD25^- cells. ^*CFSE-labeled cells in the coculture. R1 indicates nondividing cells, whereas R2 indicates proliferating cells. The same experiment in panel A was repeated 4 times and the results, expressed as percentage of proliferating cells (R2), were pooled and reported in panel B. In the left part of panel B is reported the percentage (±SD) of CFSE-labeled CD25^- proliferating T cells in the presence or absence of CD25⁺ T cells left untreated or pretreated with anti-OX40 or anti-GITR mAbs, as indicated. In the right part of panel B is reported the percentage of proliferating CFSE-labeled CD25⁺ T cells. After labeling with CFSE, CD25⁺ T cells were left untreated (NT) or were preincubated with anti-OX40 or anti-GITR mAbs, and were washed twice before seeding in the presence or absence of CD25^- T cells, as indicated.