Rapid and widespread proliferation of T reg cells after IL-2–JES6-1 treatment. Mice were injected i.p. daily for 3 d (days 0, 1, and 2) with JES6-1 mAb and/or IL-2 and analyzed on day 3 (A and B), day 5 (C–E), day 6 (F), or where indicated (G and H), usually in the spleen. Doses were 1 µg IL-2 and 5 µg mAb unless otherwise specified. (A) The effect of varying the ratio of IL-2 and JES6-1 on CD4⁺CD25⁺Foxp3⁺ (T reg cell) expansion was analyzed by injecting titrated doses (1–50 µg) of JES6-1 mAb, plus a fixed concentration (1 µg) of rmIL-2. The arrow indicates a 1:2 molar ratio of JES6-1 mAb to IL-2. (B) The effect of titrating the dose of IL-2 plus JES6-1 mAb (molar ratio constant at 2:1) was analyzed by varying the total dose from 0–24 µg per day. (C) The total numbers of T reg cells in the spleen were enumerated after treatment with JES6-1, IC mAb (rat IgG2a), IL-2, IL-2–IC mAb, or IL-2–JES6-1 mAb. Horizontal bars represent means. (D) Expansion of T reg cells in the spleen, MLN, liver, BM, PP, and small intestinal LP after injection of IL-2–JES6-1. (E) Number of Foxp3⁺ cells in the CD4⁺ single-positive and CD4⁺CD8⁺ double-positive thymocyte populations after IL-2–JES6-1 treatment. (F) Proliferation, as measured by CFSE dilution, of adoptively transferred CD4⁺CD25⁺ or CD4⁺CD25⁻ Ly5.1⁺ T cells in LNs of mice injected with IL-2–JES6-1. Percentages of donor cells divided (as determined by CFSE incorporation) are shown. (G) Kinetics of T reg cell expansion in the spleen after three injections of IL-2–JES6-1. Percentages (left) and total cell numbers (right) are shown. The percentage of Foxp3⁺ cells after 200 µg PC61 i.p. (day −1) followed by three injections of IL-2–JES6-1 is shown (arrows). (H) Expansion of cells with the βγ IL-2R, MP CD8⁺ T cells and NK cells, compared with T reg cells (αβγ IL-2R) after IL-2–JES6-1 treatment. Data are shown as a fold change for each cell type compared with untreated mice over 9 d (the dashed line indicates a fold change of 1). Data in A, B, D, and G (left) are shown as percentages of CD4⁺ T cells. Data are representative of two to five independent experiments. Data are means ± SEM. Flow cytometry plots depict log₁₀ fluorescence.