Depletion of CD25⁺ T cells restores proliferation and IL-2 production. (A) Mice were reconstituted with OVA-specific T cells and injected with αDEC-OVA or αGl117-OVA (isotype). Eight days later, OVA-specific T cells (αDEC-OVA T cells and Gl117-OVA T cells) were prepared and cocultured with OVA-pulsed BMDCs. In some experiments, as indicated, CD25⁺ T cells were depleted, and isolated CD25⁺ cells were added to T-cell preparations (1:4 ratio) derived from Gl117-OVA–injected mice. T-cell proliferation was assayed by H³-thymidine incorporation. The asterisk indicates a significant difference (α < 0.05) from proliferation obtained in samples with isotype-control–injected mice (Gl117-OVA T cells). (B) Mice were treated as in panel A, and CD25⁺ T cells were isolated after 8 days and incubated with a mixture of anti-CD3 antibodies, spleen cells, and CD4⁺ responder T cells. In controls either CD25⁺ or CD4⁺ T cells were stimulated. T-cell proliferation was determined by ³H-thymidine incorporation. The asterisk indicates a significant difference (α < 0.05) from proliferation obtained with stimulated CD4⁺ T cells only. Error bars indicate mean ± SD. (C) Mice were injected, and cells were isolated and cocultured as in panel A. Two days after coculture, cells were fixed/permeabilized and stained with FITC-labeled αCD4 and PE-labeled αIL-2 antibodies and were analyzed by FACS. Numbers shown are the percentage of cells within the designated quadrant.