CD25⁺Foxp3⁺ 2a T cells generated in CLNs have suppressing activity in vitro and in vivo. (A) CD25⁺ 2a T cells recovered from CLNs of DC-tg Rag2-deficient mice inhibit mitogen-induced proliferation of polyclonal T cells. CFSE-labeled polyclonal T cells were stimulated with an anti-CD3ϵ monoclonal antibody and syngeneic splenocytes in the presence or not of 2a T cells recovered from spleen or CLNs of DC-tg Rag2-deficient mice 4 weeks after transfer. Polyclonal T-cell proliferation was assessed by flow cytometry 72 hours later. Where indicated, recovered 2a T cells were depleted of the CD25⁺ population. Left panels: histograms of CFSE-labeled polyclonal T-cell proliferation. Right panel: quantitative analysis of the efficiency of inhibition of naive anti-CD3 stimulated T cells (Teff) cultured at the indicated ratio with CD25⁺ 2a T cells (Treg). Data are mean ± SD of 3 independent experiments. (B) IFN-γ production by 2a T cells, recovered from CLNs or spleen of DC-tg Rag2-deficient mice 3 weeks after transfer, on in vitro restimulation in the presence of splenocytes loaded or not with the Bpep. Where indicated, 2a T cells recovered from CLNs were depleted of the CD25⁺ population (CD25⁻CLN). The activation of naive 2a T cells is also shown. (C) IFN-γ production by polyclonal BALB/c T cells, recovered from CLNs or spleen of Rag2-deficient and DC-tg Rag2-deficient mice 3 weeks after transfer, on in vitro restimulation in the presence of splenocytes loaded or not with the Bpep. (D) Kinetics of CD25⁺Foxp3⁺ 2a T-cell generation in CLNs and kinetics of CD25^intFoxp3^low 2a T-cell generation in the spleen of DC-tg Rag2-deficient mice. (E) IFN-γ production by 2a T cells, recovered from CLNs or spleen of DC-tg Rag2-deficient mice 10 days after transfer, on in vitro restimulation in the presence of splenocytes loaded with the Bpep. Where indicated, 2a T cells recovered from CLNs and spleen were depleted of the CD25⁺ population (CD25⁻). (A-C,E) Data are mean ± SEM of 3 independent experiments performed by pooling cells derived from 3 mice per group. (D) Data are mean ± SEM of 3 independent experiments performed using at least 3 mice per group. (F) Top diagram: schematic representation of the adoptive transfer experiment. DC-tg Rag2-deficient mice were first transferred with naive 2a T cells; 3 weeks later, reconstituted mice were either nontreated (NT) or treated with the anti-CD25 blocking antibody, PC61 (labeled as αCD25), and one week later transferred with CFSE-labeled 2a T cells. Bottom left panels: in vivo proliferation of CFSE-labeled 2a T cells measured 48 hours after transfer. Bottom right panel: quantitative analysis of the frequency of undivided cells. Data are mean ± SD of 4 mice analyzed in 2 independent experiments. *P < .05; **P < .01; ***P < .005.