FIGURE 1.

Loss of Cbl-b partially rescues defective development of CD4⁺CD25⁺Foxp3⁺ Tregs in Cd28^−/− mice. (A) Thymocytes and splenocytes from WT, Cblb^−/−, Cd28^−/−, and Cblb^−/−Cd28^−/− mice (n=5/group; 8 wks of age) were surface-stained with anti-CD4, anti-CD8, and anti-CD25, and intracellularly stained with anti-Foxp3. The percentage of CD4⁺CD8⁻CD25⁺Foxp3⁺ cells in thymuses and CD4⁺CD25⁺Foxp3⁺ cells in spleens of different groups of mice were calculated. Statistical significance was calculated using the student t-test. *p<0.05 compared to WT, ** p<0.01 compared to WT; #p<0.001 compared to Cd28^−/−. (B) WT CD4⁺CD25⁻ T cells were incubated with CD4⁺CD25⁺ T cells isolated from WT and Cblb^−/−Cd28^−/− mice at different ratios in the presence of anti-CD3 and T-depleted APCs for 72 hrs. T cell proliferation was determined by [³H]thymidine incorporation. (C) WT and Cd28^−/− mice received daily i.v. injections of a combination of mouse IL-2 (1.5 µg/day, eBioscience) and anti-mouse IL-2 mAb (clone JES6-1A12) (50 µg/day; BioXCell) for 7–9 days. Thymuses and spleens were analyzed for Tregs on day 8–10 by flow cytometry as above. (D) Cblb^−/−Cd28^−/− mice were treated with anti-IL-2 daily by i.v. for 7–9 days, and Tregs in thymuses and spleens were analyzed by flow cytometry. *p<0.05 compared to PBS-treated controls; NS, no significance. The data shown are one representative of three independent experiments.