PD-1 deletion alters Treg homeostasis during IL-2 therapy. C57BL/6 PD-1^−/− or C57BL/6 WT mice were treated with control vehicle or IL-2 at 5000 IU once per day for 4 weeks. (A) Effect of IL-2 therapy on frequency of Tregs during treatment. Increase of percentage of Tregs during therapy from the baseline level of each group. (B-H) Spleen cells were analyzed after 1 week of IL-2 therapy. (B) pSTAT5 expression in Tregs. (C) Representative flow cytometry histograms detecting Ki-67⁺ proliferating Tregs. Percentage of Ki-67⁺ Tregs is shown for each histogram. (D) Percentage of Ki-67⁺ proliferating Tregs in PD-1^−/− and PD-1^WT mice. (E) Representative histogram identifying CD4 Tregs in PD-1^−/− and PD-1^WT mice receiving control vehicle or IL-2. (F) Frequency of CD4⁺CD25⁺Foxp3⁺ Tregs in spleen (percentage of CD4 T cells). (G) Number of CD4⁺CD25⁺Foxp3⁺ Tregs in spleen. (H-L) Spleen cells were analyzed after 2 weeks IL-2 therapy given once per day. (H) Representative histograms identify CD44^lowCD62L^high naïve, CD44^highCD62L^high central-memory, and CD44^highCD62L^low effector-memory Treg subsets after IL-2 therapy. (I) Percentage of each Treg subset after IL-2 therapy. (J) Percentage of annexin-V⁺ apoptotic cells in each Treg subset after IL-2 therapy. (K) Mean fluorescence intensity (MFI) for the ratio of Bcl-2 expression in Tregs of IL-2–treated mice:Bcl-2 expression in Tregs of control vehicle–treated mice. (L) MFI for the ratio of Fas expression in Tregs of IL-2–treated mice:Fas expression in Tregs of control vehicle–treated mice with 4 mice per group per experiment. Data are representative of (A) 2 or (B-L) 3 independent experiments and expressed as means +/– SEM. *P < .05, **P < .01, ***P < .001, ****P < .0001.