<t>PD-1</t> 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 low CD62L high naïve, CD44 high CD62L high central-memory, and CD44 high CD62L 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
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