Selective expansion of CD4 Tregs in a murine model of low-dose IL-2 therapy. (A) Representative lymphocyte gates for identification of CD4 and CD8 T-cell subsets. Within the CD4 T-cell gate, Tregs are identified as CD4⁺CD25⁺Foxp3⁺ cells and Tcons are identified as CD4⁺CD25^–Foxp3^– cells. (B) IL-2 dose-dependent phosphorylation of Stat5 in T-cell subsets. Left panel: Spleen cells (5 × 10⁵ cells per well) were cultured for 30 minutes in various concentrations of recombinant IL-2. Right panel: Wild-type C57BL/6 mice received single doses of recombinant IL-2 and spleen cells were harvested after 30 minutes. The level of intracellular pStat5 was determined by flow cytometry. (C-D) Wild-type C57BL/6 mice received control vehicle, 5000 or 20 000 IU recombinant IL-2 once per day for 14 days and spleen cells were analyzed on day 15. (C) Representative flow cytometry histograms for identification of Ki-67⁺ proliferating cells in CD8 T cells, Tcons, and Tregs. Percentage of Ki-67⁺ cells is shown for each histogram. (D) IL-2 dose-dependent increase of Ki-67⁺ proliferating cells in each T-cell subset. (E-G) Wild-type C57BL/6 mice received control vehicle or 5000 IU recombinant IL-2 subcutaneously once per day for 14 days, and spleen cells were analyzed on day 15. (E) Representative panel gated on CD4 T cells identifying CD4 Tregs (red box) in mice treated with vehicle control or IL-2. (F) Frequency of CD4⁺CD25⁺Foxp3⁺ Tregs. (G) Number of CD8 T cells, Tcons, and Tregs. (H-I) In vitro Treg suppression assay. Tcons labeled with CellTrace Violet from wild-type C57BL/6 mice were cultured at a 1:1 ratio with Tregs isolated from vehicle or IL-2–treated mice in the presence of CD3/CD28 stimulation for 3 days. (H) Representative flow cytometry histograms measuring Tcon proliferation in the presence or absence of Tregs. Percentage of divided Tcons is shown for each histogram. (I) Percentage of divided Tcons at various Tcon:Treg cell ratios. Responder Tcons (1 × 10⁵ cells per well) were cultured with various numbers of suppressor Tregs with 4 mice per group per experiment. Data are representative of (H-I) 2 or (A-G) 3 independent experiments and expressed as means +/– standard error of the mean (SEM). *P < .05, **P < .01, ***P < .001, and ****P < .0001.