Stable Tregs can produce IL-2 in vivo. (A) Representative flow plots of IL-2 expression in CD4⁺ T cells from CD4^wtIl2^fl/fl (WT) and CD4^creIl2^fl/fl mice. (B)Il2 transcript in CD4⁺ Tconv and CD4⁺ Treg cells stimulated in vitro with anti-CD3. (C) IL-2 in supernatant of CD4⁺ Tconv and CD4⁺ Treg cells stimulated in vitro with anti-CD3. (D) IL-2 expression by CD4⁺ Tregs from Il2^creRosa^RFP mice. n = 6. IEL, intraepithelial lymphocyte; LPL, lamina propria lymphocyte; mLN, mesenteric LN; PL, peritoneal lavage; PP, Peyer’s patch; Spl, spleen. (E) Frequency of CD44 and Ki67 expression in Tregs from Il2^creRosa^RFP mice. n = 7. (F) Frequency of CD44 and Ki67 expression in IL-2⁻ and IL-2⁺ Tregs from ex vivo stimulation of WT cells. n = 4. (G) Frequency of CD44^hiCD62L^lo and Ki67 expression in Tregs from Il2^GFP mice. n = 6. (H) IL-2 expression by CD4⁺ Tconv and CD4⁺ Tregs, 40 d following adoptive transfer into Rag^−/− mice. n = 3. (I and J) Representative expression of IL-2 by CD4⁺ T cells sorted from thymus (I) and spleen (J) of Foxp3-Tocky mice and stimulated ex vivo. Data are pooled from (D) or representative of (A–C, E, F, I, and J) at least two independent experiments. Significance was tested by Mann–Whitney U test (B and C), paired t test (E–G), or unpaired t test (H).