Supplementary Figure 2: Restoration of the suppressor activity of IL-2Rα-deficient T_reg cells by expression of an active form of STAT5.

(a) The schematic of Rosa26^Stat5bCA allele. CAG, cytomegalovirus early enhancer element with the promoter, the first exon and the first intron of chicken beta-actin gene and the splice acceptor of the rabbit beta-globin gene; IRES, internal ribosome entry site; EGFP, enhanced green fluorescent protein (b, c) Photographs of 4-wk-old mice. (d) The expression of Foxp3 and CD25 in CD3⁺CD4⁺ cells in the LNs and spleens of the indicated mice (4 wk old). (e) Intracellular phospho-STAT5 levels in T_reg cells from the indicated mice in vitro stimulated with rmIL-2 (1,000 U/ml) for 20 min. (f) The expression of CD62L and CD44 in CD3⁺CD4⁺Foxp3⁻ and CD3⁺CD8⁺Foxp3⁻ cells in the LNs of the indicated mice. (g) Cytokine production by splenic CD4⁺Foxp3⁻ cells stimulated for 5 hr with anti-CD3/CD28. (h) The numbers of CD3⁺CD4⁺Foxp3⁻IFN-γ⁺ (left graph) and CD3⁺CD8⁺Foxp3⁻IFN-γ⁺ (right graph) cells in the LNs of the indicated mice (4 wk old). (i) The frequencies CD44^hi cells among CD4⁺Foxp3⁻ cells (upper left graph) and the frequencies of CD62L^loCD44^hi and CD62L^hiCD44^hi cells among CD8⁺Foxp3⁻ cells (upper right graph) in the LNs of Foxp3^CreIl2ra^fl/wt, Foxp3^CreIl2ra^fl/wtRosa26^Stat5bCA, Foxp3^CreIl2ra^fl/fl, and Foxp3^CreIl2ra^fl/flRosa26^Stat5bCA mice at the indicated ages. Lower graphs show the numbers of each cell subset in the LNs of indicated mice. (j) Lifespan of Foxp3^CreIl2ra^fl/fl (solid; n=25) and Foxp3^CreIl2ra^fl/flRosa26^Stat5bCA (dotted; n=21) mice. ***, P < 0.001 (two-tailed unpaired Student’s t test). Data are from one experiment representative of two (e, i) or three (h) independent experiments with similar results with three or more mice per group in each experiment (h, i; mean ± s.e.m.) or representative data of more than six mice per group analyzed are shown (d, f, g).