Extended Data Fig. 3: Development of regulatory T cells and their function in Gch1-ablated mice.

a, b, Representative FACS plot depicting CD4⁺FoxP3⁺ regulatory T cells (T regs; a) and quantification of T-reg proportions as well as absolute numbers in the spleen (b) of control and Gch1;RORc mice (n = 6 each). Data are shown as means ± s.e.m. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant (two-tailed Student’s t-test). c, d, In vitro T-reg suppression assay, in which naive, wild-type CD4⁺ T cells were activated in the presence of varying ratios of T-reg cells from control and Gch1;RORc mice for four days. Representative histogram showing the suppressive capacity of control and Gch1;RORc T-reg cells (c) and quantification of proliferation with various ratios of T-reg cells (d). n = 4 samples. Data are shown as means ± s.e.m. *P < 0.05; **P < 0.01; NS, not significant (two-tailed Student’s t-test with multiple comparisons). Tconv, conventional CD4⁺ T cells (CD4⁺, CD25⁻ CD45RB^high). e, Naive CD4⁺ transfer colitis model, with co-transfer of FACS-purified T-reg cells from control (n = 4) and Gch1;RORc (n = 4) mice. As a control, Tconv cells (from n = 16 mice) with no co-transfer of T-reg cells were used. Changes to initial body weight (BW) were scored over five weeks. Data are shown as means ± s.e.m. *P < 0.05; ***P < 0.001; NS, not significant (two-way ANOVA with Tukey’s multiple comparison test). f, Total numbers of CD4⁺ splenic T cells at two weeks post-transfer in mice (n = 3) transferred with naive CD4⁺ cells only (‘no T regs’) and mice transferred with T regs from control or Gch1-ablated (Gch1;RORc) mice. Data are shown as means ± s.e.m. ***P < 0.001; ****P < 0.0001; NS, not significant (one-way ANOVA with Dunnett’s multiple comparison test). g, Transfer colitis model of intestinal autoimmunity. Body-weight changes are plotted relative to initial weight in mice transferred with naive CD4⁺ T cells from control or Gch1;Lck mice (n = 10 each). Data are shown as means ± s.e.m. NS, not significant (two-way ANOVA with Sidak’s multiple comparisons). h, i, Proportion of CD4⁺ T cells in the draining mesenteric lymph nodes in week 4 (h), and profiles of ntracellular cytokines (IFN-γ and IL-17) from transferred control and Gch1;Lck cells (i). Data are shown as means ± s.e.m. n = 10 for each genotype for h and n = 5 for each genotype for i. ***P < 0.001; NS, not significant (two-tailed Student’s t-test).