Extended Data Fig. 6: Enhanced superoxide levels independent of iNOS coupling observed in BH4-deficient activated T cells.

a, b, Representative FACS histogram (a) and quantification of the mean fluorescent intensity (MFI; b) showing levels of DHE (dihydroethidium, a superoxide ROS indicator) in unstimulated and 20-h anti-CD3/28-activated CD4⁺ T cells from control and GCH1;RORc mice as well as control cells treated with SPRi3 (50 μM). n = 3 samples per group. The experiment was repeated three independent times with similar results. c, d, Proliferation of control (n = 6) and Gch1;Lck (n = 9) CD4⁺ T cells and treatment with the superoxide scavenger NAC (500 μM; n = 4 each). Representative three-day proliferation histograms are shown in c; quantification is shown in d. Data are given as means ± s.e.m. Individual mice for each genotype are shown. ****P < 0.0001 (one-way ANOVA with Tukey’s multiple comparison test). e, Total iron content from unstimulated or 24-h anti-CD3/28-stimulated CD4⁺ T cells (untreated or treated with 500 μM NAC) from control (n = 17, 4, respectively) and Gch1;RORc (n = 22, 6, respectively) mice. Data are shown as means ± s.e.m. Individual mice for each genotype are shown. **P < 0.01 (two-tailed Student’s t-test with Tukey’s multiple comparisons). f, ATP measurements from stimulated wild-type CD4⁺ T cells treated with DMSO, sepiapterin and NAC for 24 h. Data are shown as means ± s.e.m. n = 5 for each genotype. *P < 0.05; **P < 0.01 (two-tailed Student’s t-test with multiple comparisons). g, Intracellular iNOS expression in purified CD4⁺ control T cells left untreated or anti-CD3/CD28-stimulated for 12 h, 24 h or 72 h. The experiment was repeated two independent times with similar results. h, i, Representative histogram showing iNOS expression in control and Gch1-ablated CD4⁺ T cells stimulated with anti-CD3/CD28 antibodies for 72 h (h) and the percentage of iNOS⁺ cells was quantified over time (i). n = 4 for each genotype. Data are shown as means ± s.e.m. NS, not significant (two-tailed Student’s t-test). j, Nitrite measurements in the supernatant of stimulated cells from i. Peritoneal, thioglycollate-elicited macrophages stimulated with LPS (100 ng ml⁻¹) for 24 h were used as a positive control. Data are shown as means ± s.e.m. n = 4 for each genotype. NS, not significant (two-tailed Student’s t-test).