Extended Data Fig. 5|. Mitochondrial dysfunction in BH4-depleted T cells after activation.

a, b, ATP measurements in control (n = 3) and Gch1;Lck (n = 3) CD4⁺ T cells (a) and in wild-type CD4⁺ T cells treated with DMSO vehicle (n = 3) or SPRi3 (50 μM; n = 3) (b), either left unstimulated or assayed at the indicated time points after T-cell activation with anti-CD3/28 antibodies. Data are shown as means ± s.e.m. n = 3 for each genotype. *P < 0.05; **P < 0.01 (two-tailed Student’s t-test with multiple comparisons). c, Metabolomic measurements of lactate and pyruvate levels in cell pellets of 16-h anti-CD3/28-activated CD4⁺ T cells from control and Gch1;Lck mice. Data are shown as means ± s.e.m. n = 4 for each genotype. *P < 0.05 (two-tailed Student’s t-test). d, Routine and total capacitance oxygen respiration in intact, 16-h anti-CD3/CD28- stimulated CD4⁺ T cells from control and Gch1;Lck mice. Data from individual mice are indicated ± s.e.m. n = 4 for each genotype. **P < 0.01 (two-tailed Student’s t-test). e, f, Oxygen uptake rate in permeabilized, 16-h anti-CD3/CD28-stimulated CD4⁺ T cells from control (n = 4) and Gch1;ROR (n = 4) mice (e) and wild-type CD4⁺ T cells treated with DMSO or SPRi3 (50 μM (n = 5 each) (f). Data from individual mice are indicated ± s.e.m. *P < 0.05; **P < 0.01 (two-tailed Student’s t-test).g, Left, representative oxygen consumption traces of complex-I-linked and complex-II-linked ETC activity from 16-h-activated wild-type CD4⁺ T cells treated with vehicle or SPRi3 (50 μM). Right, relative complex-I- and complex-II-linked activities in activated control cells treated with vehicle (n = 4) or SPRi3 (50 μM; n = 4). Data are shown as means ± s.e.m. NS, not significant; *P < 0.05 (two-tailed Student’s t-test).