Fig. 2|. Pharmacological inhibition of the BH4 pathway ameliorates T-cell-mediated inflammation.

a, BH4 production in 24-hour-activated control (n = 5) and Gch1-ablated (n = 5) CD4⁺ T cells and in wild-type cells treated with SPRi3 (50 μM; n = 4). The experiment was repeated two independent times with similar results. Data are shown as means ± s.e.m. At the top is shown the mechanism of action of SPRi3. b, Representative three-day T-cell-proliferation histogram (right) and quantification (left) of stimulated wild-type T cells (CD4⁺ and CD8⁺) treated with vehicle (n = 10) or SPRi3 (50 μM; n = 8). Data are shown as means ± s.e.m. c, Colitis model, involving transfer of wild-type CD4⁺ T cells into Rag1^−/− hosts treated with vehicle or SPRi3 (300 mg kg^–1; n = 8 each). Data (bottom left) are shown as means ± s.e.m. Right panels show representative images of intestinal immune infiltration. Scale bar represents 200 μm. d, Allergic airway inflammatory disease model in control mice treated with SPRi3 (300 mg kg^–1; n = 14) or vehicle (n = 15). Data are shown as means ± s.e.m. e, f, Proliferation of vehicle- and SPRi3-treated (50 μM) naïve human (n = 4 donors) peripheral blood mononuclear cells (PBMCs) (e) and purified effector human CD4⁺ T cells (f) re-stimulated for 3 days. Data are shown as means ± s.e.m. g, Western immunoblot of iron regulators in 24-hour-activated peripheral CD4⁺ T cells from control and Gch1;Lck mice. The experiment was repeated three independent times with similar results. HO-1, haem oxygenase-1. h, Total iron content from unstimulated and 24-hour-stimulated CD4⁺ T cells from control (n = 17) and Gch1;Lck (n = 22) mice. Data are shown as means ± s.e.m. i, Oxygen-consumption rate (OCR) in unstimulated and 16-hour-stimulated CD4⁺ T cells from control and Gch1;Lck mice (n = 6 each). Data are shown as means ± s.e.m. j, Top, dose-dependent reduction of ferri-cytochrome c (cytoc-Fe³⁺) to ferro-cytochrome c (cytoc-Fe²⁺) by BH4; and bottom, diagram of the reduction pathway. DHPR, dihydropyridine receptor; qBH2, quinonoid dihydrobiopterin. k, l, Representative oxygen uptake rate in permeabilized, 16-hour-stimulated CD4⁺ T cells from vehicle-treated and SPRi3-treated (50 μM) wild-type cells before and after the addition of cytoc-Fe²⁺ (k), and quantification of oxygen rate upon supplementation of cytoc-Fe²⁺ (n = 4 independent experiments) (l). Data are shown as means ± s.e.m. NS, not significant; *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA with Dunnett’s comparison for panel a; two-tailed Student’s t-test for panels b, d–f, h, i, l; two-way ANOVA with Sidak’s comparison for panel c).