Extended Data Fig. 10|. Overactivation of the GCH1/BH4 pathway leads to enhanced anti-tumour immunity.

a, Total iron content from 24-h anti-CD3/28 stimulated CD4⁺ T cells (untreated or treated with 5μM sepiapterin) from control (n = 5/4) and Gch1;RORc (n = 5) mice. Data are shown as means ± s.e.m. individual mice for each genotype are shown. *P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons. b, Representative FACS histogram depicting DHE levels (left) and quantification of the mean fluorescent intensity (right) in unstimulated and 20-h anti-CD3/28-activated CD4⁺ T cells from control and GOE;CD4 littermates as well as wild-type cells treated with sepiapterin (5 μM). n = 3 for each condition. Data are shown as means ± s.e.m. **P < 0.01; ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). c, ATP measurements for stimulated wild-type CD4⁺ T cells treated with DMSO, sepiapterin (5 μM) or SPRi3 (50 μM) for 24 h. 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). d, Quantification of intratumoral effector CD4⁺ T cells (CD44⁺ CD62L^lo) assayed from E0071 tumours on day 28 for vehicle- and BH4-treated mice. Data are shown as means ± s.e.m. n = 5 mice for each condition. *P < 0.05; **P < 0.01 (two-tailed Student’s t-test). e, Effect of BH4 supplementation on H-Ras- transformed TC-1 tumour growth. TC-1 tumour cells were orthotopically injected; once the tumours were palpable (day 7), BH4 (35 mg kg^–1; n = 15) or vehicle (saline; n = 10) was therapeutically administered for seven days as indicated. Data are shown for individual mice as means ± s.e.m. ***P < 0.001; ****P < 0.0001 (two-way ANOVA with Sidak’s multiple comparisons). f, Quantification of intratumoral effector CD8⁺ T cells (CD44⁺ CD62L^lo) assayed from TC-1 tumours on day 21 in vehicle- or BH4-treated mice (n = 9 mice for each genotype). Data are shown as means ± s.e.m. **P < 0.01 (two-tailed Student’s t-test). g, Effect of BH4 supplementation on TC-1 tumour growth in Rag2^−/− hosts. TC-1 tumour cells were orthotopically injected into Rag2^−/− female mice; once the tumours were palpable (day 7), BH4 (35 mg kg^–1; n = 5) or vehicle (saline; n = 5) was administered. BH4 and vehicle supplementation was carried out for seven days as indicated on the graph. Data are shown for individual mice as means ± s.e.m. NS, not significant (two-way ANOVA with Sidak’s multiple comparisons). h, Sepiapterin levels in the supernatant of wild-type CD4⁺ T cells stimulated with anti-CD3/28 antibodies for 20 h and treated with vehicle or kynurenine (KYN; 150 μM). Culture medium was also included for comparison. BQL, below quantifiable levels. Data are shown as means ± s.e.m. n = 4 independent samples for each condition. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparisons test). i, Representative histogram depicting proliferation of three-day anti-CD3/28-activated wild-type CD4⁺ T cells treated with vehicle or kynurenine (50 μM). j, Representative FACS histograms depicting DHE levels in anti-CD3/28-stimulated wild-type CD4⁺ T cells treated with vehicle (DMSO), kynurenine alone (50 μM) or kynurenine (50 μM) plus BH4 (10 μM) for 20 h. The experiment was repeated three independent times with comparable results.