Extended Data Fig. 4: Blockage of GCH1–BH4 abrogates T-cell-mediated autoimmunity.

a, OVA immunization of control and Gch1;Lck mice. T-cell-dependent IgG responses and T-cell-independent IgM responses are shown two weeks after OVA immunization (left panels, 100 μg OVA in 200 μg alum) as well as two weeks after re-challenge (right panels). n = 5 for control mice; n = 6 for Gch1;Lck mice. 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 with multiple comparisons). b, c, EAE model of autoimmunity towards the central nervous system. Data are shown as means ± s.e.m. b, EAE scores of control and Gch1;Lck mice. n = 6 for each genotype. ****P < 0.0001 (linear regression analysis was performed on the slope of each curve). c, Mean maximal EAE severity in control and littermate Gch1;Lck mice. *P < 0.05 (Mann–Whitney test). d, Schematic of the de novo, salvage and recycling arms of the BH4 pathway. The dotted arrow indicates non-enzymatic reactions; solid arrows indicate enzymatic reactions. DHFR, dihydrofolate reductase; GTP, guanosine triphosphate; PCDB, pterin-4α-carbinolamine dehydratase; PTPS, 6-pyruvoyl tetrahydropterin synthase; QDPR, quinoid dihydropteridine reductase; SPR, sepiapterin reductase. e, Representative FACS plots depicting activation marker profiles of purified wild-type control CD4⁺ T cells left unstimulated or stimulated with anti-CD3/28 antibodies for 16 h and then treated with vehicle (DMSO), SPRi3 (50 μM) or sepiapterin (5 μM). The experiment was repeated two independent times with similar results. f, Cell survival as defined by the percentage of DAPI⁻annexinV⁻ cells from purified CD4⁺ T cells stimulated for 24 h or 48 h with anti-CD3/28 antibodies and then treated with vehicle (DMSO), SPRi3 (50 μM) or sepiapterin (5 μM). The experiment was repeated two independent times with similar results. g, h, Representative FACS blots depicting EdU cell-cycle analysis after 28 hours anti-CD3/CD28 stimulation of control, Gch1;RORc and SPRi3-treated control CD4⁺ T cells. EdU was pulsed for the last 4 hours (g) and quantification of S-phase entry (h). Data from individual mice are shown ± s.e.m. ***P < 0.001 (one-way ANOVA with Dunnett’s multiple comparisons test). i, Quantification of subG1 (dead cells) populations after 24- and 48-h stimulation. EdU was pulsed for the last 4 hours of each time point. Data from individual mice are shown ± s.e.m.). **P < 0.01; NS, not significant (multiple t-test comparisons). j, k, Amino acid profiles in the supernatants (j) and cell pellets (k) from 24-h anti-CD3/CD28-stimulated CD4⁺ T cells from control and Gch1;Lck mice. n = 3 for each genotype. Data are shown as means ± s.e.m. NS, not significant (two-tailed Student’s t-test).