Extended Data Fig. 8: Loss of functional stability of T_reg cells induced by anti-CTLA-4 depends on T_reg cell glycolysis and CD28 signalling.

a, Quantification and representative plots of GlucoseCy3 staining by flow cytometry of T_reg cells activated as in Fig. 4c in the presence of 10 mM glucose ± rotenone/antimycin A or oligomycin and treated with anti-CTLA-4 or IgG control (average of two biological replicates per condition; representative of two independent experiments). b, FOXP3 expression by flow cytometry and IL-10 production by Luminex-based bead immunoassay in T_reg cells activated in the presence of 10 mM glucose ± rotenone/antimycin A or oligomycin (n = 3, representative of three independent experiments). c, d, Representative plots of in vitro assays reported in Fig. 4f, g. Representative proliferation (CellTraceViolet dilution) by flow cytometry of activated CD8⁺ T cells cultured alone or in the presence of T_reg cells at the indicated glucose concentrations and treated with anti-CTLA-4 or an IgG control (c). d, Representative CD86 staining by flow cytometry on B cells from co-cultures with CD8⁺ T cells and T_reg cells treated as in c. e, In vitro suppression assay with CD25^hi T_reg cells immunomagnetically purified from spleens of naive wild-type or CD28-knockout mice cultured for 48 h with CellTraceViolet-labelled CD8⁺ T cells and B cells and activated with anti-CD3 in the presence of anti-CTLA-4 or IgG control and the indicated glucose concentrations (n = 3 per conditions except for ‘+CD28 KO Tregs’ at 1–10 mM glucose, n = 2; representative of two independent experiments). Data are mean ± s.d. P values determined by two-sided unpaired t-test. Oligo, oligomycin.

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