Extended Data Fig. 10: Limiting T_reg cell glucose metabolism prevents anti-CTLA-4-mediated T_reg cell destabilization in glycolysis-defective tumours.

a, Foxp3^GFP-cre-ERT2;Slc2a1^fl/fl (Glut1 cKO) and Foxp3^GFP-cre-ERT2 control mice (ctrl) were implanted with B16-KD cells and treated with anti-CTLA-4 or IgG after induction of Slc2a1 deletion with tamoxifen as indicated. Tamoxifen treatment was continued throughout the treatment duration. b, Quantification of Slc2a1 mRNA relative to Actb in FOXP3–GFP⁻ T_eff cells and FOXP3–GFP⁺ T_reg cells from the spleens of control and Glut1 cKO mice at the end of treatment as in a (n = 3). c, d, Flow cytometry analysis of CD25 and CTLA-4 (c; n = 3 except for ctrl IgG, n = 1), and IFNγ and TNF expression (d; n = 2) in tumour-infiltrating T_reg cells from mice treated as in a (representative of two independent experiments). e, Ex vivo suppression assay with T_reg cells sorted from the spleens of control and Glut1 cKO mice treated with anti-CTLA-4 as in a. T_reg cell suppression of CD8⁺ T cell expansion after 48 h co-culture in 10 mM glucose and representative flow cytometry plot showing CTV dilution and generation (G) overlay of CD8⁺ T cells cultured alone (grey) or in the presence of control (black) or Glut1 cKO (red) T_reg cell (n = 3; representative of two independent experiments). f–l, Foxp3^YFP-cre;Slc2a1^fl/+ (Glut1 HET) or Foxp3^YFP-cre;Ldha^fl/fl (Ldha cKO) and Foxp3^YFP-cre mice (ctrl) were implanted with B16-KD cells and treated with anti-CTLA-4 (f). g, Quantification of Slc2a1 mRNA relative to Actb in FOXP3–GFP⁺ T_reg cells from spleens of control and Glut1 HET mice (n = 2). h, i, Quantification by flow cytometry of intra-tumour T_reg cells (h) and their expression of Ki67 (i) in control and Glut1 HET mice treated as in f (control, n = 4; HET, n = 2; representative of two independent experiments with mice carrying Glut1 HET or cKO T_reg cells). j, Quantification of Ldha mRNA relative to Actb in FOXP3–GFP⁺ T_reg cells from spleens of control and Ldha cKO mice (n = 3). k, l, Quantification by flow cytometry of intra-tumour T_reg cells (k) and their expression of Ki67 (l) in control or Ldha cKO mice treated as in f (ctrl, n = 3; Ldha cKO, n = 2; representative of two independent experiments). m, Schematic representation of the culture conditions used in n, o. CD5⁺ T cells from Ldha cKO or control mice were co-cultured for 48 h with CD45.1⁺ congenic antigen-presenting cells (either B cells or T-cell depleted splenocytes) as scaffold for soluble anti-CD3 crosslinking in low (0.5 mM) or higher (10 mM) glucose concentrations as indicated. n, Quantification by flow cytometry of Ldha cKO or control FOXP3⁺CD4⁺ T_reg cells and their expression of Ki67 after activation as in m. o, FOXP3 and CTLA-4 expression by flow cytometry (MFI) in Ki67-negative Ldha cKO or control T_reg cells from cultures as in m. Ctrl 0.5 mM glucose, n = 3 except for anti-CD28, n = 2; ctrl 10 mM glucose, n = 3; cKO, n = 4; representative of two independent experiments. aCTLA-4, anti-CTLA-4; aCD28, anti-CD28. Data are mean ± s.d. (b, e, g, j, n, o) or mean ± s.e.m. (c, d, h, i, k, l). **P < 0.01; ***P < 0.001. P values determined by two-sided unpaired t-test.

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