Extended Data Fig. 5: T_reg cell destabilization and CD8⁺ TIL activation in additional LDHA-deficient tumour models treated with CTLA-4 blockade.

a–e, Primary tumour growth and overall survival, reporting the number of tumour-free mice at the end of the experiment, in BALB/c mice implanted in the mammary fat pads with the LDHA-KD 4T1 A3-8KD cell line (10⁶ cells per mouse) treated with neoadjuvant anti-CTLA-4 (n = 9) or IgG control (n = 10), as indicated. CTLA-4 and CD25 in tumour-infiltrating T_reg cells (b), quantification of T_reg cells and CD8⁺ TILs as well as expression of the indicated markers by flow cytometry (c), and flow cytometry analysis of IFNγ expression in CTLA-4^lo and CTLA-4^hi tumour-infiltrating T_reg cells (d) from mice treated as in a (CTLA-4^lo versus CTLA-4^hi T_reg cells, two-sided paired t-test; IgG versus anti-CTLA-4 CTLA-4^lo T_reg cells, two-sided unpaired t-test). e, Pearson correlation analyses of indicated parameters in T_reg cells and CD8⁺ TILs from mice treated as in a (black, IgG; red, anti-CTLA-4). n = 9–10 mice per group; one independent experiment. f–j, 4T1-KD-bearing BALB/c mice were treated with the standard IgG2b 9D9 anti-CTLA-4 antibody (n = 10) or its IgG2a variant (n = 9) or IgG control (n = 10) (f), and overall survival (g), quantification of CTLA-4 and GITR expression in T_reg cells (h), and tumour-infiltrating T_reg cells and their expression of FOXP3 and IFNγ by flow cytometry (i) are shown. j, Pearson correlation analyses between the indicated parameters in T_reg cells and CD8⁺ TILs from mice treated as in f. n = 1 experiment with 9D9 IgG2a. k–m, LDHA protein expression by western blot (k), LDH activity (l), and glycolytic proton efflux rate (GlycoPER) (m) by Seahorse analysis in B16-KD and B16-Sc cells (n = 3, representative of 2–3 independent experiments). n–p, C57BL/6J mice were implanted with B16-KD and B16-Sc tumours and treated with anti-CTLA-4 or IgG control as indicated in n. Quantification of CTLA-4 and CD25 (o; n = 5 per group except for B16-KD IgG, n = 4) and IFNγ expression (p) in tumour-infiltrating T_reg cells by flow cytometry (B16-Sc IgG, n = 4; B16-Sc anti-CTLA-4, n = 6; B16-KD IgG, n = 4; B16-KD anti-CTLA-4, n = 3; representative of two experiments). GzmB, granzyme B; i.d., intradermal; TM, tumour. Data are mean ± s.e.m. (a–d, h–i, o–p) or mean ± s.d. (l–m). Unless stated otherwise, P values were determined by two-sided unpaired t-test (b, c, h, i, l, m, o, p), Pearson correlation coefficient (e, j) or log-rank test (g).

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