Extended Data Fig. 9: Histone demethylase JMJD3 is essential for TRAP1-mediated immunosuppression.

(a) Immunoblot analysis of JMJD3, H3K27me3, H3 and β-Actin in YUMM1.7 TCM-treated BMDMs with αTIM4 or IgG2b; representative of two independent experiments. (b) Jmjd3 expression in Trap1^+/+ and Trap1^−/− naïve BMDMs (n = 5); representative of two independent experiments. (c) CUT&Tag analysis of H3K27me3 enrichment in TCM-treated Trap1^+/+ and Trap1^−/− BMDMs. To account for the globally decreased H3K27me3 in the absence of TRAP1, Trap1^−/− track was normalized to the H3K27me3/H3 ratio from Fig. 7a, which shows the relative H3K27me3/H3 ratios are 0.817 and 0.475 for Trap1^+/+ and Trap1^−/−, respectively. Key genes shown include Arg1, Mrc1, Pparg, and Irf4. (d) Jmjd3 expression in Jmjd3^wt and Jmjd3^cKO naïve BMDMs (n = 4); representative of three independent experiments. (e) Expression of CD206 and CD301 in Jmjd3^wt and Jmjd3^cKO BMDMs co-cultured with YUMM1.7 cells for 72 h (n = 4); representative of three independent experiments. (f) Pro-inflammatory genes in TCM-treated Jmjd3^wt and Jmjd3^cKO BMDMs by RNA-seq (n = 3). (g, h) CD206 (g) and ARG1 (h) in TCM-treated Trap1^+/+ and Trap1^−/− BMDMs with or without GSK-J4 (n = 3); representative of two independent experiments. (i) Basal OCR of TAMs from Jmjd3^wt and Jmjd3^cKO tumor-bearing mice (n = 6); representative of three independent experiments. (j) ETC genes in TCM-treated Jmjd3^wt and Jmjd3^cKO BMDMs by RNA-seq (n = 3). (k) Frequencies of TAMs, TILs, neutrophils, monocytes, NKs, total DCs, DC1s, and DC2s in Jmjd3^wtand Jmjd3^cKO tumor-bearing mice on day 16 (n = 5); representative of two independent experiments. (l, m) Tumor growth (l) and weight (m) in Jmjd3^wt and Jmjd3^cKO mice subcutaneously injected with LLC cells (n = 4); representative of two independent experiments. (n) IFNγ and GZMB production in CD8⁺ T cells activated with anti-CD3/CD28, and co-cultured with TAMs from Jmjd3^wt or Jmjd3^cKO tumor-bearing mice at a 1:10 ratio for 72 h (n = 4); representative of two independent experiments. All data are mean ± s.e.m. and were analyzed using a two-tailed, unpaired Student’s t-test (b, d, k, m), one-way ANOVA with Sidak’s multiple comparison test (e, g, h, n) or two-way ANOVA with Tukey’s multiple comparison test (l).

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