Extended Data Fig. 1: KDM5B expression is negatively correlated with anti-tumor immunity, and depletion of KDM5B in mouse melanoma tumor cells induces strong anti-tumor immunity and enhances response to anti-PD-1 treatment.
From: KDM5B promotes immune evasion by recruiting SETDB1 to silence retroelements
a, Spearman correlation coefficient scores of selected genes negatively correlated with KDM5B gene expression from the melanoma TCGA dataset, categorized by their functions. b, Box plot showing expression levels of CD8A, CD8B, IFNG, TNF, CXCL9 and CXCL10 in samples from patients with melanoma from the TCGA dataset are higher in “KDM5B low” group (lowest quartile) than in “KDM5B high” (highest quartile) group. Statistical significance was computed with the unpaired two-sided Student’s t test. c, Immunofluorescence staining of pre-treatment melanoma samples with a melanoma cocktail (S100, HMB45 -green), KDM5B (red), and DAPI (blue). Images A-C are representative images from 3 independent non-responders to anti-PD-1 treatment; Images D-F are representative images from 3 independent responders. Cell-specific KDM5B expression levels of KDM5B and melanoma markers in the tumor microenvironment are displayed in 2D dot plots in non-responders (G) and responders (H). Each dot represents the mean fluorescent intensities of a single cell for KDM5B and the melanoma marker cocktail. Cells that are positive for both KDM5B and melanoma marks are in the upper right quadrants. (I) displays the frequency and distribution of expression levels of KDM5B in responders (green) and non-responders (red). MGLI = mean grey level intensity. Cutoff values for positive settings are denoted by the horizontal and vertical bars. Scale bar = 100 µm. d, Survival curves of C57BL/6 mice subcutaneously injected with 500,000 control (black) (n = 6) or Kdm5b−/− (Kdm5b sg) (red) (n = 6) YUMMER1.7 cells. Mice injected with Kdm5b−/− cells had completely regressed tumors, and were re-challenged with control tumor cells on day 45 after initial tumor injection. Data are representative of two independent experiments. Log-rank test for survival curves, **p = 0.004. e, Tumor growth curves of C57BL/6 mice injected with polyclonal YUMMER1.7 cells with stable lentiviral Ctl sg (n = 5), Kdm5b sg1 (n = 5) or Kdm5b sg2 (n = 5), with western blot analyses (insert) showing knockout efficacy of KDM5B. Data are representative of two independent experiments. Data are mean ± SEM. 2-way ANOVA analysis for comparison at day 18 after tumor injection, ****p<0.0001. For gel source data, see Supplementary Fig. 1. f, Representative images of immunofluorescent staining of CD3 (green), Melan A (red) and DAPI (blue) (left) and Quantification of CD3+ cells per view field (3-5 view fields per tumor; each dot represents the mean for each tumor; 5 tumors per group) (right) from Ctl, Kdm5b sg1 and Kdm5b sg2 YUMMER1.7 tumors harvested at day 14 after injection. scale bar = 200 µm. Quantification data are mean ± SD; Unpaired two-sided Student’s t-test, **p = 0.0081 for Ctl vs sg1; **p = 0.0063 for Ctl vs sg2. g, Quantitation of the percentages of CD4+ and CD8+ cells in total CD45+ immune cells using flow cytometry analysis of control and Kdm5b sgRNA tumors. Unpaired two-sided Student’s t-test for each comparison, *p = 0.036 for CD4+%; *p = 0.024 for CD8+%. Data are representative of two independent experiments. Data are mean± SD. h, Representative flow cytometry plots showing CD8+ T cells in Ctl and Kdm5b sg YUMMER1.7 tumors harvested at day 14 after tumor injection. i. Representative immunohistochemical staining of CD8 (CD8+ T cells) and cleaved caspase-3 in control and Kdm5b sg YUMMER1.7 tumors at day 14 after injection. scale bar = 100 µm. Data are representative of three independent experiments. j,k, Tumor growth curves (j) and survival curves (k) of C57BL/6 mice injected with single clones of Kdm5b−/− YUMMER1.7 cells, WT (n = 5), 1G8 (n = 5), 4B5 (n = 5), 4H1(n = 5) and 4H3(n = 5) are clone numbers. Statistical significance of tumor growth curves was analysed using two-way ANOVA, ****p<0.0001. Data are representative of two independent experiments. Data are mean± SEM. Statistical significance of tumor survival was analysed with log-rank (Mantel-Cox) test. **p = 0.0018. l, Tumor growth curves of wild-type C57BL/6J (B6) mice injected with control (Ctl) (n = 4) or Kdm5b−/− (Kdm5b sg) (n = 4) YUMMER1.7 cells or Rag1−/− mice injected with control (n = 5) or Kdm5b−/− YUMMER1.7 cells (n = 4). Data represent one experiment. Data are mean ± SEM. ****p<0.0001. m, Tumor growth curves of WT (n = 5) and Kdm5b−/− YUMM1.7 (n = 4) in Rag1−/− mice. Western blot of the cell lines is shown on the right. Data represent one experiment. Data are mean ± SEM. 2-way ANOVA analysis for comparison at day 25 after tumor injection, ****p<0.0001. For gel source data, see Supplementary Fig. 1. n, Tumor growth curves of C57BL/6J (B6) mice that rejected Kdm5b−/− YUMMER1.7 tumors (Challenged B6) rechallenged with YUMM1.7 (n = 6) or Kdm5b−/− YUMM1.7 (n = 5) cells compared with age matched Naïve B6 mice injected with YUMM1.7 (n = 5) or Kdm5b−/− YUMM1.7 (n = 5) cells. Data represent one experiment. Data are mean ± SEM. 2-way ANOVA analysis for comparison of tumor size at day 25. n.s., not significant; ****p<0.0001. o, Survival curves of C57BL/6J (B6) mice that rejected Kdm5b−/− YUMMER1.7 tumors (Challenged B6) rechallenged with Kdm5b−/− YUMM1.7 cells and treated with anti-CD8 antibody (n = 4) or IgG control (n = 4). Data represent one experiment. Statistical significance of tumor survival was analysed with log-rank (Mantel-Cox) test. **p = 0.0067. p, Tumor growth curves of C57BL/6 mice injected with wild-type YUMM1.7 cells treated with IgG control (n = 10) or anti-PD-1 antibody (n = 10); or Kdm5b−/− YUMM1.7 cells treated with IgG control (n = 9) or anti-PD-1 antibody (n = 10). Data are representative of two independent experiments. Data are mean ± SEM. 2-way ANOVA analysis for comparison at day 18. n.s., not significant; ****p<0.0001. q, Spider plots of tumor growth data shown in p.
