Fig 2: PD-L1 K162 hypermethylation is a predictive biomarker of PD-1/PD-L1 blockade resistance therapy.(A to D) Ten NSCLC samples and its WCE were collected; measuring PD-L1 and PD-L1 k162 methylation level by IB analysis; investigating cytotoxicity of tumor-infiltrating CD8+ T cells in tumor specimens by flow cytometry analysis. Pearson’s correlation between PD-L1 K162me/PD-L1 ratio and the number/cytotoxicity of tumor-infiltrating CD8+ T cells was shown; n = 10. (E and F) Collecting 70 clinic samples from patients with NSCLC receiving anti–PD-1 treatment, IF assays measuring PD-L1, PD-L1 K162me, SETD7, and LSD2 expression level; Pearson’s correlation between PD-L1 K162me/PD-L1 ratio and SETD7 (E) or LSD2 (F) was shown; n = 70, P = 0.021 (E), P = 0.01 (F). (G) Kaplan-Meier survival analysis was shown. Patients were grouped by PD-L1 expression level; n = 33 (high), n = 33 (low), P = 0.9345. (H) Shown is the PD-L1 relative expression level of patients with anti–PD-1 treatment resistance or sensitivity; n = 45 (sensitivity), n = 22 (resistance), P = 0.0155. (I) Shown is the PD-L1 K162me/PD-L1 relative ratio of patients with anti–PD-1 treatment resistance or sensitivity; n = 45 (sensitivity), n = 22 (resistance), P < 0.0001. (J) Receiver operating characteristic curve analysis for the indicated parameters in patients receiving anti–PD-1 treatment. (K) Shown is the PD-L1 and PD-L1 K162me expression level of patients with NSCLC receiving anti–PD-1 treatment; red (sensitivity), black (resistance); the axes were separated by the Youden index. (L) Shown is the PD-L1 and PD-L1 K162me/PD-L1 expression level of patients with NSCLC receiving anti–PD-1 treatment; red (sensitivity), black (resistance); the axes were separated by the Youden index. (M) Kaplan-Meier survival analysis was shown; patients were grouped by the PD-L1 K162me/PD-L1 ratio; n = 33 (high), n = 33 (low), P = 0.0261. (N) Working model of SETD7-catalyzed LSD2-antagonized PD-L1 K162 methylation cross-talk with host antitumor immunity and anti-PD-(L)1 treatment prognosis. Error bars are means ± SD. Statistical significance was assessed using Student’s two-tailed t test.

Fig 3: Methylation of PD-L1 K162 is required for SETD7-mediated antitumor immunity.(A) Transfecting vector or Flag-SETD7 plasmids into RKOPD-L1 WT or RKOPD-L1 K162R cells and IP and IB analysis measuring PD-1/PD-L1 interaction. (B) Activated T cells cocultured with indicated RKO cells; isolating activated T cells; measuring GZMB of T cell by flow cytometry. Statistical analysis was shown; n = 3, P = 0.0067 or 0.0002. (C and D) T cell–mediated tumor cell killing assay in indicated RKO cells; the representative images (C) and the quantitative ratio of dead cells (D) were shown; n = 3, P = 0.001 or < 0.0001. (E) The successful construction of vector or SETD7 LewishWT cells (top) and the treatment protocol were summarized (bottom). (F to H) In vitro xenograft tumor assays using indicated cells in C57BL/6hPD1 mice. The tumor images (F), tumor weight (G), and tumor growth rate (H) were shown; n = 8 or 9 or 10, P < 0.0001 (G), P < 0.05 (H). (I to K) Measuring IFN-? and GZMB expression of tumor-infiltrating CD8+ T lymphocytes by flow cytometry; representative results (I) and statistical analysis (J) and (K) were shown; n = 8 or 9 or 10, P < 0.0001 or ns (J), P = 0.0095 or < 0.0001 or not significant (ns) (K). All IBs are performed three times, independently, with similar results. Error bars are means ± SD. Statistical significance was assessed using Student’s two-tailed t test.

Fig 4: SETD7 regulates PD-L1 and PD-1 interaction and antitumor immunity.(A) IP and IB analysis measuring PD-1/PD-L1 interaction in short hairpin RNA–negative control (shNC) or short hairpin RNA–SETD7 (shSETD7) cells. (B to D) Representative images (B) showing bound PD-1/Fc fusion proteins on the membrane of shNC or shSETD7 cells. Statistical analysis was shown (C and D); n = 9, P < 0.0001 (C), P < 0.0001 (D). (E) T cell–mediated tumor cell killing assay in shNC or shSETD7 cells. The quantitative ratio of dead cells was showed; n = 3, P < 0.0001. (F) Activated T cells cocultured with shNC or shSETD7 cells; measuring GZMB of T cell by flow cytometry. Statistical analysis was shown; n = 3, P < 0.001. (G to I) In vitro xenograft tumor assays using shNC or shSETD7 cells in immunocompetent huPBMC-NCG mice. The treatment protocol was summarized (G); the tumor weight (H) and growth rate (I) were shown; n = 5, P < 0.0005 (H), P < 0.01 (I). (J) Immunohistochemistry analysis for number of intratumor CD8+ T cells. statistical analysis was shown; n = 15, P < 0.0001. (K to M) In vitro xenograft tumor assays using shNC or shSETD7 LewishWT cells in C57BL/6hPD1 mice. The treatment protocol was summarized (K); the tumor weight (L) and growth rate (M) were shown; n = 5, P < 0.0001 (L) and (M). (N and O) Measuring IFN-? and GZMB expression of tumor-infiltrating CD8+ T lymphocytes; statistical analysis was shown; n = 5, P < 0.01 (N), P < 0.05 (O). (P to R) In vitro xenograft tumor assays using vector or SETD7 LewishWT cells in C57BL/6hPD1 mice. Anti–PD-L1 antibody was used at indicated time. The treatment protocol was summarized (P). The representative bioluminescence images (Q) and tumor growth rate (R) were shown; n = 5, P < 0.0005 or = 0.0708. All IBs are performed three times, independently, with similar results. Error bars are means ± SD. Statistical significance was assessed using Student’s two-tailed t test.