Fig 2: 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 3: PD-L1 is methylated at Lys 162 by SETD7.(A) Whole cell lysis (WCL) were collected for IP with PD-L1 or SETD7 antibody, followed by IB analysis. (B) Coincubating His–PD-L1 and SETD7 proteins, followed by IB analysis. (C) Fixed RKO cells stained with PD-L1 and SETD7 antibodies, followed by immunofluorescence (IF) assays. (D) HEK293T cells transfected with HA–PD-L1, and then cotransfected with Flag-SETD7, Flag-EZH2, Flag-NSD2, or Flag-SETDB1, followed by IP and IB analysis. (E) Transfecting Flag-SETD7 or Flag-SETD7 H297A into H1975 cells, WCE were collected for IB analysis. (F) Transfecting Flag-SETD7 into RKOPD-L1 WT or RKOPD-L1 K162R cells, WCE were collected for IB analysis. (G) Silencing cellular SETD7 expression, WCE were collected for IP with PD-L1 antibody, followed by IB analysis. (H) Immunoprecipitated SETD7 WT or SETD7 H297A protein from HEK293 cells was incubated with S-adenosyl-l-methionine along with His–PD-L1 protein for in vitro methylation assay. PD-L1 methylation was analyzed by IB analysis using with PD-L1 K162 mono-methylation–specific antibody. (I) RKO cells treated by IL-6 (10 ng/ml) in a time-dependent manner, followed by IP and IB analysis. All IBs are performed three times, independently, with similar results.

Fig 4: 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.