Journal: Science Advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: ( A ) Forest plot of ORR in patients treated with anti–PD-1 or anti–PD-L1 monotherapy versus control in NSCLC. ( B ) Human embryonic kidney (HEK) 293T cell transfected with hemagglutinin (HA)– PD-L1 , and then treated with or without 3-deazaneplanocin A (Dznep), immunoprecipitation (IP) and immunoblot (IB) analysis measuring PD-1/PD-L1 interaction. ( C) Presenting potential modification sites from mass spectrometry analysis of PD-L1. ( D ) HEK293T cell transfected with various HA–PD-L variants and IP and IB analysis measuring PD-1/PD-L1 interaction. ( E ) HEK293T cells transfected with HA–PD-L wild-type (WT) or HA–PD-L1 K162R variant, followed by IP and IB analysis. ( F ) Flow cytometry (left) measuring PD-L1 or PD-1 binding on the membrane of RKO PD-L1 WT or RKO PD-L1 K162R cells. Statistical analysis for mean fluorescence intensity (MFI) was shown in the right; n = 3, P < 0.0001. ( G ) IP and IB analysis measuring PD-1/PD-L1 interaction in RKO PD-L1 WT or RKO PD-L1 K162R cells. WCL, whole cell lysis. ( H and I ) Representative images (H) for confocal image showing bound PD-1/Fc fusion proteins on the membrane of RKO PD-L1 WT or RKO PD-L1 K162R cells. Statistical analysis for relative binding was shown (I). DAPI, 4′,6-diamidino-2-phenylindole. ( J ) Ten microliters of double-distilled water containing 0.1 to 0.75 μg of different peptides was added into polyvinylidene difluoride membranes, followed by IB analysis using K162-specific mono-methylation antibody (Anti–PD-L1 K162me ). 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.

Article Snippet: Antibodies for flow cytometry were listed: phycoerythrin (PE)-conjugated anti-human PD-1 antibody (367403; BioLegend), Alexa Fluor 647– conjugated anti-human GZMB antibody (IC2906R; R&D Systems), PE-conjugated anti-human PD-L1 antibody (374512; BioLegend), antigen-presenting cell allophycocyanin (APC)–conjugated anti-mouse PD-L1 antibody (124311; BioLegend), PE-conjugated anti-mouse CD45 antibody (147711; BioLegend), APC-conjugated anti-human CD45 antibody (305011; BioLegend), fluorescein isothiocyanate (FITC)–conjugated anti-human/mouse GZMB antibody (515403; BioLegend), PE-conjugated anti-mouse IFN-γ antibody (505808; BioLegend), PerCP/Cyanine5.5–conjugated anti-human CD45 antibody (304028; BioLegend), PE-conjugated anti-human CD8 antibody (344706; BioLegend), FITC-conjugated anti-human CD3 antibody (300305; BioLegend), Zombie NIR Fixable Viability Kit (423106; BioLegend), APC-conjugated anti-mouse CD3 antibody (100236; BioLegend), PerCP/Cyanine5.5-conjugated anti-mouse CD45 antibody(147706; BioLegend), PE/Cy7-conjugated anti-mouse CD8 antibody (552877; BD Pharmingen), PE-conjugated anti-mouse CD45 antibody(147712; BioLegend), and PerCP/Cyanine5.5–conjugated anti-mouse IFN-γ antibody (505821; BioLegend).

Techniques: Transfection, Immunoprecipitation, Western Blot, Modification, Mass Spectrometry, Variant Assay, Flow Cytometry, Binding Assay, Membrane, Fluorescence, Lysis, Methylation, Two Tailed Test

Journal: Science Advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: ( A and B ) T cell–mediated tumor cell killing assay in RKO PD-L1 WT or RKO PD-L1 K162R cells. The representative images (A) and the quantitative ratio (B) of dead cells were shown; n = 3, P = 0.0015. ( C and D ) Activated T cells cocultured with pretreated RKO cells. The representative images (C) and statistical analysis (D) were shown; n = 3, P = 0.0002. Ig, immunoglobulin. ( E and F ) Measuring granzyme B (GZMB) of T cells [from experiment (A)] by flow cytometry. The representative images (E) and statistical analysis (F) were shown; n = 3, P = 0.0057. FSC-A, forward scatter area. ( G to I ) In vitro xenograft tumor assays in immunocompetent humanized peripheral blood mononuclear cell (huPBMC)-NCG mice. The treatment protocol was summarized (G); the tumor growth rate (H) and weight (I) were shown; n = 3, P = 0.0044 (H), P = 0.0013 (I). ( J and K ) Representative images for number of intratumor CD8 + T cells by immunofluorescence (IF) (J). Statistical analysis was shown (K); n = 3. ( L and M ) In vitro xenograft tumor assays in immunodeficient NCG mice. The tumor growth rate (L) and weight (M) were shown. ( N and O ) In vitro xenograft tumor assays using Lewis hWT or Lewis hK162R cells in C57BL/6 hPD1 mice; the tumor growth rate (N) and weight (O) were shown; n = 5, P = 0.0079 (N), P = 0.0079 (O). ( P and Q ) Flow cytometry measuring PD-1 + TIM3 + and GZMB expression of tumor-infiltrating CD8 + T cells; statistical analysis was shown; n = 5, P = 0.025 (P), P = 0.0089 (Q). ( R to T ) In vitro xenograft tumor assays using Lewis hWT or Lewis hK162R cells; anti-CD8 antibody to block CD8 + T cell. The treatment protocol was summarized (R); the tumor growth rate (S) and weight (T) were shown; n = 6, P = 0.0053 (O) or ns, P = 0.0067 or ns (P). Error bars are means ± SD. Statistical significance was assessed using Student’s two-tailed t test. ns, not significant.

Article Snippet: Antibodies for flow cytometry were listed: phycoerythrin (PE)-conjugated anti-human PD-1 antibody (367403; BioLegend), Alexa Fluor 647– conjugated anti-human GZMB antibody (IC2906R; R&D Systems), PE-conjugated anti-human PD-L1 antibody (374512; BioLegend), antigen-presenting cell allophycocyanin (APC)–conjugated anti-mouse PD-L1 antibody (124311; BioLegend), PE-conjugated anti-mouse CD45 antibody (147711; BioLegend), APC-conjugated anti-human CD45 antibody (305011; BioLegend), fluorescein isothiocyanate (FITC)–conjugated anti-human/mouse GZMB antibody (515403; BioLegend), PE-conjugated anti-mouse IFN-γ antibody (505808; BioLegend), PerCP/Cyanine5.5–conjugated anti-human CD45 antibody (304028; BioLegend), PE-conjugated anti-human CD8 antibody (344706; BioLegend), FITC-conjugated anti-human CD3 antibody (300305; BioLegend), Zombie NIR Fixable Viability Kit (423106; BioLegend), APC-conjugated anti-mouse CD3 antibody (100236; BioLegend), PerCP/Cyanine5.5-conjugated anti-mouse CD45 antibody(147706; BioLegend), PE/Cy7-conjugated anti-mouse CD8 antibody (552877; BD Pharmingen), PE-conjugated anti-mouse CD45 antibody(147712; BioLegend), and PerCP/Cyanine5.5–conjugated anti-mouse IFN-γ antibody (505821; BioLegend).

Techniques: Flow Cytometry, In Vitro, Immunofluorescence, Expressing, Blocking Assay, Two Tailed Test

Journal: Science Advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: ( 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 Lewis hWT cells in C57BL/6 hPD1 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-γ 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 Lewis hWT cells in C57BL/6 hPD1 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.

Article Snippet: Antibodies for flow cytometry were listed: phycoerythrin (PE)-conjugated anti-human PD-1 antibody (367403; BioLegend), Alexa Fluor 647– conjugated anti-human GZMB antibody (IC2906R; R&D Systems), PE-conjugated anti-human PD-L1 antibody (374512; BioLegend), antigen-presenting cell allophycocyanin (APC)–conjugated anti-mouse PD-L1 antibody (124311; BioLegend), PE-conjugated anti-mouse CD45 antibody (147711; BioLegend), APC-conjugated anti-human CD45 antibody (305011; BioLegend), fluorescein isothiocyanate (FITC)–conjugated anti-human/mouse GZMB antibody (515403; BioLegend), PE-conjugated anti-mouse IFN-γ antibody (505808; BioLegend), PerCP/Cyanine5.5–conjugated anti-human CD45 antibody (304028; BioLegend), PE-conjugated anti-human CD8 antibody (344706; BioLegend), FITC-conjugated anti-human CD3 antibody (300305; BioLegend), Zombie NIR Fixable Viability Kit (423106; BioLegend), APC-conjugated anti-mouse CD3 antibody (100236; BioLegend), PerCP/Cyanine5.5-conjugated anti-mouse CD45 antibody(147706; BioLegend), PE/Cy7-conjugated anti-mouse CD8 antibody (552877; BD Pharmingen), PE-conjugated anti-mouse CD45 antibody(147712; BioLegend), and PerCP/Cyanine5.5–conjugated anti-mouse IFN-γ antibody (505821; BioLegend).

Techniques: shRNA, Negative Control, Membrane, Flow Cytometry, In Vitro, Immunohistochemistry, Expressing, Plasmid Preparation, Two Tailed Test

Journal: Science Advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: ( A ) Transfecting vector or Flag- SETD7 plasmids into RKO PD-L1 WT or RKO PD-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 Lewis hWT cells (top) and the treatment protocol were summarized (bottom). ( F to H ) In vitro xenograft tumor assays using indicated cells in C57BL/6 hPD1 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-γ 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.

Article Snippet: Antibodies for flow cytometry were listed: phycoerythrin (PE)-conjugated anti-human PD-1 antibody (367403; BioLegend), Alexa Fluor 647– conjugated anti-human GZMB antibody (IC2906R; R&D Systems), PE-conjugated anti-human PD-L1 antibody (374512; BioLegend), antigen-presenting cell allophycocyanin (APC)–conjugated anti-mouse PD-L1 antibody (124311; BioLegend), PE-conjugated anti-mouse CD45 antibody (147711; BioLegend), APC-conjugated anti-human CD45 antibody (305011; BioLegend), fluorescein isothiocyanate (FITC)–conjugated anti-human/mouse GZMB antibody (515403; BioLegend), PE-conjugated anti-mouse IFN-γ antibody (505808; BioLegend), PerCP/Cyanine5.5–conjugated anti-human CD45 antibody (304028; BioLegend), PE-conjugated anti-human CD8 antibody (344706; BioLegend), FITC-conjugated anti-human CD3 antibody (300305; BioLegend), Zombie NIR Fixable Viability Kit (423106; BioLegend), APC-conjugated anti-mouse CD3 antibody (100236; BioLegend), PerCP/Cyanine5.5-conjugated anti-mouse CD45 antibody(147706; BioLegend), PE/Cy7-conjugated anti-mouse CD8 antibody (552877; BD Pharmingen), PE-conjugated anti-mouse CD45 antibody(147712; BioLegend), and PerCP/Cyanine5.5–conjugated anti-mouse IFN-γ antibody (505821; BioLegend).

Techniques: Plasmid Preparation, Flow Cytometry, In Vitro, Expressing, Two Tailed Test

Journal: Science Advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: ( A ) WCE were collected for IP with PD-L1 or LSD2 antibody, followed by IB analysis with (top) or without (bottom) PNGase F reaction. ( B ) Fixed RKO cells stained with PD-L1 and LSD2 antibodies, followed by IF assays. ( C ) Transfecting HA– PD-L1 WT or HA– PD-L1 K162R plasmids into HEK293T cells, WCE were collected for IP assays with HA antibody, followed by IB analysis. ( D ) IP and IB analysis measuring LSD-2/PD-L1 interaction in shNC or shSETD7 cells. ( E ) IP and IB analysis measuring PD-1 K162me level in vector or LSD2 cells. ( F ) WCE were collected from shNC or shLSD2 cells, followed by IB analysis. ( G ) Flow cytometry measuring PD-L1/PD-1 binding on the membrane of shNC or shLSD2 cells. ( H to J ) Representative images (H) for confocal image showing bound PD-1/Fc fusion proteins on the membrane of shNC or shLSD2 cells. Statistical analysis was shown (I and J); n = 9, P < 0.0001 (I), P < 0.0001 (J). ( K ) T cell–mediated tumor cell killing assay in indicated RKO cells; the quantitative ratio of dead cells was shown; P = 0.0004; n = 3, P = 0.0004. ( L ) Activated T cells cocultured with indicated RKO cells; measuring GZMB of T cell by flow cytometry. Statistical analysis was shown; n = 3, P < 0.01. ( M to O ) In vitro xenograft tumor assays using vector or LSD2 Lewis hWT cells in C57BL/6 hPD1 mice. Anti–PD-L1 antibody was used at indicated time. The treatment protocol was summarized (M). The representative bioluminescence images (N) and tumor growth rate (O) were shown; n = 5, P = 0.0011 or = 0.0001. 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.

Article Snippet: Antibodies for flow cytometry were listed: phycoerythrin (PE)-conjugated anti-human PD-1 antibody (367403; BioLegend), Alexa Fluor 647– conjugated anti-human GZMB antibody (IC2906R; R&D Systems), PE-conjugated anti-human PD-L1 antibody (374512; BioLegend), antigen-presenting cell allophycocyanin (APC)–conjugated anti-mouse PD-L1 antibody (124311; BioLegend), PE-conjugated anti-mouse CD45 antibody (147711; BioLegend), APC-conjugated anti-human CD45 antibody (305011; BioLegend), fluorescein isothiocyanate (FITC)–conjugated anti-human/mouse GZMB antibody (515403; BioLegend), PE-conjugated anti-mouse IFN-γ antibody (505808; BioLegend), PerCP/Cyanine5.5–conjugated anti-human CD45 antibody (304028; BioLegend), PE-conjugated anti-human CD8 antibody (344706; BioLegend), FITC-conjugated anti-human CD3 antibody (300305; BioLegend), Zombie NIR Fixable Viability Kit (423106; BioLegend), APC-conjugated anti-mouse CD3 antibody (100236; BioLegend), PerCP/Cyanine5.5-conjugated anti-mouse CD45 antibody(147706; BioLegend), PE/Cy7-conjugated anti-mouse CD8 antibody (552877; BD Pharmingen), PE-conjugated anti-mouse CD45 antibody(147712; BioLegend), and PerCP/Cyanine5.5–conjugated anti-mouse IFN-γ antibody (505821; BioLegend).

Techniques: Staining, Plasmid Preparation, Flow Cytometry, Binding Assay, Membrane, In Vitro, Two Tailed Test

Journal: Science Advances

Article Title: PD-L1 methylation restricts PD-L1/PD-1 interactions to control cancer immune surveillance

doi: 10.1126/sciadv.ade4186

Figure Lengend Snippet: ( 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.

Article Snippet: Antibodies for flow cytometry were listed: phycoerythrin (PE)-conjugated anti-human PD-1 antibody (367403; BioLegend), Alexa Fluor 647– conjugated anti-human GZMB antibody (IC2906R; R&D Systems), PE-conjugated anti-human PD-L1 antibody (374512; BioLegend), antigen-presenting cell allophycocyanin (APC)–conjugated anti-mouse PD-L1 antibody (124311; BioLegend), PE-conjugated anti-mouse CD45 antibody (147711; BioLegend), APC-conjugated anti-human CD45 antibody (305011; BioLegend), fluorescein isothiocyanate (FITC)–conjugated anti-human/mouse GZMB antibody (515403; BioLegend), PE-conjugated anti-mouse IFN-γ antibody (505808; BioLegend), PerCP/Cyanine5.5–conjugated anti-human CD45 antibody (304028; BioLegend), PE-conjugated anti-human CD8 antibody (344706; BioLegend), FITC-conjugated anti-human CD3 antibody (300305; BioLegend), Zombie NIR Fixable Viability Kit (423106; BioLegend), APC-conjugated anti-mouse CD3 antibody (100236; BioLegend), PerCP/Cyanine5.5-conjugated anti-mouse CD45 antibody(147706; BioLegend), PE/Cy7-conjugated anti-mouse CD8 antibody (552877; BD Pharmingen), PE-conjugated anti-mouse CD45 antibody(147712; BioLegend), and PerCP/Cyanine5.5–conjugated anti-mouse IFN-γ antibody (505821; BioLegend).

Techniques: Methylation, Flow Cytometry, Expressing, Two Tailed Test