Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet: Expression profile differences between PD-L1 +/hi vs. PD-L1 –/lo TAMs from human breast tumors revealed by scRNA-seq (A) scRNA-seq analysis of TAMs (n = 2,220 cells) from untreated primary breast tumor (n = 5 patients, ER + ) shown as a UMAP, highlighting identified clusters. (B) UMAP showing mutually exclusive expression of PD-L1 and SIGLEC15 in TAMs. (C) Dichotomization of TAM clusters into PD-L1 +/hi and PD-L1 –/lo subpopulations. (D) PD-L1 expression of PD-L1 +/hi and PD-L1 –/lo subpopulations. (E) Representative flow cytometry plot showing PD-L1 + % TAMs, peripheral monocytes, and intratumoral T cells from the same tumor tissue used for scRNA-seq (left panel), and PD-L1 + % quantification was compared between analysis via scRNA-seq and flow cytometry (right panel). (F) Overlay of the expression of common M1 and M2 signature genes in the PD-L1 +/hi vs. PD-L1 –/lo TAM dichotomization. (G) Volcano plot showing differentially expressed genes (DEGs) between PD-L1 +/hi vs. PD-L1 –/lo TAMs. (H and I) Expression distribution of selected genes involved in maturation, pro-inflammatory or transcriptional activator (H), and anti-inflammatory, pro-tumor, fatty acid metabolic, or extracellular matrix (I) between PD-L1 +/hi and PD-L1 –/lo TAMs. ∗∗∗∗p < 0.0001. Wilcoxon rank-sum test. (J) PD-L1 +/hi and PD-L1 –/lo TAMs were flow sorted from freshly prepared single-cell suspension of digested breast tumor (n = 4) and supernatants were collected for ELISA after 16 h. Paired t test. ∗p < 0.05, ∗∗p < 0.01.

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Expressing, Flow Cytometry, Suspension, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet: Expression profile of PD-L1 +/− TAMs from patients with TNBC revealed by published scRNA-seq data (A) Published (Pal et al. ) scRNA-seq transcriptomic clustering of TAMs (n = 4,484 cells) from untreated primary TNBC breast tumors shown as a UMAP. (B) Mutually exclusive expression of PD-L1 and SIGLEC15 in TAMs. (C) Dichotomization of TAM clusters into PD-L1 +/hi and PD-L1 –/lo subpopulations. (D) PD-L1 expression of PD-L1 +/hi and PD-L1 –/lo subpopulations. (E) Volcano plot showing differentially expressed genes (DEGs) between PD-L1 +/hi vs. PD-L1 −/lo TAMs. (F) Expression distribution of selected genes involved in maturation, pro-inflammatory, anti-inflammatory, and pro-tumor between PD-L1 +/hi and PD-L1 –/lo TAMs. (G) Published (Bassez et al. ) scRNA-seq transcriptomic clustering and dichotomization of TAMs (n = 12,952 cells) from patients with TNBC treated with neoadjuvant anti-PD1 immunotherapy into PD-L1 +/hi and PD-L1 –/lo subpopulations. (H) PD-L1 expression of PD-L1 +/hi and PD-L1 –/lo subpopulations. ∗∗∗∗p < 0.0001. Wilcoxon rank-sum test. (I) TAMs were annotated based on whether patients exhibited clonal expansion of intratumoral PD1 + T cells after the anti-PD1 treatment as expanded or non-expanded. (J) The percentages of PD-L1 +/hi TAMs were compared between tumors with anti-PD1-induced expanded vs. non-expanded clonal PD1 + T cells. ∗∗p < 0.01. Mann-Whitney test.

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Expressing, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet: PD-L1 + TAMs are associated with favorable clinical outcome in two independent cohorts of patients with BC (A and B) Kaplan-Meier relapse-free survival (RFS) curves and log rank test generated for the gene signature of PD-L1 + vs. PD-L1 – TAMs or the gene signature ratio of PD-L1 + /PD-L1 – TAMs in the luminal BC cohorts of METABRIC (n = 1098) (A) and TCGA (n = 789) (B) datasets. (C) Kaplan-Meier analyses for the M1, M2, or the ratio of M1/M2 gene signature in the luminal BC cohorts of METABRIC (n = 1098). Patients were divided into high- and low-expressing groups based on a 25% cutoff of the gene signature. (D) Schematic summarizing the histological quantification method in cohorts 1 and 2 of patients with luminal BC. (E) Representative immunofluorescence staining of PD-L1, CD68, and DAPI to identify PD-L1 + and PD-L1 – TAMs. (F–H) Using Kaplan-Meier estimate and log-rank test, relapse-free survival (RFS) was compared between patients with low and high density of PD-L1 + TAMs in cohort 1 (n = 49) (F) and in cohort 2 (n = 93) (G) or low and high density ratio of PD-L1 + /PD-L1 − TAMs in combined cohorts 1 and 2 (n = 142) (H). Median density was used as the cutoff to divide patients into low vs. high groups. (I) Univariate and multivariate analysis for the prognostic significance of the density ratio of PD-L1 + /PD-L1 – TAMs. Hazard ratio calculated with below medium vs. above medium (n = 142).

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Generated, Expressing, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet: PD-L1 + and PD-L1 – TAMs have different cell-to-cell interaction preferences (A) Multiple immunofluorescence staining and corresponding phenotype map of representative breast tumor tissue section for PD-L1 + TAMs (CD68 + PD-L1 + ), PD-L1 – TAMs (CD68 + PD-L1 – ), CD8 + T cells (CD8 + ), CD4 + T cells (CD3 + CD8 − ), and cancer cells (CK + ). (B) Whole-slide quantification of the ratio of PD-L1 + TAMs/cancer cells in total area. (C) Schematic representing the calculation of cell-cell interaction based on CD8 + T cells, CD4 + T cells, or cancer cells within a radius of 20 μm from the nuclei of PD-L1 + or PD-L1 – TAMs. (D) CD8 + T cells, CD4 + T cells, or cancer cells within a radius of 20 μm from the nuclei of PD-L1 + or PD-L1 – TAMs in untreated primary luminal breast tumors (n = 36). ∗∗∗∗p < 0.0001, ∗∗p < 0.01. Wilcoxon paired test. (E) The number of TAMs within 20 μm to PD-L1 + TAMs (left) or PD-L1 – TAMs (right) (n = 36). Paired t test. ∗∗∗∗p < 0.0001. (F) Dot plots of ligand-receptor interactions between PD-L1 + or PD-L1 – TAMs and CD4/8 + T cells (left panel), and cancer cells (right panel) based on our scRNA-seq transcriptomic analysis. Gray dot represents no significant interaction was found.

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet: PD-L1 is upregulated during the monocyte-macrophage maturation/differentiation process (A and B) Freshly isolated PBMCs from newly diagnosed patients with BC (n = 12) were ex vivo rested in RPMI 1640 with 10% FBS for 8 h. PD-L1 + % peripheral blood monocytes are shown in representative flow plots (A) and compared between fresh vs. rested monocytes (B). (C and D) PD-L1 + % between in suspension vs. adherent monocytes after 8 h resting (n = 8) are shown in representative flow plots (C) and compared (D). (E and F) PD-L1 + % monocytes between flow sorted fresh PBMCs vs. 8 h rested PBMCs (n = 5) are shown in representative flow plots (E) and compared (F). Paired t test. (G and H) MFI ratio of surface protein levels (G) and phosphorylated signal transduction protein levels (H) on PD-L1 + vs. PD-L1 – monocytes in 8 h rested PBMCs from patients with BC. Wilcoxon paired test. (I) Peripheral blood monocytes from patients with BC (n = 6) were treated with small-molecule inhibitors (Selleck) against ERK1/2 (SCH772984 at 0. 5 μM), STAT1 (fludarabine at 50 μM), Akt1/2/3 (MK-2206 2HCl at 0.5 μM), PI3Kα/δ/β (LY294002 at 5 μM), NF-κB (QNZ at 5 μM), and mTOR (rapamycin at 0.1 μM) during the 8 h resting. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗p < 0.0001. Shown are mean ± SEM.

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Isolation, Ex Vivo, Suspension, Transduction

Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet: PD-L1 upregulation in TAMs could be IFN-γ independent (A–C) PBMCs from patients with BC were rested and stimulated with IFN-γ. Representative flow plots showing IFN-γ (50 ng/mL for 15 min) induced phosphorylation of STAT1 (pY701) in peripheral monocytes (A) from patients with BC. IFN-γ signaling response (n = 28) (B) and levels of IFN-γR1 (n = 20) (C) were compared between PD-L1 –/lo vs. PD-L1 +/hi monocytes. (D and E) Rested PBMCs from patients with BC were stimulated with IFN-γ at 0.2, 1, 5, or 25 ng/mL for 16 h. Levels of PD-L1 and IFN-γR1 on monocytes or T cells are shown in the representative flow plots (D) and compared (n = 6) (E). One-way ANOVA. (F–H) Single-cell suspensions from freshly prepared primary breast tumors were stimulated with IFN-γ. Representative flow plots (F) showing IFN-γ-induced pSTAT1 (G) and levels of IFN-γR1 (H) between PD-L1 –/lo vs. PD-L1 +/hi TAMs from untreated primary breast tumors (n = 8). (I and J) Multiplex immunofluorescence staining (I) and quantification of IFN-γR1 + PD-L1 +/− cells (J) from untreated primary breast tumor tissues (n = 8). Scale bars, 100 μm. (K–M) Representative flow plots showing PD-L1 expression (K) and IFN-γ-induced pSTAT1 (L) and levels of IFN-γR1 (M) between PD-L1 –/lo vs. PD-L1 +/hi breast cancer cells (n = 8). Paired t test. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Shown are mean ± SEM.

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Multiplex Assay, Immunofluorescence, Staining, Expressing

Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet: PD-L1 + TAMs are more activated and pro-inflammatory than PD-L1 – TAMs (A and B) PBMCs from patients with BC (n = 16) were rested and the phagocytosis capacity of monocytes/macrophages were determined by using pHrodo Green E. Coli Bioparticles conjugate as shown in the representative flow plots (A) and compared between PD-L1 –/lo vs. PD-L1 +/hi monocytes/macrophages (B). Paired t test. (C and D) Freshly isolated PBMCs from patients with BC (n = 6) were rested and the PD-L1 +/− monocytes were flow sorted. CellTrace Violet dilution by CD8 + T cells determined after 4 days of TCR-stimulated coculture with autologous PD-L1 + vs. PD-L1 – monocytes/macrophages. (C) Representative flow plots showing percentage of proliferated CD8 + T cells. (D) Proliferation stimulation activity measured by cell number ratio of (CD8/CD4 + CD14)/(CD8/CD4) as the stimulatory index. (E–H) Freshly isolated PBMCs from patients with BC (n = 6) were rested and the PD-L1 +/− monocytes were flow sorted. Cytotoxic activity of CD8 + T cells determined using CD19/CD3 bispecific antibody (BiTE) after 2 days of coculture with CD19 + K562 cancer cells in the presence of autologous PD-L1 + or PD-L1 – monocytes/macrophages. (E) Schematic representing the experiment setup. (F) Representative flow plots showing PD1 and CD137 expression on CD8 + T cells. (H) Cytotoxic activity calculated by the percentage of K562 cells killed by CD8 + T cells as shown in representative flow plots (G). One-way ANOVA. ∗p < 0.05, ∗∗∗∗p < 0.0001. Shown are mean ± SEM.

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Isolation, Activity Assay, Expressing

Journal: Cell Reports Medicine

Article Title: PD-L1-expressing tumor-associated macrophages are immunostimulatory and associate with good clinical outcome in human breast cancer

doi: 10.1016/j.xcrm.2024.101420

Figure Lengend Snippet:

Article Snippet: Anti-human PD-L1 (SP142) , Abcam , Cat# ab228462; RRID: AB_2827816.

Techniques: Recombinant, Phagocytosis Assay, Software

Journal: Translational Cancer Research

Article Title: Association between clinicopathological features and prognosis significance of PD-L1 expression in small cell lung cancer patients: a systemic review and meta-analysis

doi: 10.21037/tcr-20-1512a

Figure Lengend Snippet: Main characteristics of the eligible studies

Article Snippet: Qiu ( ) , China , 2019 , IHC , 39 , 25 , LD+ED , 59 , ≥5% , SP142 (Zsbio) , C/CR. , 0.41 (0.13–1.26).

Techniques:

Journal: Translational Cancer Research

Article Title: Association between clinicopathological features and prognosis significance of PD-L1 expression in small cell lung cancer patients: a systemic review and meta-analysis

doi: 10.21037/tcr-20-1512a

Figure Lengend Snippet: Main results of meta-analysis according to the different subgroups between PD-L1 expression and clinicopathological features or survival

Article Snippet: Qiu ( ) , China , 2019 , IHC , 39 , 25 , LD+ED , 59 , ≥5% , SP142 (Zsbio) , C/CR. , 0.41 (0.13–1.26).

Techniques: Expressing

Journal: Translational Cancer Research

Article Title: Association between clinicopathological features and prognosis significance of PD-L1 expression in small cell lung cancer patients: a systemic review and meta-analysis

doi: 10.21037/tcr-20-1512a

Figure Lengend Snippet: Meta-analysis of the association between PD-L1 expression and OS: (A) overall analysis; (B) mAb and pAb subgroups analysis; (C) 2C3/28–8/SP142/SP263 clone and other clone subgroups analysis.

Article Snippet: Qiu ( ) , China , 2019 , IHC , 39 , 25 , LD+ED , 59 , ≥5% , SP142 (Zsbio) , C/CR. , 0.41 (0.13–1.26).

Techniques: Expressing

Journal: Cardiology

Article Title: The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis

doi: 10.1159/000534518

Figure Lengend Snippet: Expression profiles of inflammatory T cells, PD-1, and PD-L1 in cardiac tissues

Article Snippet: The primary antibodies were anti-CD4 rabbit monoclonal antibody (clone SP35, 518-108816, Roche Diagnostics K. K.), anti-CD8 rabbit monoclonal antibody (clone SP57, 518-101831, Roche Diagnostics K. K.), anti-FoxP3 mouse monoclonal antibody (clone 236A/E7, ab20034, Abcam, Cambridge, UK), anti-PD-1 mouse monoclonal antibody (clone NAT105, ab52587, Abcam), and anti-PD-L1 rabbit monoclonal antibody (clone SP142, ab228462, Abcam).

Techniques: Expressing

Journal: Cardiology

Article Title: The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis

doi: 10.1159/000534518

Figure Lengend Snippet: Comparison of myocardial pathology findings between patients with and without cardiac events

Article Snippet: The primary antibodies were anti-CD4 rabbit monoclonal antibody (clone SP35, 518-108816, Roche Diagnostics K. K.), anti-CD8 rabbit monoclonal antibody (clone SP57, 518-101831, Roche Diagnostics K. K.), anti-FoxP3 mouse monoclonal antibody (clone 236A/E7, ab20034, Abcam, Cambridge, UK), anti-PD-1 mouse monoclonal antibody (clone NAT105, ab52587, Abcam), and anti-PD-L1 rabbit monoclonal antibody (clone SP142, ab228462, Abcam).

Techniques: Comparison

Journal: Cardiology

Article Title: The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis

doi: 10.1159/000534518

Figure Lengend Snippet: Hematoxylin and eosin staining and immunostaining. Hematoxylin and eosin staining and immunostaining of CD4, CD8, FoxP3, PD-1, and PD-L1 in the CD8 low PD-L1 low (left side), CD8 low PD-L1 high (center), and CD8 high PD-L1 high (right side) groups. Scale bar: 200 μm.

Article Snippet: The primary antibodies were anti-CD4 rabbit monoclonal antibody (clone SP35, 518-108816, Roche Diagnostics K. K.), anti-CD8 rabbit monoclonal antibody (clone SP57, 518-101831, Roche Diagnostics K. K.), anti-FoxP3 mouse monoclonal antibody (clone 236A/E7, ab20034, Abcam, Cambridge, UK), anti-PD-1 mouse monoclonal antibody (clone NAT105, ab52587, Abcam), and anti-PD-L1 rabbit monoclonal antibody (clone SP142, ab228462, Abcam).

Techniques: Staining, Immunostaining

Journal: Cardiology

Article Title: The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis

doi: 10.1159/000534518

Figure Lengend Snippet: Event-free survival curves of patients classed as CD8 high or CD8 low based on CD8 + cell counts ( a ) and PD – L1 high or PD-L1 low based on PD-L1 expression ( b ). The Kaplan-Meier analysis showed a poorer outcome in patient classes CD8 high ( a ) and PD-L1 high ( b ).

Article Snippet: The primary antibodies were anti-CD4 rabbit monoclonal antibody (clone SP35, 518-108816, Roche Diagnostics K. K.), anti-CD8 rabbit monoclonal antibody (clone SP57, 518-101831, Roche Diagnostics K. K.), anti-FoxP3 mouse monoclonal antibody (clone 236A/E7, ab20034, Abcam, Cambridge, UK), anti-PD-1 mouse monoclonal antibody (clone NAT105, ab52587, Abcam), and anti-PD-L1 rabbit monoclonal antibody (clone SP142, ab228462, Abcam).

Techniques: Expressing

Journal: Cardiology

Article Title: The Balance of CD8-Positive T Cells and PD-L1 Expression in the Myocardium Predicts Prognosis in Lymphocytic Fulminant Myocarditis

doi: 10.1159/000534518

Figure Lengend Snippet: Event-free survival curves for the three groups (CD8 low PD-L1 low , CD8 low PD-L1 high , and CD8 high PD-L1 high ) based on the number of CD8 + T cells and PD-L1 expression. The Kaplan-Meier analysis showed the worst event-free survival in the CD8 high PD-L1 high group.

Article Snippet: The primary antibodies were anti-CD4 rabbit monoclonal antibody (clone SP35, 518-108816, Roche Diagnostics K. K.), anti-CD8 rabbit monoclonal antibody (clone SP57, 518-101831, Roche Diagnostics K. K.), anti-FoxP3 mouse monoclonal antibody (clone 236A/E7, ab20034, Abcam, Cambridge, UK), anti-PD-1 mouse monoclonal antibody (clone NAT105, ab52587, Abcam), and anti-PD-L1 rabbit monoclonal antibody (clone SP142, ab228462, Abcam).

Techniques: Expressing

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: Identification of PD-L1 C-terminal–deficient splicing variants in patients who were relapsed from PD-L1 blockade therapy. (A) Representative computed tomographic images of JFCR-119 and JFCR-151 at baseline and at the time of relapse. Bars, 5 cm. (B) Representative IHC staining of CD8 and PD-1 at baseline and in the relapsed tumor from JFCR-119. Bars, 10 µm. (C) Sashimi plot RNA-seq analysis of the PD-L1 spliced region. The figure shows representative data for PD-L1v178 (above) and PD-L1v242 (below) in MR203. (D) Integrative genomics viewer (IGV) data indicating PD-L1v242 in MR199 and MR203 are shown. (E) The PD-L1 splicing variants identified from JFCR-119 and JFCR-151. The domains are indicated as follows: signal peptide (Sig) on 1–18 aa as green; IgV domain on 19–127 aa as yellow; IgC domain on 133–225 aa as pink; transmembrane domain (TM) on 239–259 aa as blue; and cytoplasmic domain (Cyto) on 260–290 aa as gray. The red region demonstrates the additional amino acids from aberrant splicing. (F) IGV data of TARDBP on the mutated region in pretreatment and relapsed samples of JFCR-119. (G) Relapsed tumor-specific mutations in JFCR-119 analyzed by RNA-seq.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Immunohistochemistry, RNA Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: The recognition pattern of PD-L1 splicing variants by aPD-L1 antibodies. (A and B) PD-L1 mRNA levels in PD-L1 splicing variant overexpressing PC-9 (A) or SW480 (B) parental (pt) cells were quantified with real-time PCR. The results from three independent experiments are expressed as mean ± SD normalized by that of PC-9/PD-L1 or SW480/PD-L1, respectively. (C) Flow cytometric analysis of PD-L1 expression on cell surface in parental, WT, and PD-L1 variants expressing PC-9 cells. (D and F) PD-L1-WT and splicing variants with different aPD-L1 antibodies were detected by Western blotting (D) and immunofluorescence staining (F). Bars, 10 µm. (E) The epitopes of the aPD-L1 antibodies were roughly estimated by Western blotting and immunofluorescence staining. C and F were independently performed twice, yielding similar results. D was conducted once.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Variant Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunofluorescence, Staining

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: PD-L1 C-terminal–deficient splicing variants in the relapsed tumor were secreted . (A and B) WCL from PC-9 (A) and SW480 (B) cells which were overexpressed each PD-L1 variants as indicated were analyzed by Western blot. The culture supernatants (SUP) were analyzed following acetone precipitation. (C and D) Quantitative analysis of soluble PD-L1 in plasma (C) and pleural effusion (D) from healthy donors (HD), EGFR-mutated NSCLC patients, and patients with the detected PD-L1 splicing variants (JFCR-119 and JFCR-151). Each experiment was independently performed twice, yielding similar results.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Western Blot, Clinical Proteomics

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: PD-L1 C-terminal–deficient splicing variants from relapsed tumor were stable. (A and B) Glycosylation analysis of sPD-L1 variants. WCL of PC-9 parental cell (pt) and PC-9/PD-L1, or concentrated sPD-L1 variants obtained from culture supernatant (SUP) of PC-9/PD-L1v242, PC-9/PD-L1v229 (A), and PC-9/PD-L1v178 (B) were treated with N-glycanase, sialidase-A, or O-glycanase for 3 h at 37°C and then analyzed by Western blot. (C) Immunoprecipitated samples from WCL and culture supernatant with aPD-L1 antibody were analyzed by Western blot. HSP90 in WCL were used as the loading control. (D) 35 S-labeled methionine cells were cultured in a radio-free medium for the indicated period. Immunoprecipitated (IP) samples from WCL and the culture supernatant were evaluated with SDS-PAGE and visualized with a phosphor imaging scanner. (E) The remaining PD-L1 was quantified with ImageJ software based on the results of (D). (F) Phosphor imaging of culture supernatant samples immunoprecipitated by aPD-L1 antibody. C–F were independently performed twice, yielding similar results. A and B were conducted once.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Glycoproteomics, Western Blot, Immunoprecipitation, Control, Labeling, Cell Culture, SDS Page, Imaging, Software

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: sPD-L1 splicing variants bind to PD-1. (A–C) The binding of PD-L1v242 (A), PD-L1v229 (B), PD-L2, and B7-H3 to PD-1 (C) were evaluated by ELISA ( n = 3). Results are expressed as mean ± SD. (D and E) Purified Fc-tagged PD-L1v242 (D) and PD-L1v229 (E) preincubated with or without aPD-L1 or aPD-1 antibody for 30 min was incubated in PD-1 coated wells for 2 h at RT. The binding of PD-L1 variants to PD-1 was detected based on the absorbance at 450 nm ( n = 3). Results are expressed as mean ± SD. (F–I) Flow cytometry analysis for evaluating sPD-L1 splicing variants binding to PD-1. Culture supernatant from CHO parental cell (pt) and those overexpressing PD-L1v242-Fc (F and G), PD-L1v229-Fc (F and H), and PD-L1(19–239)-Fc (F and I) was incubated with Jurkat/PD-1 cells in the condition described for 1 h. Fc-tagged PD-L1 variant binding to PD-1 was evaluated with flow cytometer. Each experiment was independently performed twice, yielding similar results. The data of nonstaining and secondary antibody (2nd Ab) as negative control in F–I were the same.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Purification, Incubation, Flow Cytometry, Variant Assay, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: sPD-L1 splicing variants attenuate the neutralizing activity of aPD-L1 by trapping the antibody. (A and B) Flow cytometric analysis of aPD-L1 antibody binding to PD-L1 (A) and aPD-1 antibody binding to PD-1 (B) in the presence of indicated sPD-L1 variants molar ratio. (C) Fc-tagged PD-L1 and aPD-L1 antibodies (0.75 µg/ml) preincubated with or without PD-L1v242 as indicated were added to PD-1 precoated 96-well ELISA plates. The binding of Fc-tagged PD-L1 to PD-1 was detected based on absorbance at 450 nm; 0.2 µg/ml PD-L1v242 is equal to 0.75 µg/ml aPD-L1 antibody in molar ratio. n = 3. Results are expressed as mean ± SD. Paired two-tailed Student t test was used. *, P < 0.05; ***, P < 0.001. (D) Schematic diagram illustrating the NFAT-luc assay for evaluating TCR-mediated NFAT transduction. (E) The neutralizing activity (EC 50 ) of aPD-L1 and aPD-1 antibodies was determined using NFAT-luc assay in the presence of various molar ratios of PD-L1v242 to the antibody ( n = 3). Results are expressed as mean ± SD. (F) The correlation between PD-L1v242 and the EC 50 of the antibody according to the Lineweaver–Burk plot was analyzed based on the results of (E), which suggested that the sPD-L1 splicing variants reduce the aPD-L1 inhibitory activity competitively. Each experiment was independently performed twice, yielding similar results.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Transduction

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: sPD-L1 splicing variants contribute the resistance to PD-L1 blockade in WT-1 tumor antigen–specific iPSC-derived CD8 T cell model. (A) Schematic illustration of apoptosis assay for testing whether PD-L1v242 attenuates the blockade effect of aPD-L1 antibody. (B) iPSC-derived WT-1–specific T cells overexpressing PD-1 were cocultured with THP-1 cells overexpressing PD-L1 for 18 h in the presence of aPD-L1 antibody (1 µg/ml) or PD-L1v242 (2 µg/ml). The dead cell ratio was flow cytometrically measured using propidium iodide staining, and bars represent the proportion of live T cells in comparison with those before coculture. 2 µg/ml of PD-L1v242 was approximately eight times more than 1 µg/ml aPD-L1 antibody in molar ratio. The results are representative from three independent experiments and are shown as mean ± SD ( n = 3). Paired two-tailed Student t test was used. ***, P < 0.001. The experiment was independently performed twice, yielding similar results.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Derivative Assay, Apoptosis Assay, Staining, Comparison, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: sPD-L1 splicing variants mediate the resistance to PD-L1 blockade in MC38 syngeneic mouse model. (A) C57BL/6 mice bearing MC38/cont ( n = 6), MC38/mPD-L1 ( n = 6), MC38 ( n = 10), MC38/mPD-L1v242 ( n = 10), and MC38/mPD-L1v178 ( n = 5) were intraperitoneally administrated 50 µg/mouse control IgG or 35 µg/mouse aPD-L1 antibody. The schedules for treatment are indicated by black arrows (control IgG) and red arrows (aPD-L1). Tumor volume is plotted individually. (B) Kaplan–Meier survival curves for mice bearing MC38 or MC38/mPD-L1v242. The survival curves were compared by applying the Gehan–Breslow–Wilcoxon test. ***, P < 0.001. (C and D) Representative IHC staining of mouse CD8, PD-1, and granzyme B was performed on day 21 for MC38/mPD-L1 (C) and MC38/mPD-L1v242 (D) xenograft tumors. Bars, 20 µm. Each experiment was independently performed twice, yielding similar results.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Control, Immunohistochemistry

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: aPD-1 treatment overcame the resistance to PD-L1 blockade induced by sPD-L1 splicing variants in MC38 syngeneic mouse model. (A) C57BL/6 mice bearing MC38/mPD-L1 ( n = 7), MC38/mPD-L1v242 ( n = 7), 10% MC38/mPD-L1v242 + 90% MC38/mPD-L1 ( n = 7), and 1% MC38/mPD-L1v242 + 99% MC38/mPD-L1 ( n = 7) were intraperitoneally administered 35 µg/mouse control IgG or 35 µg/mouse aPD-L1 antibody. The schedules for treatment are indicated by black arrows for control IgG and red arrows for aPD-L1. The plot shows the tumor volumes for each mouse. (B) The plasma levels of soluble PD-L1 in mice bearing MC38/mPD-L1 and 1% MC38/mPD-L1v242 were sequentially evaluated by ELISA. To remove the exosome fraction, the plasma was ultracentrifuged at 100,000 g for 90 min. (C) C57BL/6 mice bearing MC38/mPD-L1 and MC38/mPD-L1v242 were intraperitoneally administrated either 100 µg/mouse control IgG ( n = 8) or aPD-1 antibody at doses of 100 µg/mouse ( n = 8), 35 µg/mouse ( n = 8), or 20 µg/mouse ( n = 4). The treatment days are indicated by black arrows for control IgG and red arrows for aPD-1. The plot shows the tumor volumes for each mouse. A was independently performed twice, yielding similar results. B and C were conducted once.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: Secreted PD-L1 variants mediate resistance to PD-L1 blockade therapy in non–small cell lung cancer

doi: 10.1084/jem.20180870

Figure Lengend Snippet: The proposed model for the sPD-L1 splicing variants associated with resistance to aPD-L1 antibody treatment.

Article Snippet: The cells were labeled with aPD-L1 antibodies (E1J2J, Cell Signaling Technology; 22C3, Dako; and SP142, Spring Bioscience) in buffer containing 0.3% Triton X-100 and 1× Blocking One solution (Nacalai Tesque) overnight at 4°C.

Techniques: