human b7 h1 pdl1 human pd1 duoset elisa  (R&D Systems)

Journal: Nature Communications

Article Title: PD-1/CD80 + small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression

doi: 10.1038/s41467-024-48200-9

Figure Lengend Snippet: a A TEM image of circulating sEVs from HNSCC patients, showing the cup-shape spherical morphology of sEVs. n = 3 biologically independent samples. Scale bar, 200 nm. sEVs small extracellular vesicles, TEM transmission electron microscope, HNSCC head and neck squamous cell carcinoma. b Determination of particle size distribution in purified sEVs using nanoparticle tracking analysis. c Heat map illustrating the levels of immune checkpoint proteins (CD80, PD-1, PD-L1, CTLA-4, CD86, LAG-3 and TIM-3) on circulating sEVs from healthy donors (HDs, n = 36) and HNSCC patients (n = 46) were shown on the left. Blue and red hatches indicate HD and HNSCC patients, respectively. Bar plots show bootstrap importance scores based on random forest model that distinguish HDs and HNSCC patients, higher values (red) represent stronger association with HNSCC (right). Nanoparticle flow cytometry analysis of pretreatment levels of immune checkpoint proteins PD-L1 ( d ), PD-1 ( e ), and CD80 ( f ) on circulating sEVs from responders (R, n = 12) and non-responders (NR, n = 11). Overall survival for HNSCC patients with high and low levels of circulating sEV PD-1 ( g ) and CD80 ( h ). Log-rank test. i Pie Chart showing the proportion of circulating sEV PD-1 High , sEV CD80 High , and sEV PD-1 High CD80 High in HNSCC patients (n = 46). j Nanoparticle flow cytometry analysis of PD-1 and CD80 expression in sEVs after purification. Left, the gating strategy. Right, the Venn diagram illustrating the percentages of PD-1 + sEVs, CD80 + sEVs and PD-1 + CD80 + sEVs in HNSCC patients. For ( d , e , f) Data were presented as mean ± s.d.; Two-sided t -test. The relevant raw data are provided as a file.

Article Snippet: The plates were then incubated with capture antibody against PD-L1 (Clone 5H1-A3, Millipore) and biotinylated monoclonal PD-L1 antibody (Clone MIH1, eBioscience). sEV PD-1 and sEV CD80 were quantified using the human PD-1 and CD80 ELISA kit (for PD-1 catalogue number DY1086, for CD80 catalogue number DY140) from R&D Systems (Minneapolis, MN).

Techniques: Transmission Assay, Microscopy, Purification, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: PD-1/CD80 + small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression

doi: 10.1038/s41467-024-48200-9

Figure Lengend Snippet: a Scheme for the experimental setup of tumour xenograft mice model. 5 × 10 5 MC38 cells or 1.5 × 10 5 B16F10 cells were subcutaneously injected into flanks of 8-week-old female C57BL/6 mice. Tail vein injections of sEVs (100 μg) were performed every 2 days from days 12 to 22. For blocking PD-1 or CD80, sEVs were pretreated with corresponding blocking antibodies. IgG isotype was used as control. b Growth curve of MC38 tumours in C57BL/6 mice with indicated treatments (n = 6 mice per group). c Weights of MC38 tumours in C57BL/6 mice with indicated treatments (n = 6 mice per group). d Overall survival for C57BL/6 mice bearing MC38 tumours with indicated treatments (n = 6 mice per group). Log-rank test. e Scheme for the experimental setup of the MC38 tumour xenograft mice model with anti-PD-1 blockade antibodies treatment. Anti-PD-L1 was intraperitoneally administered (200 μg) every 2 days from days 13 to 19. An isotype IgG antibody was used as the control. f Growth curve of MC38 tumours in C57BL/6 mice with indicated treatments (n = 7 mice per group). g Overall survival of C57BL/6 mice bearing MC38 tumours with indicated treatments. Log-rank test. h , i Immunohistochemistry analysis of CD8 + T cell infiltration in MC38 tumour sections from C57BL/6 mice with indicated treatments. Representative images ( h ) of CD8 staining in tumour sections. Scale bar, 100 µm. Quantification analysis ( i ) of infiltrated CD8 + T cells in MC38 tumours (n = 6 mice per group). TILs, tumour infiltrating lymphocytes. For ( b , f ) data were presented as mean ± s.d.; Two-way ANOVA. For ( c , i ) data were presented as mean ± s.d.; Two-sided t -test. Source data are provided as a file.

Article Snippet: The plates were then incubated with capture antibody against PD-L1 (Clone 5H1-A3, Millipore) and biotinylated monoclonal PD-L1 antibody (Clone MIH1, eBioscience). sEV PD-1 and sEV CD80 were quantified using the human PD-1 and CD80 ELISA kit (for PD-1 catalogue number DY1086, for CD80 catalogue number DY140) from R&D Systems (Minneapolis, MN).

Techniques: Injection, Blocking Assay, Control, Immunohistochemistry, Staining

Journal: Nature Communications

Article Title: PD-1/CD80 + small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression

doi: 10.1038/s41467-024-48200-9

Figure Lengend Snippet: a Immunohistochemistry analysis of PD-L1 expression in HNSCC patient with PD-L1 positive or negative tumours. Left, representative images of PD-L1 staining in biopsies of HNSCC patients. Scale bar, 50 µm. Right, pie chart showed the proportion of patients with either PD-L1 negative tumour cells (green, n = 30) and patients with PD-L1 positive tumour cells (red, n = 22). Pearson correlation analysis of the level of circulating PD-L1 + sEVs with circulating sEV PD-1 and CD80 in patients with PD-L1 positive (n = 22) ( b ) and PD-L1 negative ( c ) tumour cells (n = 24). d Flow cytometry profiles (left) and quantification of relative fluorescence intensity (RFI) (right) of circulating sEV PD-L1 levels in mice MC38-bearing C57BL/6 mice with indicated treatment (n = 6 mice per group). e , f Immunohistochemistry analysis of PD-L1 expression in MC38 tumour sections from C57BL/6 mice with indicated treatments. Representative images ( e ) of PD-L1 staining in tumour cells. Scale bar, 100 µm. Quantification analysis ( f ) of membrane expression of PD-L1 in MC38 tumour cells (n = 6 mice per group). For ( d , f ) data were presented as mean ± s.d.; Two-sided t -test. Source data are provided as a file.

Article Snippet: The plates were then incubated with capture antibody against PD-L1 (Clone 5H1-A3, Millipore) and biotinylated monoclonal PD-L1 antibody (Clone MIH1, eBioscience). sEV PD-1 and sEV CD80 were quantified using the human PD-1 and CD80 ELISA kit (for PD-1 catalogue number DY1086, for CD80 catalogue number DY140) from R&D Systems (Minneapolis, MN).

Techniques: Immunohistochemistry, Expressing, Staining, Flow Cytometry, Fluorescence, Membrane

Journal: Nature Communications

Article Title: PD-1/CD80 + small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression

doi: 10.1038/s41467-024-48200-9

Figure Lengend Snippet: An acceptor-photobleaching FRET assay showing the molecular nearness of PD-1 ( a ) or CD80 ( b ) on sEVs surface and PD-L1 on the tumour cell membrane. Left, scheme for experimental setup. Middle, Confocal microscopy images showing PD-1 ( a ) or CD80 ( b ) (green) on sEVs and PD-L1 (red) on CAL27 cells. The difference in fluorescence intensity of the energy donor before and after photobleaching was evaluated as the level of FRET efficiency. Right, the quantification analysis of the difference in fluorescence intensity (FI). Scale bar, 10 µm. c Flow cytometry profiles (left) and quantification of mean fluorescence intensity (MFI) (right) of membrane PD-L1 levels in CAL27 cells with or without aT-sEV treatment. d , e Immunofluorescence staining of PD-L1 in CAL27 cells after incubation with CFSE-labelled aT-sEVs for 0, 15 and 30 min ( d ). Scale bar, 10 µm. Quantification analysis of the relative levels of cytoplasmic PD-L1 in CAL27 cells after incubation with CFSE-labelled aT-sEVs ( e ). f Immunofluorescence images of CAL27 cells staining with PD-L1 (red) and EEA or RAB7 (green) treated with or without aT-sEVs. The fluorescence intensity profiles are plotted on the right. The nuclei were counter-stained with DAPI (blue). n = 3 biologically independent samples. Scale bar, 10 μm. g Confocal images of CAL27 cells staining with PD-L1 (red) and CD63 (green) treated with or without aT-sEVs. The nuclei were counter-stained with DAPI (blue). The fluorescence intensity profiles are plotted on the right. n = 3 biologically independent samples. Scale bar, 10 μm. h Flow cytometry profiles (left) and quantification of proportion (right, top) and relative fluorescence intensity (RFI) (right, bottom) of sEV PD-L1 in supernatants from CAL27 cells with or without aT-sEV treatment. i Western blot analysis of PD-L1 expression level in WCL and sEVs from CAL27 cells after aT-sEV treatment (top). Quantification analysis of relative PD-L1 levels in WCL and sEVs (bottom). WCL whole cells lysate. The samples derive from the same experiment and that gels/blots were processed in parallel. For ( a , b , c , e , h , i ) data were presented as mean ± s.d.; n = 3 biologically independent samples; Two-sided t -test. For ( e ) data were presented as mean ± s.d.; Two-way ANOVA. The relevant raw data and uncropped blots are provided as a Source Data file.

Article Snippet: The plates were then incubated with capture antibody against PD-L1 (Clone 5H1-A3, Millipore) and biotinylated monoclonal PD-L1 antibody (Clone MIH1, eBioscience). sEV PD-1 and sEV CD80 were quantified using the human PD-1 and CD80 ELISA kit (for PD-1 catalogue number DY1086, for CD80 catalogue number DY140) from R&D Systems (Minneapolis, MN).

Techniques: Membrane, Confocal Microscopy, Fluorescence, Flow Cytometry, Immunofluorescence, Staining, Incubation, Western Blot, Expressing

Journal: Nature Communications

Article Title: PD-1/CD80 + small extracellular vesicles from immunocytes induce cold tumours featured with enhanced adaptive immunosuppression

doi: 10.1038/s41467-024-48200-9

Figure Lengend Snippet: a ROC curve analysis of circulating sEV PD-L1, PD-1, and CD80 in clinical responders (n = 12) and non-responders (n = 11) (top). AUC area under curve. Bottom, detailed data associated with the ROC curve analysis. b ORR of immunotherapy in patients with high (n = 10) and low (n = 13) pretreatment levels of circulating sEV PD-L1 (left), high (n = 18) and low (n = 5) pretreatment levels of circulating sEV PD−1 (middle), high (n = 12) and low (n = 11) pre-treatment levels of circulating sEV CD80 (right). ORR, objective response rate. c Tracking the levels of circulating sEV PD-L1 and PD-1/CD80 stratified responders to anti-PD-1 therapy (green) from non-responders (red) (left). HNSCC patients were divided into three quadrants (Q1-Q3) based on the levels of circulating sEV PD-L1 and sEV PD-1/CD80 (top). Detailed information of ORR in the three subtypes of patients (bottom). d Bar plots showing importance scores of different indexes based on random forest machine learning for distinguish responders to immunotherapy from nonresponders. TB, tumour burden. e Analysis of ORR in patients with PD-L1 negative tumour cells (n = 2) and patients with PD-L1 positive tumour cells (n = 6). All six patients were extracted from Q3 in ( c ), in which high levels of sEV PD-1/CD80 and low levels of sEV PD-L1 were detected in patients. Representative histochemistry images of PD-L1 staining in patient with PD-L1 positive or negative tumours (left). Right, ORR was plotted bar diagram. f Scheme of the strategy for predicting patients’ immunotherapy response. For ( b , d ) Data were represented as mean ± s.d.; Two-sided Fisher’s exact test.

Article Snippet: The plates were then incubated with capture antibody against PD-L1 (Clone 5H1-A3, Millipore) and biotinylated monoclonal PD-L1 antibody (Clone MIH1, eBioscience). sEV PD-1 and sEV CD80 were quantified using the human PD-1 and CD80 ELISA kit (for PD-1 catalogue number DY1086, for CD80 catalogue number DY140) from R&D Systems (Minneapolis, MN).

Techniques: Staining