14-5983-80

antibody from Invitrogen Antibodies

Targeting: CD274 B7-H, B7-H1, B7H1, PD-L1, PDCD1LG1, PDL1

Provider product page for 14-5983-80

Western blot

Immunocytochemistry

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Antibody data

Antibody Data
Antigen structure
References [102]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Flow cytometry [1]
Other assay [89]

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Product number: 14-5983-80 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH1), eBioscience™
Antibody type: Monoclonal
Antigen: Other
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: MIH1
Vial size: 25 µg
Concentration: 0.5 mg/mL
Storage: 4° C

CD274 protein structure - 14-5983-80 shown in red.

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Experimental details: Figure 6 IL-25-Mediated PD-L1 Expression in Human Monocytes and hMSCs Is Mediated through JNK and STAT3, with STAT3 Involved in Transcriptional Control of PD-L1 (A) Human PBLs were pretreated with inhibitors of STAT3 (WP1066; 2.5 muM), JNK (SP600125; 25 muM), or MEK1 (PD98059; 20 muM) prior to 100 ng/ml rhIL-25 for 18 hr, with subsequent flow cytometric analysis for PD-L1 surface expression on monocytes, gated using FSC and SSC. Filled histograms represent isotype control; unfilled histograms represent PD-L1 antibody staining. (B-D) Pooled data (three donors) are shown (B) with bars representing MFI. hMSCs were treated with inhibitors of STAT3 (C; WP1066, 2.5 muM) and JNK (D; SP600125, 25 muM) for 6 hr, and subsequently assessed by flow cytometric analysis for PD-L1 surface expression. Pooled data (all three donors) for each respective inhibitor are provided (left charts) with bars representing MFI. (E) Putative GAS elements (STAT-binding sites) in the proximal promoter region of human PD-L1 gene (700 bp region upstream from the transcription start site), as determined with TFSearch web-based software. (F) Binding of STAT3 or IgG (negative control) in hMSCs was analyzed by chromatin immunoprecipitation (ChIP) with promoter-specific primers for region 1 and region 2. The input samples (positive control) represent 1% starting chromatin. (G) Schematic shows a model of hMSC-mediated suppression of Th17 responses involving the IL-25/STAT3/PD-L1 axis.

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Experimental details: Figure 2 IL-27 increases PD-L1 surface protein and mRNA expression in EOC cells in vitro A. FACS analysis of surface PD-L1 expression in four EOC cell lines, cultured in the presence of medium (control), IL-27 or IFN-gamma. Dotted lines are isotype-matched unrelated Ig staining controls. Numbers in brackets are Median Fluorescence Intensity (MFI) values calculated as median PD-L1 minus median Ig control. Data are representative of two independent experiments showing similar results. B. QRT-PCR analysis of PDL1 mRNA expression in five IL-27-stimulated EOC cells relative to untreated cells. Data are the mean (+-SD) of three independent experiments. C. Comparative analysis of IDO1 and PDL1 mRNA up-regulation by IL-27 or IFN-gamma in a representative EOC cell line (CAOV3). Data are the mean of two independent replicates and are expressed as DeltaDeltaCT-fold change. Error bars represent the minimum and maximum.

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Experimental details: Figure 1 Mesenchymal stromal cell (MSC) cell surface expression and secretion of PD-L1 and PD-L2 are potentiated by pro-inflammatory cytokines, IFNgamma and TNFalpha. MSCs ( n = 4) were exposed to 100 U/ml IFNgamma and 10 ng/ml TNFalpha for 3 days in culture. Cell surface expression (MFI) of (A) PD-L1 and (B) PD-L2 was assessed by flow cytometry. Secretion of (C) soluble (s)PD-L1 and (D) sPD-L2 within the conditioned media of stimulated cells was assessed by ELISA. Bar charts indicate mean +- SEM. Transcriptional regulation of (E) PD-L1 and (F) PD-L2 were assessed by qRT-PCR. mRNA data are expressed as fold change compared to unstimulated, resting MSCs +- SEM. *, p < .05; **, p < .01. Abbreviations: IFNgamma, Interferon gamma; MFI, mean fluorescence intensity; PD-L1 and PD-L2, programmed death 1 ligands 1 and 2; TNFalpha, tumor necrosis factor alpha.

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Experimental details: Figure 4 RRV-RSV-miRPDL1 and RRV-H1-miRPDL1 Exhibit Comparable PDL1 Downregulation Activity as RRV-miRPDL1 (A) Replication kinetics of RRV-RSV-miRPDL1 and RRV-H1-miRPDL1. The viral genome in the supernatants of infected LN-18 cells (MOI of 0.1) at indicated time points were quantified by qRT-PCR using primer set targeted to the env region ( Figure 1 ). RRV-yCD2 and RRV-miRPDL1 (indicated as U6-miRPDL1 in the graph) were included as positive controls. (B) Vector stability of RRV-RSV-miRPDL1 and RRV-H1-miRPDL1 in LN-18 cells was analyzed by endpoint PCR at 14 and 30 days post infection. Lanes 1 and 6: DNA molecular marker (1 Kb Plus marker, Invitrogen); lanes 2, 4, and 7 are positive controls using the corresponding plasmid DNA as the templates; lane 3: RRV-H1-miRPDL1; lane 5: RRV-RSV-miRPDL1; and lane 8: RRV-miRPDL1. The arrow indicates the expected size of the PCR products. (C) LN-18 cells infected with RRV-miRPDL1 (U6), RRV-H1-miRPDL1 (H1), and RRV-RSV-miRPDL1 (RSV) were stained for PDL1 cell surface expression with PDL1 antibody and analyzed by flow cytometry.

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Experimental details: Fig 6 Representative dot matrices for apoptotic and necrotic cell death of experimental groups are shown under panel A while bar graphs (panel B) show percent of each type of damaged/dead or normal cells for each group. Data are means +- SEM; n = 6 hearts/group for normoxic control, IRI and IRI; Anti-PD-L1 and n = 4 hearts for IRI; isotype matching control. Also shown are representative caspase 3 immunostaining for experimental groups (panel C). 400x. 7AAD: 7-Amino Actinomycin D. a: necrotic; b: apoptotic/necrotic; c: early apoptotic. IRI: Ischemia Reperfusion Injury. * p

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Experimental details: Figure 6 Cutaneous (MEL13.03, MEL93.05 and A375) and conjunctival melanoma (CRMM1, CRMM2 and CM2005.1) cell lines express various levels of PD-L1 and PD-1 MEL13.03 is the positive control cell line for both PD-L1 and PD-1. Representative histograms show ( A ) PD-L1 and PD-1 ( B ) expression in cell lines with or without IFN-gamma (100 IU/ml) exposure for 48 h. Pink, blue and brown shaded histograms represent unstained, PD-L1 (PD-1) staining, and the effect of IFN-gamma stimulation on PD-L1 and PD-1, respectively.

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Experimental details: Figure 2 Common periodontal pathogens induce the expression of PD-L1 on PDLCs. (A) Flow cytometry histogram overlays of PDLCs co-cultured with P.g , F.n and P.i . (B) Comparison of expression levels of PD-L1 induced by P.g, F.n and P.i. Data are expressed as the mean +- standard error of the mean of three independent experiments. All three strains significantly increased the expression of PD-L1 on the PDLCs, * P

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Experimental details: Figure 4. Levels of PD-L1, p-JAK1, p-JAK2 and p-STAT3 are suppressed in C4-2-ATMSi and CWR22Rv1-ATMSi cells compared with in the control cells, and JAK inhibitor 1 significantly suppresses the expression of PD-L1 in ATM knockout groups and control groups. (A) A significant decrease in PD-L1 expression was revealed in experimental groups by reverse transcription-quantitative polymerase chain reaction and western blotting. (B) Decreased levels of p-JAK1, p-JAK2 and p-STAT3 were revealed in the experimental groups by western blotting. (C) JAK inhibitor 1 and Stattic were used to treat all cell groups. Downregulation of JAK significantly reduced PD-L1 expression, whereas Stattic had no significant effect on PD-L1 expression. **P

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Experimental details: Figure 4 Medulloblastoma cell lines express PD-L1 in a subgroup dependent manner PD-L1 expression in low and high MYC MB cell lines. Paraffin embedded cell line pellets stained for PD-L1 in DAOY ( A ) and D425-MED ( D ). Histograms of PD-L1 expression by flow cytometry using phycoerythrin (PE) conjugated MIH1 clone of PD-L1 (EBioscience) with and without IFN-gamma stimulation in DAOY ( B ), UW228 ( C ), D425-MED ( E ), and D283-MED ( F ).

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Experimental details: Figure 5 MYC overexpression in DAOY (YM21) does not alter PD-L1 expression Dot Plots of PD-L1 expression by flow cytometry using PE-conjugated MIH1 clone of PD-L1 (EBioscience) in YM21 construct made by overexpressing MYC in DAOY via stable lentiviral transfection.

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Experimental details: Figure 6 Radiation induces PD-L1 in most medulloblastoma cell lines Bar graph depicts percent PD-L1 positive cells above baseline as determined by flow cytometry using PE-conjugated MIH1 clone of PD-L1 (EBioscience) in DAOY ( A ), UW228 ( B ), D283-MED ( C ), and D425-MED ( D ) after irradiation with 2, 5, or 10 Gy at 2, 4, 8, 24, and 48 hours. IFN-gamma values were obtained without radiation. Each bar graph depicts findings from 2 different experiments where all data points were repeated. Error bars represent standard deviation of the mean. Levels of PD-L1 that were comparable to those induced by IFN-gamma included UW228 hours 2-24 at 2, 5 and 10 Gy and D283-MED at hour 8, 10 Gy. All other values were significantly less than those induced by IFN-gamma as determined by 2-way ANOVA corrected for multiple comparisons. Full statistical analysis available in Supplementary Table 1-1-1-4 .

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Experimental details: Figure 6 Chromatin immunoprecipitation (ChIP) assays of the CD274 promoter in primary human monocytes. Cells were treated with or without LPS for 1 h. ChIP assays were carried out using an anti-p65 antibody. IgG, anti-beta-actin and anti-STAT1 antibodies were used as negative controls. Relative enrichment of each transcription factor-bound DNA was detected by qPCR using ChIP primers. All the results were normalized to input DNA.

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Experimental details: Figure 1 LPS treatment regulates CD274 mRNA levels mainly via the NF-kappaB signaling pathway in primary human monocytes. CD274 mRNA levels were determined by quantitative real-time PCR and are shown as fold changes in arbitrary units. (A) LPS treatment regulates CD274 mRNA levels (n = 3, repeated three times, *P

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Experimental details: FIGURE 1 Human Prostate Cancer Cell Lines Display Varying Expression of PD-L1 in Response to IFN-gamma Histograms representing PD-L1 surface expression as detected by flow cytometry in human prostate cancer cell lines with and without exposure to IFN-gamma. Open histograms represent cells stained with an isotype IgG1 antibody tagged with a matching PE fluorochrome. Shaded histograms represent PD-L1. Quantitative measurements reflect mean fluorescence index. LNCaP does not express PD-L1 at rest or in response to IFN-gamma. DU145 expresses PD-L1 at rest and has little response to IFN-gamma indicating an innate immune resistance phenotype. PC3 expresses PD-L1 at rest but robustly up-regulates this expression in response to IFN-gamma displaying a more classic adaptive immune resistance phenotype.

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Experimental details: FIGURE 2 Human Prostate Cancer Cell Lines Display No Change in PD-L1 Expression in Response to Bicalutamide Histograms representing PD-L1 surface expression as detected by flow cytometry in human prostate cancer cell lines with and without exposure to bicalutamide. Open histograms represent cells stained with an isotype IgG1 antibody tagged with a matching PE fluorochrome. Shaded histograms represent PD-L1. Quantitative measurements reflect mean fluorescence index. LNCaP, DU145, and PC3 displayed no changes in PD-L1 expression upon exposure to bicalutamide.

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Experimental details: FIGURE 4 Loss of PTEN Does Not Correlate with PD-L1 Expression in Resected Primary Prostate Carcinomas PD-L1 and PTEN staining in tumor lesions from two different patients. A. Case number 8 showing focally positive PD-L1 area. Arrow indicates positive tumor cell staining which is predominantly on the plasma membrane. B, similar region from adjacent section of case number 8 showing intact PTEN staining in all tumor cells (arrow shows a group of tumor cells with intact PTEN staining). C, case number 9 showing negative staining for PDL1 in all tumor cells (arrow shows tumor cells without PD-L1 staining). D, adjacent region showing PTEN loss in nearly all of the tumor cells (arrow shows tumor cells with negative PTEN staining; arrowhead shows stromal element with strongly positive PTEN staining). All images taken at 100 X original magnification.

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Experimental details: Figure 7 IL-27 induces PD-L1 and/or IDO expression in human PC3 prostate and A549 lung cancer cells and adherent PBMC A. Western blot analysis of IDO expression in human adherent PBMC, non-adherent PBMC and in PC3 and A549 cells treated with the indicated cytokines or medium only for 48 hours. alpha-tubulin is used as loading control. Similar results were observed in two additional experiments. B. QRT-PCR analysis of IDO1 and PDL1 mRNA expression in cytokine-stimulated adherent or non-adherent PBMC, PC3 and A549 cells relative to untreated cells. Data are expressed as DeltaDeltaCT-fold change. Mean values of three independent experiments. Error bars represent SD. C. FACS analysis of surface PD-L1 in IL-27-treated or untreated PC3 and A549 cell lines and adherent PBMC. Dotted lines are isotype-matched unrelated Ig staining controls. Similar results were obtained in five different experiments (PC3: MFI 5.5 +- 2.7 vs 1.5 +- 0.6, mean +- SD in IL-27-treated vs un-stimulated cells, P = 0.03 by paired Student's t test; A549: MFI 2 +- 1 vs 0.43 +- 0.4, P = 0.01).

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Experimental details: Figure 4 Expression of PD-L1 on PDLCs improves survival of PDLCs. Flow cytometry histrograms of (A) PDLCs, (B) PHA-activated PBMCs, (C) PDLCs co-cultured with activated PBMCs, (D) PDLCs pretreated with TNF-alpha and co-cultured with activated PBMCs, and (E) PDLCs pretreated with TNF-alpha, and incubated with activated PBMCs and anti-PD-L1 antibodies. (F) Comparison of PDLC survival, according to the percentages of CFSE + /PI - cells. a, c, d and e represent the PDLC control, untreated control, TNF-alpha induced and TNF-alpha induced+anti-PD-L1 groups, respectively. Data are expressed as the mean +- standard error of the mean of three independent experiments. Co-culturing the activated PMBCs with untreated PDLCs resulted in a significant decrease in viable PDLCs ( * P

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Experimental details: Figure 5 Expression of PD-L1 is correlated with the severity of periodontitis in the experimental periodontitis model. (A) Flow cytometry histograms of the expression of PD-L1 on the surface of periodontal tissue cells from healthy mice, and mice with mild periodontitis and severe periodontitis. (B) Expression of PD-L1 in periodontal tissues from the three groups, detected by immunohistochemical staining (magnification, x400). (C) Comparison of the expression of PDL-1 in periodontal tissues between the three groups ( ++ P

Supportive validation

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Experimental details: Figure 5 RRV-RSV-yCD2-miRPDL1 and RRV-RSV-yCD2-U6-miRPDL1 Express yCD2 Protein and Exhibit PDL1 Downregulation Activity (A) Replication kinetics of RRV-RSV-yCD2-miRPDL1 and RRV-RSV-yCD2-U6-miRPDL1. The viral genome in the supernatants of infected LN-18 cells (MOI of 0.1) at indicated time points were quantified by qRT-PCR using primer set targeted to the env region ( Figure 1 ). A paired t test was performed and showed no statistically significant difference in replication kinetics between RRV-RSV-yCD2-miRPDL1 versus RRV-RSV-yCD2 (p = 0.0649) and RRV-RSV-yCD2-U6-miRPDL1 (p = 0.0801). RRV-yCD2, RRV-RSV-yCD2, and RRV-miRPDL1 (indicated as U6-miRPDL1 in the graph) were included as positive controls. (B) Vector stability of RRV-RSV-yCD2-miRPDL1 and RRV-RSV-yCD2-U6-miRPDL1 in LN-18 cells was analyzed by endpoint PCR at 14 days post infection. Lane 1: DNA molecular marker (1 Kb Plus marker, Invitrogen); lanes 2, 4, and 6 are positive controls using the corresponding plasmid DNA as the templates; lane 3: RRV-RSV-miRPDL1; lane 5: RRV-RSV-yCD2-miRPDL1; and lane 7: RRV-RSV-yCD2-U6-miRPDL1. The arrows indicate the expected size of the PCR products (844 bp for RRV-RSV-miRPDL1; 1,326 bp for RRV-RSV-yCD2-miRPDL1; and 1,591 bp for RRV-RSV-yCD2-U6-miRPDL1). (C) yCD2 protein expression in LN-18 cell infected with RRV-yCD2, RRV-RSV-yCD2, RRV-RSV-yCD2-miRPDL1, RRV-RSV-yCD2-U6-miRPDL1, and naive cells. GAPDH is included as loading control. The numbers shown on the bottom of the immunoblot indica

Supportive validation

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Experimental details: Figure 2 Interferon gamma responsiveness in mouse and human NSCLC cell lines. In A-C, cultured cell lines (CMT167, LLC, EA1, EA2) were treated with vehicle or 100 ng/mL IFNgamma for 48 hours, and analyzed by flow cytometry for cell surface expression of (A) PD-L1, (B) MHC I, or (C) MHC II. Data for PD-L1 and MHC I are defined by median fluorescent intensity (MFI) of all cells, given the uniform induction of these proteins ( online supplementary figure 1 ). MHC II expression is defined as the frequency of positive cells, based on MHC II induction in a subset of cells ( online supplementary figure 2 ). Statistical analysis in (A, B) defined between vehicle and IFNgamma treated samples using an unpaired T-test (statistical significance identified by *p

Supportive validation

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Experimental details: Figure 6 SHP-1 Can Replace SHP-2 for PD-1 Coinhibition in Jurkat T Cells (A) Immunoblot analysis of equal amounts of proteins from total lysates of Raji (WT), Raji PD-L1, Raji SHP-2 - , and Raji PD-L1 SHP-2 - cells probed with antibodies to SHP-1 (right panel), SHP-2 (left panel), or VAV1 (loading control). Left margin, molecular size in kilodaltons. Data are representative of two independent experiments. (B) Raji, Raji PD-L1, Raji SHP-2 - , and Raji PD-L1 SHP-2 - cells were analyzed using flow cytometry for expression of HLA-DR, PD-L1, PD-L2, CD80, CD86, and HVEM. Gray shaded curves correspond to isotype-matched control antibody (negative control), and data are representative of two independent experiments. (C) Jurkat-PD-1 OST and Jurkat-PD-1 OST SHP-2 - cells were stimulated with Raji SHP-2 - cells or Raji PD-L1 SHP-2 - cells that have been preincubated in the absence (-) or presence (+) of SEE and lysed for 2 min after the initial contact. Immunoblot analysis of equal amounts (90%) of lysates from the specified conditions subjected to affinity purification (AP) on Strep-Tactin-Sepharose beads, followed by elution of proteins with D-biotin, and probed with antibody to anti-SHP-1, anti-SHP-2, and phosphorylated proteins (Anti-p-Tyr). Also shown is immunoblot analysis of equal amounts (10%) of total lysates of the specified cells probed with anti-PD-1 antibody (loading control). Left margin, molecular size in kilodaltons (kDa). Data are representative of two independent exper

Supportive validation

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Experimental details: Figure 2 PD-1 and PD-L1 were upregulated on T cells and monocytes in septic shock patients . Blood samples were obtained from 19 septic shock patients and 22 healthy controls and were stained for programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) gated on CD4 + T cells, CD8 + T cells, and CD14 + monocytes. (a) to (c) Percentage of PD-1 expression on (a) CD4 + T cells and (b) CD8 + T cells, and (c) percentage of PD-L1 expression on CD14 + monocytes. Each dot represents one individual. Data are mean +- standard error of the mean (SEM) of three independent experiments. ** P < 0.01 compared with healthy controls. (d) to (f) Mean fluorescence intensity (relative fluorescence units) of PD-1 expression on (d) CD4 + T cells, (e) PD-1 expression on CD8 + T cells, and (f) PD-L1 expression on CD14 + monocytes Each dot represents one individual. Data are mean +- SEM of three independent experiments. * P < 0.05 compared with healthy controls. (g) Representative PD-1 expression levels on CD4 + T cells and CD8 + T cells, and PD-L1 expression on CD14 + monocytes. Values in the upper-right quadrant indicate the percentage of cells that express PD-1 or PD-L1.

Supportive validation

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Experimental details: Figure 1 Effects of IFN-gamma on PD-L1 and signaling pathways. (A) PD-L1 expression was measured by flow cytometry in the cell lines at 48 h following treatment with DMSO, which was used as a vehicle control, 50 u M PD98059 (MAPK inhibitor), 1 u M wortmannin (PI3K-AKT inhibitor), 1 u M lapatinib (combined epidermal growth factor receptor/human epidermal growth factor receptor 2 tyrosine kinase inhibitor), and 10 ng/ml IFN-gamma. Error bars represent the means +- SEM. ** P

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Experimental details: Figure 3 PKD3 overexpression enhances IFN-gamma-induced PD-L1 expression in DOK cells. (A and B) The levels of PKD3 and PD-L1 protein were examined by western blot analysis and immunofluorescence. The PKD3 overexpression plasmid was transfected into DOK cells using Lipofectamine 2000. (C and D) Cells transfected with the PKD3 overexpression plasmid and cells transfected with control shRNA were cultured in the presence or absence of IFN-gamma (20 ng/ml) for 24 h. The expression levels of PKD3 and PD-L1 were determined by western blot analysis. In addition, the cell surface expression of PD-L1 was analyzed by flow cytometry. Data are presented as the means +- SD (n=3). *** P

Supportive validation

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Experimental details: Figure 3 Example flow cytometry plots demonstrating gating for PD-L1 positive cells THP-1 cells were (A and C) treated with IFN-gamma (50 ng/mL, approximate EC20 of PD-L1 activation), (B) left untreated, or (D) treated with IFN-gamma and a compound which we have identified that increases PD-L1 expression (unpublished data). Cells represented in (A) were not stained with the PE conjugated anti-PD-L1 antibody, whereas (B, C and D) were. All four groups of cells were otherwise stained (including fixable viability dye) and analyzed by flow cytometry as described in this protocol. All plots depict viable cells, gated as seen in Figure 2 .

Supportive validation

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Experimental details: Figure 5 Representative control and compound treated wells Flow cytometry histogram plots showing PD-L1 expression (PE fluorescence signal) for wells treated with either No IFN-gamma, IFN-gamma and vehicle, or IFN-gamma and one of two compounds from a screening library which were identified as modulating PD-L1 expression.

Supportive validation

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Experimental details: Fig. 3 Apigenin and curcumin suppress IFN-gamma-induced PD-L1 expression in human melanoma cells. a - c indicated melanoma cells were pretreated with DMSO, curcumin (25 muM), or apigenin (30 muM) for 4 h and then treated with IFN-gamma (10 ng/ml) for 24 h. Cells were lysed and PD-L1 expression was detected by Western blotting using a PD-L1 antibody. GAPDH was used as a loading control. Column charts below show quantitation data of relative PD-L1 expression. Error bars represent the mean +- S.D. ( n = 3, * P < 0.05 and ** P < 0.01). d and e cells were treated as described above and processed for immunofluorescence analysis. Representative micrographs show PD-L1 staining from different groups with same intensity settings on a fluorescent microscope (Olympus, Japan). DAPI stains nucleus. Scale bar = 25 mum. f and g , A375 and A2058 cells were treated as described above, and cell surface PD-L1 expression was determined by flow cytometry

Supportive validation

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Experimental details: Figure 3 Phorbol 12-myristate 13-acetate (PMA) treatment induces PD-L1 (CD274) and PD-1 (CD279) expression in THP-1-differentiated macrophages. According to the experimental design shown in Figure 1A , immunostaining followed by flow cytometric histogram and dot-plot analysis showed the expression of PD-L1 (CD274) and PD-1 (CD279) in PMA-treated THP-1 cells ( A ) without or ( B ) with the gating of CD68 - CD11b - and CD68 + CD11b + cells. Representative data were selectively obtained from three individual experiments, and the percentage of positive cells is shown.

Supportive validation

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Experimental details: Figure 5 Different expression of PD-L1 (CD274) and PD-1 (CD279) in THP-1-differentiated macrophages. ( A ) In PMA-stimulated THP-1-differentiated M 0 macrophages, cells were then treated with LPS (1 mug/mL)/IFN-gamma (10 ng/mL) and IL-4 (25 ng/mL)/IL-13 (25 ng/mL) for polarization of M 1 and M 2 , respectively, in the absence and presence of neutralizing antibodies (5 mug/mL) against CD274 (alpha-PD-L1) and CD279 (alpha-PD-1) according to the experimental design. ( B ) For immunostaining, cells were stained with CD80 and CD209 for dissecting M 1 and M 2 , respectively. Immunostaining followed by flow cytometric histogram analysis showed the expression of CD274 and CD279 in these cells. ( C ) Furthermore, the expression of CD80 and CD209 in M 1 and M 2 without or with the blockade of CD274 and CD279 were shown. For all flow cytometric analysis, representative data were selectively obtained from three individual experiments, and the percentage of positive cells is shown. THP-1 (black); THP-1 M 0 (red); THP-1 M 1 (blue); THP-1 M 2 (green).

Supportive validation

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Experimental details: Figure 2 Correlation of PD-L1 cell surface amount with the transcript levels. The amount of PD-L1 on acute myeloid leukemia (AML) blast surface was determined using flow cytometry and expressed as the percentage of positive cells (left column) or as the mean fluorescence intensity (MFI) of the blast population (right column). The relative levels of v1 and v2 transcripts were measured by PCR and normalized to GAPDH.

Supportive validation

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Experimental details: Figure 5 Correlation of PD-L1 and PD-1 expression. PD-L1 expression on AML blasts and PD-1 expression on AML blasts ( a ) or on the autologous T-cells ( b ) was determined by flow cytometry. Left: positive cell fractions, right: mean fluorescence intensity (MFI).

Supportive validation

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Experimental details: 1 Fig Expression of programmed cell death ligand-1 (PD-L1) is higher in patients with active multiple myeloma (MM) as compared to those with complete remission (CR). ( A ) Quantified polymerase chain reaction analysis of PD-L1 gene expression and ( B ) flow cytometric analysis of PD-L1 protein expression in bone marrow mononuclear cells (BMMCs) from patients with active MM. PD, progressive disease.

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Experimental details: 5 Fig Ruxolitinib (RUX) reduces programmed cell death ligand-1 (PD-L1) expression in human multiple myeloma (MM) LAGlamda-1 cells. Flow cytometric analysis of percentage of PD-L1-postive cells in human MM xenograft LAGlamda-1 tumour cells treated with increasing concentrations of RUX for 2 or 7 days. All experiments were n = 3 (technical replicates).

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Experimental details: Figure 1 Azacytidine alters gene expression in NSCLC cell lines for multiple immune related pathways (A) Top panel: Gene Set Enrichment Analysis (GSEA) for pathways up-regulated by azacytidine. Normalized enrichment scores are plotted as a heat map. Bottom panel: boxplot showing degree of demethylation in each cell line, as measured by the difference in beta values between the AZA and mock-treated cells immediately after drug withdrawal and 7 days later. (B) FACS analysis shows increased level of cell surface PD-L1 after AZA treatment by day 10 in NSCLC lines H838 and H1299. (C) to (J) AZA-mediated expression changes at day 10 in key genes from pathways outlined in (A). Y axis = Ratio of expression values (log2) of AZA -treated vs. mock-treated cells; X-axis = gene names.

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Experimental details: Figure 3 TCR + PD-1 + 2D3 cells as a model assay for evaluation of involvement of PD-1 signaling in cell-mediated antigen-specific T-cell activation ( A - C ) WT1 (A, B) and gp100 (A, C) specific T-cell activation expressed as percentage viable CD8 + eGFP + PD-1 - and PD-1 + 2D3 cells (+- SEM) after 24 hours co-culture with different PD-L1 + stimulator cells. Neutralizing antibody against PD-1 (alphaPD-1; A, B; 15 ug/mL in WT1 model (A, B), 5 ug/mL in gp100 model (A)) or PD-L1 (alphaPD-L1; C) was added to cells 1 hour prior to co-culture to verify PD-1-mediated signaling, where indicated. (A) PD-1-dependent stimulating capacity of two differently generated peptide-pulsed mature monocyte-derived dendritic cells (WT1 (4 DC donors tested in two independent experiments); gp100 (4 DC donors in four independent experiments)). (B)Impact of induced PD-L1 expression on peptide-pulsed THP-1 leukemic cells on WT1-specific T-cell activation (6 replicate experiments). (C) gp100-specific PD-1 + T cell-activating capacity of peptide-pulsed wild-type or stably transduced PD-L1 + MCF-7 breast carcinoma cells (4 replicate experiments). Data information: in A, the horizontal line represents the median percentage eGFP expression ( n = 4). * P

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Experimental details: Figure 1. Wnt-induced beta-catenin activation results in PD-L1 up-regulation in tumor cells. (B, D, F, and G) Immunoblotting analyses were performed with the indicated antibodies. (E-G) Data represent the means +- SD of three independent experiments. (A) IHC staining of 20 diffuse astrocytoma (grade II), 20 anaplastic astrocytoma (grade III), and 50 GBM (grade IV) specimens was performed with the indicated antibodies. Representative images of IHC staining from the specimens are shown. Scale bar, 50 um. Red arrows point to CD8 + cells. (B) The indicated tumor cells were serum starved for 12 h and then stimulated with Wnt3A (20 ng/ml) for the indicated periods of time. Immunoblotting analyses were performed. (C) U87/EGFR cells were treated with or without Wnt3A (20 ng/ml) for 12 h. A cell surface analysis of PD-L1 protein was performed using a flow cytometer. (D) Serum-starved U87/EGFR cells were pretreated with or without actinomycin D (1 ug/ml) for 2 h and then stimulated with or without Wnt3A (20 ng/ml) for 12 h. (E) The indicated tumor cells were serum starved for 12 h and then stimulated with or without Wnt3A (20 ng/ml) for the indicated periods of time. Real-time PCR analyses were performed. *, P < 0.0001, on the basis of Student's t test. (F) U87/EGFR cells with stable expression of beta-catenin shRNA or a control shRNA were treated with or without Wnt3A (20 ug/ml) for 12 h. A real-time PCR analysis (top panel) and immunoblotting analyses (bottom panel) were performed. b

Supportive validation

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Experimental details: Extended Data Figure 5 TASL mirrors SLC15A4 requirement for TLR7/8 activation. ( a ) Fraction of R848-induced genes affected by SLC15A4 and TASL knockout, related to Fig. 2b . ( b ) Upset plot representing number of R848-induced genes commonly affected by indicated sgRNAs, related to Fig. 2b . ( c ) TASL gene expression levels in indicated THP1 cells, related to Fig. 2b . Bar graphs shown mean (n=3 biological replicates), error bars show 95 % confidence interval of mean. ( d ) Flow cytometry of PD-L1 surface expression in indicated unstimulated (ns) or R848-stimulated (5 mug/ml, 24h) THP1 cells. ( e ) Immunoblots of indicated THP1 DUAL cells. Lysates treated with PNGase F as indicated. ( f-i,k ) Indicated THP1 DUAL cells were (co-)treated for 24 hours with R848 (5 mug/ml), CL075 (5 mug/ml), ssRNA40/LyoVec(tm) (5 mug/ml) or inactive control ssRNA41/LyoVec(tm) (5 mug/ml), C12-iE-DAP (5 mug/ml), MDP (10 mug/ml), murabutide (10 mug/ml), Pam3CSK4 (0.1 mug/ml), Flagellin (0.1 mug/ml), cGAMP (3 mug/ml) or interferon beta (20 ng/ml). ( h ) CRISPR/Cas9 editing efficiency (%) estimated by TIDE. ( j ) Indicated THP1 DUAL cells were primed or not with interferon gamma (0.1 mug/ml) for 24h, washed and stimulated or not with MDP (10 mug/ml, 24h). ( f-k ) Supernatants were analyzed for ISRE and NF-kappaB reporter activity. Mean +- s.d. (n=3 biological replicates). ( l ) Relative mRNA expression of SLC15A4 , TASL or MYD88 in siRNA-transfected CD14 + monocytes in comparison to control (si CTR

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Experimental details: Fig. 4 NEK2 positively correlates and interacts with PD-L1 in pancreatic cancer. a Western blot analysis of NEK2 and PD-L1 in clinical pancreatic tissue samples from patients ( n = 20) (N: Normal pancreatic tissue; T: Pancreatic tumor tissue). b - d Representative images and statistical results of IHC staining of NEK2 and PD-L1 in a tissue microarray ( n = 156). e - g Cell lysates from SW1990, KPC, and CFPAC-1 separately analyzed by IP and Western blotting using the antibodies indicated. Representative image is shown n = 3 independent experiments. h GST-pull down assay of NEK2-His and GST-PD-L1 protein. Representative image is shown n = 3 independent experiments. i Representative images of individual immunofluorescence staining of NEK2 and PD-L1 interaction in KPC cells by Duolink assay. The red dots (NEK2/PD-L1 interaction) indicate their interaction. Representative image is shown n = 3 independent experiments. The Spearman correlations and p -values by Spearman's test are indicated in d .

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Experimental details: Figure 2 IL-27 upregulates PD-L1 molecule expression and release by MM cells. ( A ) Flow cytometry analysis of membrane PD-L1 expression in MSTO, MPP89, and IST-MES1 MM cell lines cultured with medium alone (baseline), IL-6, sIL-6R/IL-6 chimera, or IL-27 (induced). Dotted line shows isotype-matched Ig control. Numbers in brackets represent Median Fluorescence Intensity (MFI) values calculated as median anti-PD-L1 mAb minus median Ig control. Data are representative of two independent experiments yielding similar results. ( B ) qRT-PCR analysis of CD274 (PD-L1) mRNA expression in the same three MM cell lines stimulated with IL-6, sIL-6R/IL-6 chimera, IL-27, or IFN-gamma relative to untreated cells. Data are calculated with the DeltaDeltaCT method and expressed as fold change versus untreated control. Error bars represent SD of triplicates. LGALS3 (Galectin 3) and GBP1 (Guanylate Binding Protein 1) mRNA levels are shown, respectively, as negative and positive controls of IL-27 activity. ( C ) Evaluation by ELISA of soluble (s)PD-L1 release in the conditioned media of the indicated MM cells treated with IL-6, sIL-6R/IL-6 chimera, or IL-27. Data are expressed as a percent of untreated control and are the mean of two independent experiments, run in duplicates. Error bars represent the minimum and maximum values (* p < 0.001, Student's t -test versus untreated control).

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Experimental details: Figure 3 TLR2 depletion attenuates IFNgamma-mediated upregulation of PDL1 in UM-SCC-49 cells. ( A ) UM-SCC-49 cells were treated with siRNA as indicated, and knockdown of TLR2 and GAPDH RNA expression was tested by qPCR. ( B ) Cells were transfected with non-targeting (NT), GAPDH, or TLR2 siRNA for 24 h were then treated -/+ 10 ng/mL IFNgamma for 48 h, and protein expression was assessed by immunoblot as indicated. The uncropped Western blots have been shown in Figure S3 .

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Experimental details: Figure 7 AIM binds directly to PSA, and the ectopic (re)expression of AIM enhances the anticancer therapeutic effect of Pembrolizumab against PCa cells. 2D visualization of the auto-generated ( A ) CD5L-KLK3 interactome showing complicity in the immune landscape, macrophages, EMT and CSC regulation, ( B ) CD5L interactome, or KLK3 interactome. ( C ) Three-dimensional visualization of the direct complex formation by AIM/CD5L (surface model) and PSA/KLK3 (ribbon model). ( D ) Line graph showing the effect of CD5L_OE with or without 5 muM Pembrolizumab on PC3 cell proliferation. ( E ) Representative photomicrographs ( upper ) and histograms ( lower ) showing the effect of CD5L_OE with or without 5 muM Pembrolizumab on PC3 cell invasion. ( F ) Representative photomicrographs ( upper ) and quantitative chart ( lower ) showing the effect of CD5L_OE with or without 5 muM Pembrolizumab on the tumorsphere formation capability of PC3 cells. ( G ) Representative IHC photomicrographs ( upper ) and histograms ( lower ) of the immunoreactivity of AIM, PSA, and PDL1 in pembro_responders or pembro_non-responders from the TMU-SHH cohort. ( H ) Histograms showing the differential CD8 + T-cell count/field in pembro_responders or pembro_non-responders from the TMU-SHH cohort. WT, wild type; OE, overexpression; * p < 0.05; ** p < 0.01; *** p < 0.001.

Supportive validation

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Experimental details: Figure 1 Expression of PD-L1 in breast cancer cell lines and macrophages. (A) The representative western blot analysis of PD-L1 expression in triple-negative (MDA-MB-231, HCC-1806), ER-positive (MCF-7, ZR-75-1, T47D), and HER-2-positive (SKBR-3) breast cancer cell lines (upper panel). beta-actin was used as a loading control. The experiment was repeated three times. Bands were quantified by densitometry; the signal for PD-L1 band was normalized to the corresponding actin band (lower panel). (B) Representative density plots and histogram overlays illustrating PD-L1 expression (red) against a background from isotype control (gray) for MCF-7 (upper panel) and MDA-MB-231 (lower panel) breast cancer cell lines using flow cytometry. The staining was performed using an anti-PD-L1 antibody (cat. no. 12-5983-42, eBioscience, clone MIH1, dilution 1:100). Numbers on the density plots indicate the percentage of PD-L1-positive cells. The experiment was repeated at least three times. (C) PD-L1 expression in macrophage subpopulations (M0, M0+IFNgamma, M2) detected by immunocytohistochemistry assay using Cytation 1 Cell Imaging Multi-Mode Reader (BioTek, Agilent). PD-L1 positively stained cells were detected using an anti-PD-L1 antibody (clone MIH1, cat. no. 14-5983-82, eBioscience, dilution 1:100) and are marked in green; red shows CD206-positive cells (cat. no. AF2534, R&D Systems, dilution 1:100). The signal was developed using AF488-conjugated or AF647-conjugated secondary antibody, resp

Supportive validation

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Experimental details: Figure 5 Induction of PD-L1 expression on the target cells. (A) PD-L1 expression induced by supernatant from activated CAR T or CAR-NK-92 cells on cancer cells was assessed by flow cytometry. The control (only medium) and conditioned supernatants from the 24 hours coincubation cultures of control (unmodified) or PD-L1-CAR T/NK-92 cells with the target MDA-MB-231 cells were transferred onto the culture of MCF-7 and incubated for 48 hours. Next, PD-L1 surface presence was assessed using anti-PD-L1 antibody (clone MIH1) by flow cytometry. The experiment was repeated three times. (B) Representative images of different subpopulations of macrophages (M0, M0+IFNgamma, M2) stained for PD-L1 assessed by immunocytochemistry assay using Cytation 1 Cell Imaging Multi-Mode Reader (BioTek, Agilent). Macrophages were incubated with 10 ng/mL IFNgamma or IL-4 and IL-10 (for M2) for 5 days before staining (every 2-day fresh portion of cytokines was added). The control (only medium) and conditioned supernatants from the 24 hours coincubation cultures of control (unmodified) or PD-L1-CAR T cells with the target MDA-MB-231 cells were transferred onto different subpopulations of macrophages and incubated for 48 hours. Next, PD-L1 surface presence was assessed using anti-PD-L1 antibody (clone MIH1, cat. no. 14-5983-82, eBioscience, diluted 1:100). The signal was developed using AF488-conjugated secondary antibody (green), and nuclei were counterstained with DAPI (blue), scale bar: 100 um. The backg

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Experimental details: Figure 6 Expression of PD-L1 and PD-L1-CAR T mediated cytotoxicity in non-malignant cells. (A) IFNgamma induced expression of PD-L1 on HEK293T cell line derived from human embryonic kidney cells and non-malignant cells (HMEC, MCF-10A, and bone marrow-derived mesenchymal stem cells (BM-MSC)) assessed by flow cytometry as presented on the left panel. PD-L1 staining was performed using anti-PD-L1 antibody (clone MIH1). The representative western blot analysis of PD-L1 expression in human embryonic kidney HEK293T cells and non-malignant mammary epithelial HMEC and MCF10A cells, and BM-MSC (right panel). beta-actin was used as a loading control. The experiment was repeated three times. (B) PD-L1 expression induced on MCF-10A cells by activated CAR T cells (left panel) and RTCA-monitored cytotoxic activity of PD-L1 CAR T cells toward MCF-10A cells (right panel). The control (only medium) and conditioned supernatants from the 24 hours coincubation cultures in the presence of control (unmodified) T cells or PD-L1-CAR T cells with the target MDA-MB-231 cells were transferred onto the cultures of MCF-10A cell line and incubated for 48 hours. Next, PD-L1 surface presence was assessed by flow cytometry using anti-PD-L1 antibody (clone MIH1). Cytotoxic activity of PD-L1-CAR T cells against MCF-10A non-malignant cell line was measured by impedance analysis at the E:T ratios of 1:1 and 2:1. Samples were internally normalized for the cell index value measured before PD-L1-CAR T cells additio

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Experimental details: 10.1371/journal.pone.0124059.g007 Fig 7 Panels A-E show representative histograms for proliferative pattern of T lymphocytes (i.e., CD71+). Panel A shows T lymphocytes alone while panel B shows T lymphocytes co-cultured with antigen presenting cells (APCs). On the other hand, panel C shows the pattern for co-culturing of T lymphocytes with normoxic cardiac cells (CCs) while panels D and E show patterns for T lymphocyte co-cultured with ischemic-reperfused CCs in the absence and presence of the PD-L1 blocking antibody, respectively. Panel F shows the percent of proliferating T lymphocytes under each condition; data are average of the triplicate samples for each heart (n = 3 hearts/group/condition). IRI: Ischemia Reperfusion Injury. * p

Supportive validation

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Experimental details: Figure 5 IL-25 Induces PD-L1 Surface Expression on hMSCs and Human Monocytes (A) PD-L1 in siCtrl MSCs (left) and siPD-L1 MSCs (right) was analyzed by surface staining. (B) Freshly isolated human PBLs were co-cultured without (left) or with siCtrl MSCs (middle) or siPD-L1 MSCs (right) for 3 days, followed by PMA/ionomycin stimulation for 6 hr. IL-17A production in CD3 + T cells was assessed by intracellular staining. Representative data are shown with numbers in the top right quadrants representing the percentages of IL-17A-producing CD3 + T cells. (C) Pooled data from PBLs (n = 4) and two hMSC donors (donors A and B) are shown. (D) Folds of reversed phenotypes of siIL-25 and siPD-L1 are shown. (E) PD-L1 expression on siCtrl hMSCs (left) and siIL-25 hMSCs (right) was assessed by cell surface staining. Filled histograms represent isotype control; unfilled histograms represent PD-L1 antibody staining. (F) Pooled data of PD-L1 expression (indicated by fold change in MFI) on siIL-25 hMSCs and siPD-L1 hMSCs (all three donors) are shown. PD-L1 expression levels were compared between hMSCs silenced for the target gene (IL-25 or PD-L1) and the respective siCtrl. (G) hMSCs were treated with the indicated doses of rhIL-25 for 18 hr and assessed for cell surface PD-L1 expression by cell surface staining. Pooled data (all three donors) are shown in chart to the right with bars representing MFI. (H) Human PBLs were treated with the indicated doses of rhIL-25 for 18 hr and assessed for cell

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Experimental details: Figure 7 Blockade of APC-Intrinsic PD-1 Enhances the Synaptic Enrichment of T Cell-Intrinsic PD-1 and Inhibits T Cell-Mediated Cytotoxicity (A) Left: cartoon showing a Jurkat cell expressing PD-1-mGFP (shown as PD-1 in green) conjugated with a Raji cell co-transduced with PD-L1-mCherry and unlabeled PD-1. Right: representative confocal images of the conjugate at the indicated channel acquired 2 min after cell-cell contact. Scale bars, 10 um. (B-D) Same as (A), except that Raji (PD-L1+/PD-1+) cells (B), Jurkat (PD-1-mGFP) cells (C), or both (D) were preincubated with pembrolizumab and washed extensively prior to conjugation. Scale bars, 10 um. (E) Bar graph comparing the interface enrichment indices (calculated as described in STAR Methods) of the four conditions shown in (A)-(D). Data are expressed as mean +- SEM; n = 40 cells from three independent experiments. (F) Cartoon illustrating the cytotoxicity assay, in which EL4 cells were used as the target for OT-I cytotoxic T cells. (G) Bar graph summarizing the OT-I cytotoxicity under indicated conditions, with PD-1 blockade antibody J43 or isotype antibody (Iso) preincubated with neither cell, only EL4, only OT-I, or both cell types. See STAR Methods for details. Cytotoxicity was normalized to the no blockade antibody condition (i.e., EL4 and OT-I preincubated with the isotype control). n.s., not significantly different. Data are presented as mean +- SEM from three independent replicates. (H) Left: FACS histograms showing PD-1

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Experimental details: FIGURE 2 Expression of PD-L1 on human HCC PLC/PRF/5 cells. (A) Human HCC PLC/PRF/5 cells were cultured alone in the absence of GPC3-CAR T cells in RPMI 1640 medium containing 10% FBS. (B) Human HCC PLC/PRF/5 cells were cocultured with GPC3-CAR T cells at an effector:Target ratio of 1:1 for 18 h in RPMI 1640 medium containing 10% FBS. PD-L1 was determined by flow cytometry in the CD3-negative gate, and the fixable, viable stain 780 was used for discriminating live from dead cells.

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Experimental details: Figure 5 The activities of the tested molecules in the cell-based in vitro immune checkpoint blockade assay. ( A ) Western blot analysis of the expression of the human PD-L1 protein in the hPD-L1 aAPCs (artificial antigen-presenting cells, CHO-K1 TCRact/PD-L1) in comparison to the original CHO-K1 cells (CHO-K1 wt) and CHO-K1 overexpressing only the TCR Activator molecule (CHO-K1 TCRact). ( B ) Flow cytometry analysis of the expression of human PD-L1 on the surface of the hPD-L1 aAPCs. ( C ) Comparison of bioactivities of the known PD-L1-targeting molecules: nivolumab, peptide-57 , BMS-1166 , and the tested molecules, CA-170 and AUNP-12 in the hPD-1/hPD-L1 immune checkpoint assay.

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Experimental details: Figure 3 Detection of scFv PD-L1 binding to PD-L1 by competitive ELISA Wells in a 96-well microtiter plate were coated with ( A ) recombinant human or ( B ) mouse PD-L1-Fc followed by co-incubation with His-tagged recombinant PD-1-Fc in competition with 25, 50 or 100 uL of supernatant of undefined scFv PD-L1 (scFv) and scFvFc PD-L1 (scFvFc) protein concentration collected from CT26 cells maximally infected with RRV-scFv-PDL1 and RRV-scFvFc-PDL1, respectively. Anti-human or mouse PD-L1 antibody was included as positive control (indicated as alpha-hPD-L1 and alpha-mPD-L1). Anti- His tag antibody was used to detect bound His-tagged PD-1-Fc. Optical density was measured at 450 nm. The percentage of inhibition was calculated with respect to the supernatant from CT26 maximally infected with RRV-GFP (non-scFv-PD-L1) used in the competition. Error bars indicate the standard deviation of the dataset. ( C ) Wells were coated with recombinant human PDL1-Fc followed by co-incubation with His-tagged recombinant PD-1-Fc in competition with purified scFv PD-L1 or anti-human PD-L1 antibody range from 0.01-100 nM. IC50 values were determined using best-fit values from non-linear three-parameters logistic.

Supportive validation

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Experimental details: Figure 3 HPV-positive HNSCCs increase PD-L1 and PD-L2 on fibroblasts. PD-L1 and PD-L2 expression on primary BJ human fibroblasts, HPV-positive (SCC154), and HPV-negative (SCC099) HNSCC cell lines (HNSCCs) was detected by flow cytometry. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts (black histograms), HPV-positive (red histograms), or HPV-negative (blue histograms) HNSCCs (A) . Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 5) in fibroblasts, HPV-positive, and HPV-negative HNSCCs (B) . Fibroblasts were cultured alone or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs. Fibroblasts were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 ( C ; red histograms) or HPV-negative SCC099 ( D ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 5) on fibroblasts cultured alone (w/o) or co-cultured directly with HNSCC cells (E) . HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with fibroblasts. HNSCCs were identified in co-cultures by EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured directly with fibroblasts ( F ; red histograms). Graphs show PD-L1 and PD-

Supportive validation

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Experimental details: Figure 4 PD-L1 and PD-L2 expression in co-cultures of macrophages and HPV-positive or HPV-negative HNSCCs. Macrophages were cultured alone (w/o) or co-cultured in direct contact (direct) with HPV-positive (SCC154) or HPV-negative (SCC099) HNSCC cell lines (HNSCCs). Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone ( A ; black histograms). Macrophages were identified in co-cultures by lack of EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on macrophages cultured alone (black histograms) or co-cultured directly with HPV-positive SCC154 ( B ; red histograms) or HPV-negative SCC099 ( C ; blue histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 4) on macrophages cultured alone (w/o) or co-cultured directly with HNSCCs (D) . HPV-positive (SCC154) or HPV-negative (SCC099) HNSCCs were cultured alone or co-cultured in direct contact (direct) with macrophages. HNSCCs were identified in co-cultures by EpCAM expression. Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured directly with macrophages ( E ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 3) on HPV-positive SCC154 cultured alone or co-cultured directly with macrophages (F) . Illustrative histograms show PD-L1 and PD-L2 expression on HPV-negative SCC099 cultured alone (black histograms) or co-cultured directly with macrophages ( G ; blue hist

Supportive validation

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Experimental details: Figure 5 Conditioned medium from HPV-positive HNSCCs up-regulates PD-L1 and PD-L2 on fibroblasts. Fibroblasts were cultured alone or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or co-cultured with conditioned medium from HPV-positive SCC154 ( A ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 13) on fibroblasts cultured alone (w/o), co-cultured directly with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM) (B) . HPV-positive (SCC154) HNSCCs were cultured alone or co-cultured in direct contact with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM). Illustrative histograms show PD-L1 and PD-L2 expression on HPV-positive SCC154 cultured alone (black histograms) or co-cultured with conditioned medium from fibroblasts ( C ; red histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 4) on HPV-positive SCC154 cultured alone (w/o), co-cultured directly with fibroblasts (Fibro direct) or with conditioned medium from fibroblasts (Fibro CM) (D) . Macrophages were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (direct) or with conditioned medium from HPV-positive SCC154 (CM). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 3) on macrophages for the in

Supportive validation

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Experimental details: Figure 6 Blockade of IFN-gamma, TNF-alpha, or CD81 does not affect PD-L1 and PD-L2 up-regulation by HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) as indicated. Graphs (A) show IFN-gamma and TNF-alpha levels in culture supernatants (mean +- SEM; n = 4). The dashed red line indicates the lowest value (15.6 pg/ml) of the dynamic range for the ELISA assays used. Neutralizing antibodies anti-IFN-gamma (B,C) , anti-TNF-alpha (D,E) , or anti-CD81 (F,G) were added to the cultures as indicated. Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 or with conditioned medium from HPV-positive SCC154 alone (red histograms) or in the presence of blocking antibodies (green histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 3) on fibroblasts for the indicated treatments. ns, not significant (one-way ANOVA with Bonferroni correction for multiple comparisons) Numbers adjacent to plots represent MFI values; dashed histograms show control staining with isotype-matched antibodies. MFI, mean fluorescence intensity.

Supportive validation

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Experimental details: Figure 7 The TLR9 antagonists ODN TTAGGG and chloroquine inhibit PD-1 ligands up-regulation on fibroblasts co-cultured with HPV-positive HNSCCs. Fibroblasts were cultured alone (w/o) or co-cultured in direct contact with HPV-positive SCC154 (SCC154 direct) or with conditioned medium from HPV-positive SCC154 (SCC154 CM) in the presence or absence of the TLR9 antagonists ODN TTAGGG (ODN) or chloroquine (CHQ). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), co-cultured directly with HPV-positive SCC154 (red histograms) or co-cultured directly with HPV-positive SCC154 in the presence of ODN (A) or CHQ (H) (green histograms). Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms), cultured with conditioned medium from HPV-positive SCC154 (red histograms) or with conditioned medium from HPV-positive SCC154 in the presence of ODN (B) or CHQ (I) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 6) on fibroblasts for the indicated treatments (C,J) . Illustrative histograms show PD-L1 and PD-L2 expression on fibroblasts cultured alone (black histograms) or in the presence of ODN (D) or CHQ (K) (green histograms). Graphs show PD-L1 and PD-L2 expression (mean +- SEM; n = 4) on fibroblasts for the indicated treatments (E,L) . HPV-positive SCC154 were cultured alone or co-cultured in direct contact (direct) with fibroblasts in the presence

Supportive validation

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Experimental details: Figure 5. Exosomal PD-L1 Suppresses Tumor Progression in Syngeneic Colorectal Cancer Model (A) Western for PD-L1 in WT and Rab27a MC38 100k g extracellular fraction. (B) Flow cytometry for surface PD-L1 on MC38 cells. (C) Cell counts over time for Rab27a versus WT MC38 cells. n = 3. Error bars represent SD. (D) Cell counts over time for Pd-l1 versus WT MC38 cells. n = 3. Error bars represent SD. (E) Tumor growth over time following subcutaneous injection of 1 x 10 6 WT, Rab27a , or Pd-l1 MC38 cells into immunocompetent B6 mice. n = 5 for each genotype. Error bars represent SEM. MC38 WT versus MC38 Rab27a , p < 0.05. MC38 WT versus MC38 Pd-l1 , p < 0.05. MC38 WT versus MC38 Pd-l1 ; Rab27a , p < 0.05 (two-way ANOVA test). (F) Mouse survival curve following injection of cells like in (D). n = 10 for each genotype. MC38 WT versus MC38 Rab27a , p < 0.001. MC38 WT versus MC38 Pd-l1 , p < 0.001. MC38 WT versus MC38 Pd-l1 ; Rab27a , p < 0.001 (log rank test). (G) Survival curve for mice injected with WT, Rab27a , or Pd-l1 MC38 cells followed by treatment with either anti-PD-L1 or isotype control antibody. n = 5 for each condition. MC38 WT isotype versus MC38 WT anti-PD-L1, p < 0.01. MC38 Rab27a isotype versus MC38 Rab27a anti-PD-L1, p < 0.05. MC38 Pd-l1 isotype versus MC38 Rab27a anti-PD-L1, ns. (log rank test). See also Figure S7 .