16-5983-82

antibody from Invitrogen Antibodies

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

Provider product page for 16-5983-82

Flow cytometry

Blocking/Neutralizing

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Antibody data

Antibody Data
Antigen structure
References [68]
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Validations
Flow cytometry [2]
Other assay [43]

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Product number: 16-5983-82 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH1), Functional Grade, eBioscience™
Antibody type: Monoclonal
Antigen: Other
Description: Description: The MIH1 monoclonal antibody reacts with human B7-H1, also known as PD-L1. B7-H1, a member of the B7 family, has a predicted molecular weight of approximately 40 kDa and belongs to the Ig superfamily. B7-H1 is expressed on a majority of leukocytes. B7-H1 is a ligand for PD-1. Interaction of PD-1 with either PD-L1 (B7-H1) or PD-L2 (B7-DC) results in inhibition of T and B cell responses. MIH1 is reported to be a blocking antibody. Applications Reported: The MIH1 antibody has been reported for use in flow cytometric analysis. It has also been reported in blocking in in vitro functional assays. Applications Tested: The MIH1 antibody has been tested by flow cytometric analysis of normal human peripheral blood cells. This can be used at less than or equal to 1 µg per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test. It is recommended that the antibody be carefully titrated for optimal performance in the assay of interest. Storage and handling: Use in a sterile environment. Filtration: 0.2 µm post-manufacturing filtered. Purity: Greater than 90%, as determined by SDS-PAGE. Endotoxin Level: Less than 0.001 ng/µg antibody, as determined by LAL assay. Aggregation: Less than 10%, as determined by HPLC.
Reactivity: Human
Host: Mouse
Isotype: IgG
Antibody clone number: MIH1
Vial size: 100 μg
Concentration: 1 mg/mL
Storage: 4°C

CD274 protein structure - 16-5983-82 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.

Supportive validation

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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.

Supportive validation

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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 .

Supportive validation

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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.

Supportive validation

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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.

Supportive validation

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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).

Supportive validation

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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

Supportive validation

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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 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 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

Supportive validation

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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

Supportive validation

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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.