12-5983-42

antibody from Invitrogen Antibodies

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

Provider product page for 12-5983-42

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References [72]
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Flow cytometry [1]
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Product number: 12-5983-42 - Provider product page
Provider: Invitrogen Antibodies
Product name: CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH1), PE, 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.
Conjugate: Yellow dye
Antibody clone number: MIH1
Concentration: 5 µL/Test

CD274 protein structure - 12-5983-42 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: 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 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 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 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
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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
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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.
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Experimental details: Figure 5 Genetic targeting of PD-L1 expression on cancer cells reverses the CD8 + TIL dysfunction and suppresses metastasis ( a ) Cell surface expression of PD-L1 on 344SQ PD-L1 knockdown (344SQ-shPD-L1) vs 344SQ scramble control (344SQ-scr) cells by FACS (red line, isotype control staining; blue line, anti-PD-L1 staining). The measurement was independently repeated at least three times. ( b ) Primary tumor mass (top left) and lung metastases (top right) in 129/Sv mice (n = 10) injected subcutaneously with 344SQ-shPD-L1 or 344SQ-scr cancer cells. Micrometastases (bottom) observed in hematoxylin and eosin-stained lung tissue sections are indicated by yellow arrows. Scale bar, 2mm. Samples were obtained 6 weeks post-injection. The data from two independent experiments were pooled. Data are shown as mean +- s.e.m. t -test was used to analyze. P values are shown in the graphs. ( c , d ) FACS analysis of ( c ) surface PD1, LAG3, and TIM3 marker expression levels on CD8 + T cells; ( d ) CD8 + TIL frequency for primary tumors in 129/Sv mice (n = 5) injected subcutaneously with 344SQ-shPD-L1 (shPD-L1) or 344SQ-scr control (Control) cancer cells and necropsied 2 weeks later. The analyses were independently repeated three times. Data are shown as mean +- s.e.m. t -test was used to analyze. P values are shown in the graphs.
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Supportive validation

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Experimental details: Figure 7 PD-L1 expression on tumor cells is critical to the repression of anti-tumor immunity ( a ) The knockdown (KD) efficiency of PD-L1 in LLC-JSP murine lung cancer cells measured by FACS. Representative histograms (left), and statistical analysis (right). The measurement was independently repeated at least three times. ( b ) Representative FACS histogram of PD-L1 expression on myeloid cells (CD11b + ) in PD-L1 KO or WT mice (n = 3). ( c ) Tumor growth in PD-L1 KO or WT mice (n = 6) of subcutaneously injected LLC-JSP cells (10, 000 cells with 100 mul of PBS per mouse) with differing PD-L1 knockdown (Vector, PD-L1 KD vector control; PD-L1 int , PD-L1 intermediate KD; PD-L1 hi , PD-L1 high level KD). The data is shown from two independent experiments. Data are shown as mean +- s.e.m. ( d ) FACS analysis of CD8 + TIL frequency and T cell exhaustion marker expression levels on CD8 + T cells in subcutaneous primary tumors of LLC-JSP vector control (Vector ctrl) and LLC-JSP intermediate PD-L1 knockdown (Intermediate KD) in PD-L1 WT (WT) and PD-L1 KO (KO) mice (n = 5, from two independent experiments). Analysis was done 3 weeks post-tumor cell injection (20, 000 cells with 100 mul of PBS per mouse). t -test was used to analyze the data. p < 0.0001. ( e ) PD-L1 expression levels on LLC-JSP-shPD-L1 cells (the high knockdown efficiency cells) after reconstitution of PD-L1. The measurement was independently repeated at least three times. Control, LLC-JSP-shPD-L1 + vector control; PD
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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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