Three different, previously characterized, and well-validated antibodies (E1L3N, SP263, and 28-8) were used in addition to the MIH1 antibody. The E1L3N clone (Cat# 13684, Cell Signaling Technology, Danvers, MA, United States) was chosen due to its higher reported sensitivity compared with other clones [15 (link)]. On the other hand, SP263 (Cat# 07494190001, Ventana, Oro Valley, AZ, United States) was chosen as the standard anti-PD-L1 antibody used by Ventana BenchMark autostainer, available in our pathology department. On the other hand, the 28-8 clone (Cat# ab205921, Abcam, Cambridge, United Kingdom) is a companion test used to detect PD-L1 for the therapeutic anti-PD-L1 Nivolumab® (Bristol Meyers Squibb, BMS, New York, NY, USA). The MIH1 (Cat# 14-5983-82, eBioscience, San Diego, CA, USA) was the primary antibody used in our previous studies [6 (link),7 (link)].
Clone 28 8
Manufactured by Abcam
Sourced in United Kingdom
Clone 28-8 is a monoclonal antibody targeting the CD28 receptor. It is commonly used in research applications for the detection and study of the CD28 protein.
Automatically generated - may contain errors
Lab products found in correlation
Clone e1l3n, by Cell Signaling Technology (2 mentions)
Sp263, by Roche (1 mentions)
Ventana benchmark xt, by Roche (1 mentions)
Bond max, by Leica (1 mentions)
Dako autostainer link 48, by Agilent Technologies (1 mentions)
E1l3n, by Cell Signaling Technology (1 mentions)
Tris borat edta, by Roche (1 mentions)
Ventana benchmark ultra, by Roche (1 mentions)
Clone sp263, by Roche (1 mentions)
Zli 9017, by OriGene (1 mentions)
Pv 6001, by OriGene (1 mentions)
Cd45 efluor450, by Thermo Fisher Scientific (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Pg m1, by Agilent Technologies (1 mentions)
Histofine sab po kit, by Nichirei Biosciences (1 mentions)
Envision plus system, by Agilent Technologies (1 mentions)
High ph target retrieval solution, by Agilent Technologies (1 mentions)
Lumiglo reagent, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
10 protocols using clone 28 8
1
Comparative Assessment of PD-L1 Antibody Clones
2
Immunohistochemical Scoring of PD-L1 Expression
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IHC was performed according to IVD protocols for the 22C3 Ab on the Dako Autostainer Link 48 (Agilent, Santa Clara, CA), and the SP142 and SP263 clones on the Ventana BenchMark XT (Roche Ventana, Tuscon, AZ) (Supplementary Table 1 ). The remaining assays were conducted as LDTs. IHC using the 28-8 clone (concentration 1:500; Abcam, Cambridge, UK) and E1L3N (1:200; Cell Signaling Technologies, Beverly, MA) was performed on the BOND-MAX autostainer (Leica, Wetzlar, Germany). Positive and negative controls were included and consisted of placental tissue and sections stained without primary Ab, respectively.
IHC stains were independently scored by three pathologists (BRA, MCH, SH), according to vendors’ recommendations. Tumor staining was scored according to the extent of cell staining (0%, 1%, 5%, 10%, and then nearest 5% from 11-100%) at each level of staining intensity from 0-3 (0, none; 1, partial; 2, compete and faint; 3 complete and strong). IC were scored as previously described [10 (link), 22 (link)], specifically as IC0 (<1% of IC staining at any intensity), IC1 (1-4%), IC2 (5-9%), and IC3 (≥10%). Raw scores were then converted to comply with vendor specifications. To facilitate analysis, the scores from a single pathologist (BRA) were used, following confirmation of an adequate agreement in scores between pathologists (Cohen's kappa test, p<0.05).
IHC stains were independently scored by three pathologists (BRA, MCH, SH), according to vendors’ recommendations. Tumor staining was scored according to the extent of cell staining (0%, 1%, 5%, 10%, and then nearest 5% from 11-100%) at each level of staining intensity from 0-3 (0, none; 1, partial; 2, compete and faint; 3 complete and strong). IC were scored as previously described [10 (link), 22 (link)], specifically as IC0 (<1% of IC staining at any intensity), IC1 (1-4%), IC2 (5-9%), and IC3 (≥10%). Raw scores were then converted to comply with vendor specifications. To facilitate analysis, the scores from a single pathologist (BRA) were used, following confirmation of an adequate agreement in scores between pathologists (Cohen's kappa test, p<0.05).
3
Immunohistochemical Evaluation of PD-L1
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PD-L1 expression analysis was performed using three different antibodies against PD-L1 (clone 28–8 (Abcam plc, Cambridge, UK), clone SP142 (Linaris GmbH, Dossenheim, Germany), and clone SP263 (Roche AG, Rotkreuz, Switzerland)). In brief, 3 μm sections of the TMA were deparaffinized, pre-treated with an antigen retrieval buffer (Tris/Borat/EDTA, pH 8.4; Ventana, Roche) and stained using an automated device (Ventana Benchmark Ultra, Roche). Dilutions were as follows: 1:100 for antibody 28–8, 1:25 for antibody SP142, and a ready-to-use kit for antibody SP263. Tumor cells and surrounding tumor stroma, including inflammatory infiltrates, were scored separately. The number of cells showing membranous staining was evaluated in percentage. According to the German consensus recommendations for immunohistochemical evaluation of PD-L1, any positivity was defined as ≥1% of positive cells having at least weak membranous staining [10 (link)]. Tumor cells with pure cytoplasmic staining were scored negatively. Figures were created using Inkscape (v.0.91, Free Software Foundation, Inc., Boston, USA) and R (www.r-project.org , v.3.2.5, Free Software Foundation).
4
Immunohistochemical Analysis of PD-L1 Expression
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Representative 4 μm slices were prepared from FFPE tissue blocks. In short, all tissue slices were exposed to 3% hydrogen peroxide (OriGene; PV-6001) for 10 mins to inhibit the activity of endogenous peroxidase, and heat-mediated antigen retrieval was performed through Tris/EDTA buffer with 8 pH (OriGene; ZLI-9066). Afterwards, 200 μL PD-L1 antibody (Abcam, clone 28–8, monoclonal, 1:400, anti-rabbit) and tissue slices were incubated in a wet box at 4°C overnight, and then HRP-coupled goat anti-rabbit IgG secondary body (OriGene, PV-6001) was added. After addition, they were placed at 37°C for 30 mins, and incubated with 3, 3-diamino benzidine (DAB) (OriGene; ZLI-9017) at indoor temperature for 5 m. In the end, the slices were dried out and immobilized. Negative (no primary antibody) and positive (amniotic membrane roll) controls were adopted in each running. The staining results were interpreted by professional pathologists according to antibody specification. The specific positive PD-L1 was located in cell membrane and cytoplasm. Tumor cells with positive expression rate of PD-L1 ≥1％ were considered as positive tumor cells, and recorded as TC1, and those with the rate <1％ were considered as negative tumor cells, and recorded as TC0.20 (link)
5
PD-L1 Expression Analysis in Tumor Xenografts
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Immunofluorescence staining was performed on SW1990 cells transfected with siMTHFD2 and PD-L1 plasmid. Cells were scratched from the culture dish and washed with PBS. Each sample was incubated with PD-L1 antibody (abcam, clone28-8) for 30 min on ice in dark. The immunofluorescence of PD-L1 in SW1990 was analyzed by flow cytometry.
Xenografts from C57BL/6J mice were removed and minced into 1 mm3 pieces in the solution of 0.2% collagenase IV at 37 °C for 4 h with mild shake. Cells dissociated from the tissues were sieved through a 40-μm mesh and diluted to 106 cells/mL in PBS. Samples were incubated with CD45 eFluor 450 (eBioscience) for 30 min in dark. Cells were acquired in a FACS Verse flow cytometer (BD), and data were analyzed using FlowJo software (Tree Star).
Xenografts from C57BL/6J mice were removed and minced into 1 mm3 pieces in the solution of 0.2% collagenase IV at 37 °C for 4 h with mild shake. Cells dissociated from the tissues were sieved through a 40-μm mesh and diluted to 106 cells/mL in PBS. Samples were incubated with CD45 eFluor 450 (eBioscience) for 30 min in dark. Cells were acquired in a FACS Verse flow cytometer (BD), and data were analyzed using FlowJo software (Tree Star).
6
IHC Analysis of PD-L1 and Immune Cells
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Sections of resected specimens were fixed in 10 per cent buffered formalin, embedded in paraffin, and stained using the peroxidase‐labelled streptavidin–biotin technique with the Histofine® SAB‐PO kit (Nichirei Biosciences, Tokyo, Japan)14 . The primary antibody used was an antihuman PD‐L1 rabbit monoclonal antibody (clone 28‐8, dilution 1 : 450; Abcam, Cambridge, UK). Tumour cells showed membranous staining for PD‐L1, which was evaluated as positive staining. The proportion of PD‐L1‐positive tumour cells was estimated as the percentage of total tumour cells. Because the distribution of PD‐L1‐positive cells was focal, positivity for PD‐L1 was defined as more than 1 per cent of tumour cells stained for PD‐L115 . Sections from human placenta were used as positive controls. Immunohistochemical (IHC) staining was performed using anti‐CD8 (C8/144B; DAKO, Carpinteria, California, USA) and anti‐CD68 (PG‐M1, DAKO) antibodies. Serial sections were treated before incubation with primary antibodies in a microwave oven for 20 min, reacted with the antibodies. The numbers of cells with cytoplasm or membrane staining in five high‐power fields (HPFs) were counted. The CD8+/CD68+ cell ratio was calculated from the median numbers of CD8+ and CD68+ cells.
7
Immunohistochemical Staining of Breast Tumors
8
Immunohistochemical Staining of PD-L1
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The rabbit monoclonal anti-human PD-L1 antibodies used were clone 28-8 (Abcam) and clone SP142 (Spring Bioscience). Heat-induced epitope retrieval with High pH Target Retrieval Solution (DAKO) was performed. Endogenous peroxidase activity was blocked by incubating in 0.3% hydrogen peroxidase and 0.1% sodium azide contained 0.01 M phosphate-buffered saline, and the EnVision plus system (DAKO) was used for secondary detection. The final product was visualized by 3-3′ diaminobenzidine.
9
Quantification of PD-L1 Protein Levels
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Cell pellets were lysed in a whole cell extract lysis (WCE) buffer that includes 1 M HEPES, 0.4 mM NaCl, 25% glycerol, 1 mM EDTA, 15 mM NaF, and 0.1% NP-40, supplemented with protease inhibitors (Thermo Scientific) and phosphatase inhibitors (Roche PhosSTOP) for 30 min at 4 °C. Cell lysates were assayed for protein concentration using the BCA protein assay (Thermo Fisher Scientific, USA). Whole cell extracts were subjected to 10% SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. Ponceau S staining was performed to validate efficient transfer. Membranes were placed together with the following primary antibodies: antihuman PD-L1 (1:100; clone 28-8, Abcam) and antihuman vinculin (1:1000; clone EPR 8185, Abcam). Binding of primary Abs was detected with HRP-conjugated antirabbit (clone Ab 205718, Abcam) (1:1000). LumiGLO reagent (Cell Signaling Technology) was used for enhanced chemiluminescence detection. The blots were visualized using a chemiluminescent western blotting detection system (ChemiDoc MP imaging system; Bio-Rad) and analyzed by Bio-Rad Image Lab software.
10
Comparative Analysis of PD-L1 Staining
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FFPE tissue specimens were collected from 315 patients, and three 5-μm consecutive sections were cut from each specimen. PD-L1 staining using three primary antibodies was compared: clone SP142 (1:100; Spring Bioscience Corp, rabbit IgG), clone 28–8 (1:300; Abcam, rabbit IgG) and clone E1L3N (1:200; Cell Signaling Technology Inc., rabbit IgG). PD-L1 antibody clone SP142 applied using the Ventana Benchmark platform and clones 28–8 and E1L3N were applied using the Leica Bond platform. Bond™ Epitope Retrieval ER2 Solution and ER1 Solution were used for E1L3N and 28–8 antigen retrieval, respectively. The normal tonsil tissue was used as a positive control and the isotype control was a rabbit IgG monoclonal antibody applied to sections.
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