Formalin-fixed, paraffin-embedded (FFPE) tumor biopsies (3 biopsies at distant sites per patient) were sectioned at a thickness of 3 μm and stained on positively charged glass slides. Deparaffinization, rehydration, and antigen retrieval were performed by CC1 (prediluted; pH 8.0) antigen retrieval solution (Ventana Medical Systems, Inc.) on the Ventana BenchMark ULTRA automated slide stainer for 32 min at 100 °C. Specimens were incubated with primary antibodies anti-CD3 (clone LN10; Leica; dilution 1:100), anti-CD8 (clone C8/144B; Dako, dilution 1:25), and anti-CD20 (clone L26; Diagomics; dilution 1:150) followed by visualization with the Ultraview DAB IHC Detection Kit. The specimens were then counterstained with hematoxylin II and bluing reagent (Ventana) and coverslipped. Each IHC run contained a positive control.
Anti cd3 clone ln10
Manufactured by Leica
Sourced in Denmark, United Kingdom, Germany
The Anti-CD3 (clone LN10) is a monoclonal antibody used in laboratory settings for the detection and analysis of CD3-positive cells. It binds specifically to the CD3 complex, which is expressed on the surface of mature T cells and is a key component of the T cell receptor complex. This antibody can be utilized in various immunological techniques, such as flow cytometry, to identify and characterize T cell populations.
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Lab products found in correlation
Anti cd8 clone c8 144b, by Agilent Technologies (3 mentions)
Antigen retrieval solution, by Roche (1 mentions)
Hematoxylin 2 and bluing reagent, by Roche (1 mentions)
Benchmark ultra, by Roche (1 mentions)
Pd l1 clone e1l3n, by Cell Signaling Technology (1 mentions)
Mlh1 clone es05, by Leica (1 mentions)
Pms2 clone m0r4g, by Leica (1 mentions)
Ventana benchmark, by Roche (1 mentions)
Ki 67 clone mib 1, by Agilent Technologies (1 mentions)
Cd68 clone kp1, by Agilent Technologies (1 mentions)
Foxp3 clone 236a e7, by Abcam (1 mentions)
Cd8 clone 4b11, by Leica (1 mentions)
Cd20 clone l26, by Leica (1 mentions)
Nkp46 clone 195314, by R&D Systems (1 mentions)
Msh2 clone fe11, by Agilent Technologies (1 mentions)
Y5200, by Agilent Technologies (1 mentions)
Ndp view2, by Hamamatsu Photonics (1 mentions)
Anti pd l1 clone e1l3n, by Cell Signaling Technology (1 mentions)
Dako autostainer universal staining system, by Agilent Technologies (1 mentions)
Chemmate dako envision detection kit, by Agilent Technologies (1 mentions)
8 protocols using anti cd3 clone ln10
1
Multiplex IHC for Tumor Immune Profiling
2
Evaluating Immunologic Response and PD-L1 Expression
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Potential local immunologic response after treatment was evaluated. Analysis of PD-L1 expression was performed to identify potential targets for anticancer treatment. Serial sections of FFPE blocks of 4 µm were cut. The first slide was stained with hematoxylin and eosin (HE). Immunohistochemical staining was performed using anti-PD-L1 clone 22C3 (Agilent/Dako, Glostrup, Denmark, cat# M3653), anti-CD3 clone LN10 (Leica/Triolab AS, Broendby, Denmark, cat# NCL-L-CD3–565) and anti- CD8 clone C8 /144B (Agilent/Dako, cat# GA623). All staining was performed as double-labelling with anti-Cytokeratin clone BS5 (Nordic Biosite Aps, Copenhagen K, Demmark, cat# BSH-7124–1) on the automated instrument Omnis (Agilent/Dako), and details of protocols are outlined in
Supplementary 1 . For PD-L1, a previously published detailed protocol was followed
16 (link)
.
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Potential local immunologic response after treatment was evaluated. Analysis of PD-L1 expression was performed to identify potential targets for anticancer treatment. Serial sections of FFPE blocks of 4 µm were cut. The first slide was stained with hematoxylin and eosin (HE). Immunohistochemical staining was performed using anti-PD-L1 clone 22C3 (Agilent/Dako, Glostrup, Denmark, cat# M3653), anti-CD3 clone LN10 (Leica/Triolab AS, Broendby, Denmark, cat# NCL-L-CD3–565) and anti- CD8 clone C8 /144B (Agilent/Dako, cat# GA623). All staining was performed as double-labelling with anti-Cytokeratin clone BS5 (Nordic Biosite Aps, Copenhagen K, Demmark, cat# BSH-7124–1) on the automated instrument Omnis (Agilent/Dako), and details of protocols are outlined in
16 (link)
.
3
Comprehensive Immunophenotyping of FFPE Samples
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FFPE blocks were obtained from the Pathology Department of Tokyo Metropolitan Bokutoh Hospital. Immunohistochemistry was performed using the Ventana BenchMark automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) with labeled streptavidin–biotin and visualized with 3,3′-diaminobenzidine. The primary antibodies used were anti-CD3 (clone LN10, Leica), -CD4 (clone SP35, Ventana), -CD8 (clone 4B11, Leica), -CD45RO (clone UCHL-1, Ventana), -FOXP3 (clone 236A/E7, Abcam), -CD20 (clone L26, Leica), -NKp46 (clone #195314, R&D), -CD68 (clone Kp-1, Dako), -CD163 (clone 10D6, Leica), -CD204 (clone SRA-E5, Transgenic), -Ki-67 (clone MIB-1, Dako), -PD-L1 (clone E1L3N, Cell Signaling), -MLH1 (clone ES05, Leica), -MSH2 (clone FE11, Dako), -MSH6 (clone Polyclonal (Rabbit), GeneTex) and -PMS2 (clone M0R4G, Leica). EBV-encoded small RNA in situ hybridization (EBER-ISH) was performed on paraffin sections using a fluorescein isothiocyanate (FITC)-labeled peptide nucleic acid probe (Y5200; Dako, Glostrup, Denmark) and anti-FITC antibody (V0403, Dako). Slides were digitized with a Nanozoomer 2.0-HT virtual slide scanner (Hamamatsu Photonics, Hamamatsu, Japan) and observed in the NDP.view2 software (Hamamatsu Photonics). The density of immune cells was analyzed by Tissue Studio 2.0 software (Definiens, Munich, Germany).
4
Immunohistochemical Analysis of PD-L1 and CD3
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Immunohistochemistry was performed on cases with tissue available for further analysis. The following antibodies were used: anti-PD-L1 (clone E1L3N, Cell Signaling Technologies) and anti-CD3 (clone LN10, Leica Biosystems).
5
Glioma Molecular Profiling and TILs Analysis
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We obtained a total of 1,149 glioma cases of the brain (619 GBM cases and 530 low-grade glioma [LGG] cases) with known mRNA expression data from TCGA database (https://gdc.cancer.gov/about-data/publications/pancanatlas and https://www.cbioportal.org/ ) [12 (link)]. Normal samples as well as tumor samples with missing data were excluded from analysis. The analysis was finally performed on 525 cases with both virtual histopathological slides and clinical data (from a total of 619 GBM samples). We present the raw data of our study in Supplementary Data 1.
Immunohistochemical staining was performed to evaluate the presence or absence of tumor-infiltrating lymphocytes (TILs) in GBM human tissue diagnosed at Hanyang University Guri Hospital. Haematoxylin and eosin (H and E)-stained slides were reviewed by at least two pathologists for each case (Min and Kim). In non-necrotic tissue with TILs, immunostaining for anti-CD3 (clone LN10 Leica Biosystems, Newcastle, UK), anti-CD8 (clone 4B11 Leica Biosystems, Newcastle, UK) and anti-CD4 (clone 4B12 Leica Biosystems was performed using the Dako Autostainer Universal Staining System (DakoCytomation, Carpinteria, CA, USA) and the ChemMate™ Dako EnVision™ Detection Kit.
Immunohistochemical staining was performed to evaluate the presence or absence of tumor-infiltrating lymphocytes (TILs) in GBM human tissue diagnosed at Hanyang University Guri Hospital. Haematoxylin and eosin (H and E)-stained slides were reviewed by at least two pathologists for each case (Min and Kim). In non-necrotic tissue with TILs, immunostaining for anti-CD3 (clone LN10 Leica Biosystems, Newcastle, UK), anti-CD8 (clone 4B11 Leica Biosystems, Newcastle, UK) and anti-CD4 (clone 4B12 Leica Biosystems was performed using the Dako Autostainer Universal Staining System (DakoCytomation, Carpinteria, CA, USA) and the ChemMate™ Dako EnVision™ Detection Kit.
6
Duplex CD3/CD19 IHC Assay for Melanoma
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A duplex CD3/CD19 IHC assay was developed. CD3 and CD19 were selected as pan–T-cell and B-cell markers to assess both immature and mature cellular patterns. Duplex CD3/CD19 IHC staining (n = 59) using anti-CD3 (clone LN10) and anti-CD19 (clone BT51E; both of Leica Biosystems) was performed on the Leica Bond Rx autostainer. Slides were scanned at 20× magnification using Aperio AT2, Leica digital whole-slide scanner. Digital image analysis (IA) was performed using the multiplex IHC module, HALO v2.3 software platform (Indica Labs). Analysis enabled clear distinction of melanoma tumor nests (or tumor compartment) and intratumoral and peritumoral stroma (or stroma compartment) and excluded lymphoid tissue (Supplementary Fig. S1); melanoma lesions were annotated by a pathologist in collaboration with imaging scientists. IA was performed blinded to clinical information to avoid unintentional bias. A specific IA algorithm was developed to quantitatively assess the percentage of CD3- and CD19-positive cells within melanoma tumor and stroma compartments. To evaluate the extent of direct T-cell/B-cell interactions, proximity analysis between CD3- and CD19-positive cells was performed with the spatial analysis module using a 10-μm radial distance setting to assess close T-cell/B-cell interactions in tumor and stroma classified regions, respectively.
7
Quantifying CD3+ Lymphocytes in Paraffin-Embedded Tissues
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Formalin-fixed tissues embedded in paraffin were mounted and de-paraffinized according to standard laboratory procedure. The number of individuals in whom paraffin tissues were available; au-CC: 20, rb-CC: 18, HC: 16, ref-CC: 14. Consecutive slides were stained with anti-CD3 (clone LN10, 1/200 dilution) (Leica Biosystems, Wetzlar, Germany) while haematoxylin was used as a counterstain. The Nikon E800 microscope (Nikon instruments Inc. Tokyo, Japan), equipped with a x 40 objective lens, and connected to the software NIS elements (Nikon instruments Inc. Tokyo, Japan) was used to acquire digital photos. The ImageJ programme (https://imagej.nih.gov/ ) was used in order to manually quantify the numbers of CD3+ lymphocytes in a minimum of eight unique fields of vision (Supplementary Image 1
8
Immune Checkpoint and Infiltration Analysis in CLL and RS
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Expression of the immune checkpoint molecules in CLL and RS lymph node samples was analyzed by IHC staining using antibodies specific for PD-L1 and PD1. Immune cell infiltration in CLL and RS lymph node samples was assessed by IHC staining using antibodies specific for CD3, CD8, FOXP3, and CD163. IHC staining was done on 4-μm FFPE sections on DAKO Autostainer Plus (Agilent, Santa Clara, CA) using standard protocol17 (link),20 (link). The antibodies used include anti-PD-L1 (clone SP263, Ventana Medical Systems, Inc., Tucson, AZ), anti-PD1 (clone NAT105, Abcam, Inc., Cambridge, MA), anti-CD3 (clone LN10, Leica Biosystems, Newcastle Upon Tyne, UK), anti-CD8 (clone C8/144B, Dako, Carpenteria, CA), anti-FOXP3 (clone 236A/E7, Abcam, Inc., Cambridge, MA), and anti-CD163 (clone10D6, Leica Biosystems, Newcastle Upon Tyne, UK). IHC images were taken using whole slide imaging technology with MoticEasyScan Pro (Motic digital pathology, San Francisco, CA), and saved in tagged image file format. Percentage of expression for each individual antigen was calculated by dividing number of cells with positive staining by number of total cells in the image using the Image-Pro premier 3D 9.1.4 software (Media Cybernetics, Silver Spring, MD).
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