Anti pd 1 clone eh12.2h7

Manufactured by BioLegend

Sourced in United States

The Anti-PD-1 (clone EH12.2H7) is a lab equipment product offered by BioLegend. It is a monoclonal antibody that binds to the PD-1 (Programmed Cell Death 1) protein.

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Close spelling variants of this product

Anti-PD-1-APC (clone EH12.2H7, PE-labeled anti-PD-1 (Clone EH12.2H7) Anti-PD-1 BV421 (clone EH12.2H7), Anti-PD-1 antibody (clone EH12.2H7) Anti-human PD-1 (clone EH12.2H7, PD-1 (clone EH12.2H7), Anti-PD-1 (EH12.2H7, PD-1 (EH12.2H7, Clone EH12.2H7 PD-1 (EH12.1)

9 protocols using anti pd 1 clone eh12.2h7

Evaluating Immune Checkpoint Expression

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Mouse spleen cells were washed with PBS containing 1% BSA (staining buffer), blocked with mouse IgG in staining buffer, then stained with conjugated antibodies. Antibodies used were anti-PD-1 (clone 29F.1A12, Biolegend, San Diego, CA), anti-TIGIT (clone 1G9, Biolegend), anti-GITR (clone yGITR765, Biolegend), anti-CD25 (clone PC61, Biolegend), anti-PD-L1 (clone 10F.9G2, Biolegend), anti-CTLA4 (clone UC10-4B9, Biolegend), anti-2B4 (clone M2B4(B6)458.1, Biolegend), anti-VISTA (clone MIH63, Biolegend), anti-LAG3 (clone C9B7W, Biolegend), anti-TIM3 (clone RMT3-23, Biolegend), and anti-CD4 (clone RM4-5, Biolegend).
Human PBMCs were stained with anti-CD4 (clone OKT4, Biolegend), anti-CD25 (clone BC96, Biolegend), anti-FoxP3 (clone 236A/E7, Biolegend), anti-PD-1 (clone EH12.2H7, Biolegend), anti-TIGIT (clone A15153G, Biolegend). Prior to anti-FoxP3 staining, the cells were fixed for 18 hours and permeabilized.

Muhammad F., Wang D., McDonald T., Walsh M., Drenen K., Montieth A., Foster C.S, & Lee D.J. (2020). TIGIT+ A2Ar-Dependent Anti-Uveitic Treg Cells are a Novel Subset of Tregs Associated with Resolution of Autoimmune Uveitis. Journal of autoimmunity, 111, 102441.

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Modulation of CD4+ T Cell Activation by ox-LDL

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CD4⁺T cells from HCs were stimulated with different concentrations of ox-LDL (0, 1, 10, 50, and 100 µg/ml) for 48 h. Some CD4⁺T cells were also cultured with a plate-bound anti-CD3 antibody (OKT-3; 5 μg/ml) plus anti-CD28 antibody (28.2; 1 μg/ml) to get activated.
For Tim-3- and PD-1-targeting experiments, arterial CD4⁺T cells were cultured (5 × 10⁵ per well) in the presence of 10 μg/ml anti-Tim-3 (clone F38-2E2; BioLegend San Diego, CA, USA) and anti-PD-1 (clone EH12.2H7, BioLegend San Diego, CA, USA), or isotype control. After 48 h, the culture supernatants were collected and further analyzed by flow cytometry (FCM).
For intracellular cytokine analysis, brefeldin A (a Golgi inhibitor) (10 mg/ml, Biolegend, San Diego, CA, USA) was used for 4 h (at the end of the culture) to block the secretion of cytokines into the media after the activation of cells by phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, 12 h) and ionomycin (1 μg/ml, 12 h). Cells were harvested and analyzed by FCM for intracellular cytokine production.

Cui L., Chen L., Dai Y., Ou J., Qiu M, & Wang S. (2022). Increased Level of Tim-3+PD-1+CD4+T Cells With Altered Function Might Be Associated With Lower Extremity Arteriosclerosis Obliterans. Frontiers in Immunology, 13, 871362.

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Immune Checkpoint Blockade Modulation

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PBMCs were stimulated with PFA-fixed E. coli in the presence of anti–PD-1 (clone EH12.2H7; BioLegend), anti–LAG-3 (clone 17B4; Abcam), anti-IFNAR2 antibody (clone MMHAR-2; PBL Assay Science), or purified anti–IL-10R antibody (clone 3F9; BioLegend) at 10 μg/ml. Mouse IgG2a κ and rat IgG2a κ isotype control antibodies were used as controls.

Tang X., Zhang S., Peng Q., Ling L., Shi H., Liu Y., Cheng L., Xu L., Cheng L., Chakrabarti L.A., Chen Z., Wang H, & Zhang Z. (2020). Sustained IFN-I stimulation impairs MAIT cell responses to bacteria by inducing IL-10 during chronic HIV-1 infection. Science Advances, 6(8), eaaz0374.

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IVS Assays for Immune Checkpoint Inhibition

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IVS assays were performed as described previously (8 (link)). Briefly, PBMCs were stimulated for 6 d in the presence of 10 μg/ml anti-IL-10R and/or anti-PD-1 (clone EH12.2H7, Biolegend) blocking mAb or isotype control antibodies prior to intracellular cytokine staining. Alternatively, cells were harvested and then surface stained with APC-labeled A2/NY-ESO-1 tetramers, and subsequently with CD8-PECy7 and Annexin V-FITC (ApoScreen Annexin V-FITC Apoptosis Kit, Beckman Coulter).

Sun Z., Fourcade J., Pagliano O., Chauvin J.M., Sander C., Kirkwood J.M, & Zarour H.M. (2015). IL-10 and PD-1 cooperate to limit the activity of tumor-specific CD8+ T cells. Cancer research, 75(8), 1635-1644.

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CFSE Proliferation Assay for Tumor-Infiltrating T Cells

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Brain tumor tissue was prepared as for bulk RNA-seq. Fresh or thawed dissociated tumor cells (1 × 10⁶ cells per ml) were stained with 1 µl ml^–1 CFSE (Thermo Fisher Scientific, C34554) in protein-free medium. The cells were stained for 20 min in the dark at RT. Following this incubation period, the remaining CFSE was quenched using complete medium. CFSE-labeled cells (1 × 10⁵) were plated on a flat-bottom, 96-well plate in 100 µl of complete medium. Where indicated, 40 µg ml^–1 of the blocking anti-PD1 (clone EH12.2H7, BioLegend, 329926) or isotype control (BioLegend, 401401) was added. The cells were incubated at 37 °C and 5% CO₂ for 96 h. Afterward, cells were stained with anti-CD45, anti-CD11B and anti-CD3 (ref. ^{27 (link)}), and CFSE dilution in T cells was recorded. All antibodies used in this study are summarized in Supplementary Table 5a. Samples were acquired using a BD LSR Fortessa II, and data analysis was performed with FlowJo software version 10.8.1 (FlowJo).

Wischnewski V., Maas R.R., Aruffo P.G., Soukup K., Galletti G., Kornete M., Galland S., Fournier N., Lilja J., Wirapati P., Lourenco J., Scarpa A., Daniel R.T., Hottinger A.F., Brouland J.P., Losurdo A., Voulaz E., Alloisio M., Hegi M.E., Lugli E, & Joyce J.A. (2023). Phenotypic diversity of T cells in human primary and metastatic brain tumors revealed by multiomic interrogation. Nature Cancer, 4(6), 908-924.

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Multiparameter Flow Cytometry Analysis of T Cell Subsets

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The following mouse anti-human monoclonal antibodies (MAb) were used: anti-PD1/clone EH12.2H7 (BioLegend Inc., San Diego, CA, USA), anti-2B4/clone C1.7 (Beckman Coulter, Fullerton, CA, USA) as wells as anti-Tim3/clone 344823, and anti-human IFNγ/clone 25723 (R&D Systems, Minneapolis, MN, USA). Anti-CD14/clone M5E2, anti-CD19/clone 5J25C1, anti-CD56/clone B159, anti-CD107b/clone H4B4, anti-TNF/clone MAb11, anti-CD8/clone SK1, and mouse IgG isotype controls were all obtained from BD Pharmingen (Becton Dickinson, Heidelberg, Germany). Tetramer and antibody stainings were performed as detailed previously (19 (link)). Cells were acquired using a BD FACSCanto II flow cytometer (Becton Dickinson, Heidelberg, Germany) and analyzed with FlowJo Software (TreeStar Inc., San Diego, CA, USA). A multimer-specific CD8⁺ T cell population was considered detectable if the frequency was at least 0.1% of total CD8⁺ T cells. Exclusion of unspecific events was done using a dump channel (CD14⁺CD19⁺CD56⁺). Respective FMO controls were included to enable gating of cell populations.

Owusu Sekyere S., Suneetha P.V., Hardtke S., Falk C.S., Hengst J., Manns M.P., Cornberg M., Wedemeyer H, & Schlaphoff V. (2015). Type I Interferon Elevates Co-Regulatory Receptor Expression on CMV- and EBV-Specific CD8 T Cells in Chronic Hepatitis C. Frontiers in Immunology, 6, 270.

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Multiparametric Flow Cytometry for Immune Profiling

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Single cell suspensions isolated from PB, SN and tumour were stained for surface and intracellular markers for flow cytometry analysis. Briefly, cells were stained with fixable live/dead dye (Life Technologies), followed by surface marker staining using anti‐CD8 (clone RPA‐T8; BD Biosciences), anti‐CD56 (clone B159; BD Biosciences), anti‐CD103 (clone Ber‐ACT8; BioLegend), anti‐CCR7 (clone 150503; BD Biosciences), anti‐CD45RA (clone HI100; BD Biosciences) and anti‐PD‐1 (clone EH12.2H7; BioLegend) antibodies. The cells were then fixated and permeabilized using the forkhead box P3 (FOXP3) transcription factor kit (eBioscience, San Diego, CA, USA). Next, the cells were stained for intracellular marker using: anti‐perforin (clone δG9; BD Biosciences), anti‐granzyme B (clone GB11; BD Biosciences), anti‐T‐bet (clone 4B10; BioLegend) and anti‐Ki‐67 (clone 20Raj1; eBioscience) antibodies. Isotype control was used to ascertain the correct gating for the following markers: perforin, granzyme B, T‐bet, Ki‐67 and PD‐1. Flow cytometry data were acquired using an LSR Fortessa instrument (BD Biosciences) and analysed with FlowJo version 10 (TreeStar, Inc., Ashland, OR, USA).

Hartana C.A., Ahlén Bergman E., Broomé A., Berglund S., Johansson M., Alamdari F., Jakubczyk T., Huge Y., Aljabery F., Palmqvist K., Holmström B., Glise H., Riklund K., Sherif A, & Winqvist O. (2018). Tissue‐resident memory T cells are epigenetically cytotoxic with signs of exhaustion in human urinary bladder cancer. Clinical and Experimental Immunology, 194(1), 39-53.

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Comprehensive Immune Cell Profiling

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The following monoclonal antibodies and reagents were used: for surface staining, anti-CD25 (clone M-A251), anti-CD45RA (clone 5H9), anti-CXCR3 (clone 1C6/XCXR3), anti-CCR6 (clone 11A9) and anti-CTLA-4 (clone BNI3) from BD Biosciences, anti-CD127 (clone 17-1278) from eBioscience, anti-TIM-3 (clones F38-2E2 and 344823 from Biolegend and R&D systems respectively), anti-PD-1 (clone EH12.2H7) from Biolegend, anti-LAG-3 (polyclonal) from R&D systems and Live/Dead fixable dead cell staining kit from Invitrogen; for intracellular staining, anti-FoxP3 (clone PCH101) from eBioscience and anti-pSTAT3 (pY705) from BD Biosciences.

Gautron A.S., Dominguez-Villar M., de Marcken M, & Hafler D.A. (2014). Enhanced Suppressor Function of TIM-3+ FoxP3+ Regulatory T cells. European journal of immunology, 44(9), 2703-2711.

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PD-1 and PD-L1 Expression Analysis

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PD-1 and PD-L1 expression was detected with biotin-coupled anti–PD-1 (clone EH12.2H7, BioLegend) and anti-PD-L1 (clone 29E.2A3, BioLegend) antibody staining followed by streptavidin-allophycocyanin (APC) (BioLegend) staining. CD3 surface expression was detected by APC-conjugated anti-CD3ε antibody (clone OKT3, BioLegend). Cells were analyzed by flow cytometry using a Gallios flow cytometer (Beckman Coulter). Cell sorting was performed using a BD FACS Aria III cell sorter (Becton Dickinson). Data were analyzed using the software FlowJo v10 (TreeStar Inc.).

Marasco M., Berteotti A., Weyershaeuser J., Thorausch N., Sikorska J., Krausze J., Brandt H.J., Kirkpatrick J., Rios P., Schamel W.W., Köhn M, & Carlomagno T. (2020). Molecular mechanism of SHP2 activation by PD-1 stimulation. Science Advances, 6(5), eaay4458.

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