Clone eh12.2h7

Manufactured by BioLegend

Sourced in United States

Clone EH12.2H7 is a monoclonal antibody produced by BioLegend. It is designed for use in flow cytometry applications.

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PE-labeled anti-PD-1 (Clone EH12.2H7) PD-1-BV421 (clone EH12.2H7) Anti-PD-1 BV421 (clone EH12.2H7), PD-1 APC (clone EH12.2H7; PD-1 BV605 (clone EH12.2H7; Anti-PD-1 antibody (clone EH12.2H7) FITC) (clone EH12.2H7, CD279 (clone EH12.2H7) CD279 (PD1; clone EH12.2H7; PD-1 (clone EH12.2H7),

5 protocols using clone eh12.2h7

Multiparameter Flow Cytometry Assay

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Flow cytometry was performed as described previously (6 (link)). Isolated cells were incubated with predetermined optimized concentrations of anti-CD3-Alexa fluor 700 (clone SP34-2, BD Pharmingen), anti-CD8-Pacific Blue or PE-CF594 (clone RPA-T8, BD Pharmingen), anti-CD20-APC-Cy7 (clone L27, BD Pharmingen), anti-CXCR5-phycoerythrin (PE) (Clone MU5UBEE, eBioscience), anti-CD200-APC (clone OX104, eBioscience), anti-CD4-PerCP (clone L200, BD Pharmingen), anti-CD95-PE-Cy5 (clone DX2, BD Pharmingen), anti-CCR7-APC-Cy7 (clone 3D12, Biolegend), anti-PD-1-PE-Cy7 (clone EH12.2H7, Biolegend), anti-ICOS-Pacific Blue (C398.4A, Biolegend) and Live/Dead dye-Alexa430 (Invitrogen). After staining the cell surface, cells were permeabilized and fixed by BD perm wash (BD Pharmingen) and washed twice. Finally, cells were incubated with anti-Ki67-FITC (B56, BD Pharmingen), washed twice and diluted in 1% PFA. Data were acquired on a LSRII flow cytometer (BD Biosciences) and the data analyzed using FlowJo software (version 9.2 Tree Star).

Hong J.J., Amancha P.K., Rogers K.A., Courtney C.L., Havenar-Daughton C., Crotty S., Ansari A.A, & Villinger F. (2014). Early lymphoid responses and germinal center formation correlates with lower viral load set points and better prognosis of SIV infection. Journal of immunology (Baltimore, Md. : 1950), 193(2), 797-806.

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Modulating CAR T Cell Cytotoxicity

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To assess the effect of drugs on CAR T cell cytotoxicity, cancer cells were plated at 5,000/well concentration in 96 well plates in triplicate. Next day, cells were treated with drugs (1-Methyl-d-tryptophan (1-MT, #452483, Sigma), indomethacin (#17378, Sigma), gemcitabine hydrochloride (#G6423, Sigma), paclitaxel (Taxol equivalent, #P3456, Invitrogen) and 5-Fluorouracil (#F6627, Sigma)) at indicated concentrations for 24 h. After 24 h, the drug was removed, cells were washed and further incubated with mock or CAR T cells at T:E ratio of 1:10 for 72 h. Thereafter, the percentage of surviving cancer cells were measured using the MTT assay. For Gal-9 and PD-1 blocking experiments, cancer cells were plated at 10,000 cells/well concentration in 96 well plates in triplicate. After 24 h, mock or CAR T cells were added at T:E ratio of 1:10. 0.1, 1, and 10 μg/mL of Gal-9 (clone 9M1-3, BioLegend, San Diego, CA, USA) or PD-1 blocking Ab (clone EH12.2H7, BioLegend) was added to the media and incubated at 37 °C for 72 h. Survival was measured using the MTT assay.

Yazdanifar M., Zhou R., Grover P., Williams C., Bose M., Moore L.J., Wu S.T., Maher J., Dreau D, & Mukherjee P. (2019). Overcoming Immunological Resistance Enhances the Efficacy of a Novel Anti-tMUC1-CAR T Cell Treatment against Pancreatic Ductal Adenocarcinoma. Cells, 8(9), 1070.

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Phenotypic Characterization of Activated T Cells

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PBMC were purified as indicated above and activated with TransAct™ (1:100, Miltenyi-Biotec 130-111-160) and recombinant human IL2 (100 IU/ml; PeproTech). Then at days 0 and days 2-4-6 post-stimulation, the cells were stained with Fixable Viability Stain 450 (250 ng/mL; BD Biosciences), next with anti-CD3-PE-Cy5 (1/30; clone HIT3a; BD Biosciences), anti-CD4-PE-Vio770 (1/100; clone rea623; Miltenyi Biotec), anti-CD8-APC-Cy7 (1/30; clone RPA-T8; Biolegend), anti-CD25-Alexa fluor 488 (1/30; clone M-A251; Biolegend), anti-CD45RA-APC (1/30; clone HI100; Biolegend), anti-CCR7-PE (1/50; clone G043H7; Biolegend), anti-Granzyme-B-PE (1/50; clone GB12; Invitrogen), anti-PD1-Alexa 488 (1/50; clone EH12-2H7; Biolegend) and anti-KLRG1-APC (1/100; clone Rea261; Miltenyi Biotec). FACS analyses were performed on a MACSQuant Analyzer (Miltenyi Biotec) and data analyzed using FlowJo 10 software (FlowJo, LLC). All samples were gated on forward and side scatter, for singlets and for live cells.

Cuche C., Mastrogiovanni M., Juzans M., Laude H., Ungeheuer M.N., Krentzel D., Gariboldi M.I., Scott-Algara D., Madec M., Goyard S., Floch C., Chauveau-Le Friec G., Lafaye P., Renaudat C., Le Bidan M., Micallef C., Schmutz S., Mella S., Novault S., Hasan M., Duffy D., Di Bartolo V, & Alcover A. (2023). T cell migration and effector function differences in familial adenomatous polyposis patients with APC gene mutations. Frontiers in Immunology, 14, 1163466.

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Validating Anti-PD-1 and Anti-PD-L1 Antibodies

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To confirm specificity of commercially acquired anti-PD-1 mAb, clone J43 (eBioscience, San Diego, CA, United States) and clone EH12.2H7 (BioLegend, San Diego, CA, United States), as well as anti-PD-L1 mAb, clone MIH5 (eBioscience) and clone 29E.2A3 (BioLegend), HEK293T cells transiently transfected with recombinant woodchuck PD-1 (wcPD-1) and woodchuck PD-L1 (wcPD-L1) were examined as additional test targets. For this purpose, sequences encoding wcPD-1 and wcPD-L1 were determined, expression vectors constructed using pCDNA 3.1(+), and HEK293T cells transfected using PolyJet DNA transfection reagent (SignaGen Laboratories LLC, Gaithersburg, MD, United States). Surface and intracellular expression of the transfected protein was determined by flow cytometry and compared to non-transfected cells and an isotype control.

Corkum C.P., Wiede L.L., Ruble C.L., Qiu J., Mulrooney-Cousins P.M., Steeves M.A., Watson D.E, & Michalak T.I. (2022). Identification of antibodies cross-reactive with woodchuck immune cells and activation of virus-specific and global cytotoxic T cell responses by anti-PD-1 and anti-PD-L1 in experimental chronic hepatitis B and persistent occult hepadnaviral infection. Frontiers in Microbiology, 13, 1011070.

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Decidual CD8+ T Cell Immune Modulation

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Decidual immune CD8⁺ T cells were cultured (5 × 10⁵ per well) in the presence of antibodies against Tim-3 (10 μg/ml, clone F38-2E2, BioLegend), PD-1 (10 μg/ml, clone EH12.2H7, BioLegend), both Tim-3 and PD-1, or isotype control. After 48 h, the culture supernatant was collected and analyzed by flow cytometry or cytotoxicity assay.

Wang S.C., Li Y.H., Piao H.L., Hong X.W., Zhang D., Xu Y.Y., Tao Y., Wang Y., Yuan M.M., Li D.J, & Du M.R. (2015). PD-1 and Tim-3 pathways are associated with regulatory CD8+ T-cell function in decidua and maintenance of normal pregnancy. Cell Death & Disease, 6(5), e1738-.

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