Pd 1 eh12.2h7

Manufactured by BioLegend

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PD-1 (EH12.2H7) is a monoclonal antibody that recognizes the programmed cell death 1 (PD-1) receptor. PD-1 is an inhibitory receptor expressed on activated T cells, B cells, and myeloid cells. The EH12.2H7 clone of the PD-1 antibody can be used for flow cytometry and other immunological applications to detect and study PD-1 expression.

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EH12.2H7 (13 citations) PD-1 (EH12.1) (4 citations) Anti-human PD-1 (clone EH12.2H7, (4 citations) Anti-PD-1 BV421 (clone EH12.2H7), (3 citations) PD-1 (FITC; clone: EH12.2H7; (2 citations) PD‐1 BV421 (EH12.2H7; (2 citations) PD-1 BV605 (clone EH12.2H7; (2 citations) Anti-PD-1 PerCP-Cy5.5 (EH12.2H7, (2 citations) PD-1 APC (clone EH12.2H7; (2 citations) PD-1-PE (EH12.2H7, (2 citations)

20 protocols using pd 1 eh12.2h7

Innate and Adaptive Immune Phenotyping

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Monoclonal antibodies used for staining of PBMCs for innate cell phenotyping were CD3 (UCHT1, #557943), CD19 (HIB19, #557921), CD14 (M5E2, #565283), HLA-DR (G46-6, #560651), CD11c (O33-782, #561355), and CD123 (7G3,554529) from BD Biosciences (Franklin Lakes, NJ), CD56 (MEM188,17-0569) and CD16 (CB16,47-0168) from eBiosciences (San Diego, CA); for cT_FH phenotyping CD3 (SP34-2, #562877), CD4 (L200, #560836), CXCR5 (RF8B2), and CXCR3 (IC6/CXCR3) BD Biosciences, ICOS (C398.4 A) and PD-1 (EH12.2H7) from Biolegend (San Diego, CA) (Supplementary Fig. 2). Data were collected on an LSRII (BD Biosciences) and data were analyzed using R 3.6.3 software. Sample sizes available for statistical analysis differed based on sample availability (monocytes—MNP: n = 7; IM = 10, cT_FH—MNP: n = 8; IM: n = 9).

Rouphael N.G., Lai L., Tandon S., McCullough M.P., Kong Y., Kabbani S., Natrajan M.S., Xu Y., Zhu Y., Wang D., O’Shea J., Sherman A., Yu T., Henry S., McAllister D., Stadlbauer D., Khurana S., Golding H., Krammer F., Mulligan M.J, & Prausnitz M.R. (2021). Immunologic mechanisms of seasonal influenza vaccination administered by microneedle patch from a randomized phase I trial. NPJ Vaccines, 6, 89.

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Detailed T-cell Activation Assay Protocol

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RPMI 1640 (31870074), Dynabeads Human T-Activator CD3/CD28 for T cell expansion and Activation kit (1132D) were purchased from Thermofisher Scientific. Hyclone Fetal bovine serum was obtained from Fisher Scientific. Poly-L-lysine solution 0.1% (w/v) in H₂O was obtained from Sigma (25988-63-0). The following anti-human antibodies were purchased from Biolegend, CD62L (DREG-56), CCR7 (G043H7), CD3 (UCHT-1), CD4 (A161A1), CD8 (HIT8a), CD2 (RPA2.10), CD28 (CD28.2), PD-1 (EH12.2H7), CD11a (TS2/4), mouse IgG1 isotype control (MOPC-21), CD58 (TS2/9), mouse IgG1 isotype control (MG1-45; 401402), CD45 (HI30), CD127 (A019D5), HLA-A2 (BB7.2), CD45RO (UCHL1). The following anti-human antibodies were purchased from BD Bioscience, CD45RA (HI100), CD127 (HIL-7R-M21), CD58 (1C3), CD2 (CD2.1) clone was a gift of D. Olive (Aix Marseille University). The following anti-human antibodies were purchased from eBioscience, CD8 (OKT8) and CD28 (CD28.2). Flow cytometry mAbs were used between 1-5ug/ml depending on how many cells were staining (i.e. between 5x10³- 1x10⁶). The following antibodies were purchased from Cell Signaling, pPLCγ1(pY783), pLAT(pY171), pSFK (pY416) (D49G4) and used at dilutions recommended by the company. The following anti-mouse antibodies were purchased from Biolegend, CD4 (RM4- 5), CD3 (145-2C11), TCRβ (H57-97). TCRβ (H57-97) Fab was obtained from Bio X Cell.

Demetriou P., Abu-Shah E., Valvo S., McCuaig S., Mayya V., Kvalvaag A., Starkey T., Korobchevskaya K., Lee L.Y., Friedrich M., Mann E., Kutuzov M.A., Morotti M., Wietek N., Rada H., Yusuf S., Afrose J., Siokis A., Meyer-Hermann M., Ahmed A.A., Depoil D, & Dustin M.L. (2020). A dynamic CD2 rich compartment at the outer edge of the immunological synapse boosts and integrates signals. Nature immunology, 21(10), 1232-1243.

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Multiparameter Flow Cytometry for Lymphocyte Phenotyping

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Flow cytometry for surface staining was performed using standard protocols (Xu et al. 2015 ). Cells from lymph nodes and blood were stained with: CD3 (SP34), CD4 (SK3), CD8 (SK1), CD95 (DX2), CD45RA (L48) and CD28 (CD28.2) (all from BD Biosciences Pharmingen, San Diego, CA), CXCR5 (MU5UBEE, eBioscience), PD-1 (EH12.2H7, BioLegend), CCR7 (GO43H7) and LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, Grand Island, NY). For assessing proliferation, PBMC was surface stained, treated with FACS lysing solution, washed, and intracellularly stained with anti-Ki67 (Clone B56). Isotype-matched controls were included in all experiments. Samples were resuspended in BD Stabilizing Fixative (BD Biosciences) and acquired on a FACS FORTESSA (Becton Dickinson, San Jose, CA). Data were analyzed with Flowjo software (Tree Star, Ashland, OR).

Xu H., Andersson A.M., Ragonnaud E., Boilesen D., Tolver A., Jensen B.A., Blanchard J.L., Nicosia A., Folgori A., Colloca S., Cortese R., Thomsen A.R., Christensen J.P., Veazey R.S, & Holst P.J. (2017). Mucosal Vaccination with Heterologous Viral Vectored Vaccine Targeting Subdominant SIV Accessory Antigens Strongly Inhibits Early Viral Replication. EBioMedicine, 18, 204-215.

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Multiparametric Flow Cytometry Analysis

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The following fluorochrome-conjugated antibodies were used: CD4 (RPA-T4, Biolegend), CD8 (SK1, Biolegend), CD19 (HIB19, Biolegend), CD69 (FN50, Biolegend), CD25 (BC96, Biolegend), CXCR5 (J252D4, Biolegend), PD-1 (EH12.2H7, Biolegend), IFNγ (4S.B3, Biolegend), IL-4 (MP4-25D2, Biolegend), IL-17A (BL168, Biolegend), FOXP3 (PCH101, Invitrogen), and EZH2 (11/EZH2, BD Biosciences). For surface staining, cells were stained with antibodies at 4 °C in the dark for 30 min. For intracellular cytokine staining, cells were stimulated with a leukocyte activation cocktail containing GolgiPlug (BD Bioscience) for 4 h, then fixed and permeabilized using Cytofix/Cytoperm (BD Bioscience), followed by incubation with antibodies. For FOXP3 and EZH2 staining, cells were fixed and permeabilized using the FOXP3 staining kit (Thermo Fisher) according to the instructions of the manufacturer. Cells were analyzed with a BD Aria II Flow Cytometer (BD Bioscience) and data were processed using FlowJo X (Tree Star).

He C., Yang Y., Chen Z., Liu S., Lyu T., Zeng L., Wang L., Li Y., Wang M., Chen H, & Zhang F. (2022). EZH2 Promotes T Follicular Helper Cell Differentiation Through Enhancing STAT3 Phosphorylation in Patients With Primary Sjögren’s Syndrome. Frontiers in Immunology, 13, 922871.

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Immune Cell Profiling by Flow Cytometry

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For biomarker analysis, 15 mL of blood was collected at baseline and at the time of each disease evaluation. Peripheral blood mononuclear cells (PBMCs) were obtained by standard density gradient centrifugation. To evaluate the prognostic implications of immune cells, multicolour flow cytometry analyses using the CytoFLEX flow cytometry platform (Beckman Coulter, Brea, CA) were performed to determine the proportions of different immune cell populations in the PBMCs. Panels for multicolour flow cytometry included CD3 (UCHT1; BioLegend, San Diego, CA, USA), CD4 (OKT4; BioLegend), CD8 (SK1; BioLegend), CD14 (63D3; BioLegend), CD11c (3.9; BioLegend), CD56 (5.1H11; BioLegend), γδ TCR (B1; BioLegend), HLA-DR (L243; BioLegend), CD69 (FN50; BioLegend), FoxP3 (236A-E7; eBioscience, San Diego), PD-1 (EH12.2H7; BioLegend), LAG-3 (11C3C65; BioLegend), CTLA-4 (L3D10; BioLegend), and TIGIT (A15153G; BioLegend).

Yoo C., Lee S.S., Song K.B., Jeong J.H., Hyung J., Park D.H., Song T.J., Seo D.W., Lee S.K., Kim M.H., Lee S.S., Kim J.H., Jin H.S., Park J.H., Hwang D.W., Lee J.H., Lee W., Chang H.M., Kim K.P., Ryoo B.Y, & Kim S.C. (2020). Neoadjuvant modified FOLFIRINOX followed by postoperative gemcitabine in borderline resectable pancreatic adenocarcinoma: a Phase 2 study for clinical and biomarker analysis. British Journal of Cancer, 123(3), 362-368.

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Flow Cytometry Protocol for Tumor-Infiltrating Lymphocyte Analysis

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Flow cytometry was performed using CytoFLEX (Beckman Coulter, Brea, CA, USA). Data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). For immunolabeling TILs, fluorophore-conjugated monoclonal antibodies against the following proteins were used: CD8 (RPA-T8, Cat# 301048, Biolegend, San Diego, CA, USA, 1:50), CD3 (SK7, Cat# 344808, Biolegend, San Diego, CA, USA, 1:100), PD-1 (EH12.2H7, Cat# 329933, Biolegend, San Diego, CA, USA, 1:20), CD103 (Ber-ACT8, Cat# 350230, Biolegend, San Diego, CA, USA, 1:20), CD39 (A1, Cat# 328210, Biolegend, San Diego, CA, USA, 1:20), GZMB (QA16A02, Cat# 372214, Biolegend, San Diego, CA, USA, 1:50), and CD4 (RPA-T4, Cat# 560837, BD Biosciences, San Diego, CA, USA, 1:50). The LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit was from Invitrogen (L34973, Waltham, Massachusetts, USA, 1:100). The sequential gating strategies used for analysis for data obtained from flow cytometry are described in Supplementary Fig. S15.
For t-distributed stochastic neighbor embedding (tSNE) visualization of flow cytometry data, we used three samples for each group (EGFR-WT and EGFR-MT). Each sample was down-sampled to 7000 randomly selected CD3⁺ live and singlet-gated cells, yielding 42,000 cells. A tSNE plot for the merged 42,000 cells was constructed using FlowJo (v10.6.2) with default settings (3000 iterations and perplexity = 100).

Cho J.W., Park S., Kim G., Han H., Shim H.S., Shin S., Bae Y.S., Park S.Y., Ha S.J., Lee I, & Kim H.R. (2021). Dysregulation of TFH-B-TRM lymphocyte cooperation is associated with unfavorable anti-PD-1 responses in EGFR-mutant lung cancer. Nature Communications, 12, 6068.

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CFSE-labeled Cell Cytotoxicity Assay

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The day prior to the assay, 3×10⁴ JEG-3 cells were labeled with 1 µM of CFSE (CellTrace, Thermo Fisher) and seeded in flat-bottom 96-well microplates. On the day of the assay, CAR-T cells were added at various E:T ratios to either the plated CFSE-labeled JEG-3 cells or 3×10⁴ CFSE-labeled K562/K562-HLA-G1 cells. After 24 hours incubation, medium was collected and cells recovered, washed and labeled with antibodies against CD4 (clone RPA-T4, Biolegend), CD8 (clone RPA-T8, Biolegend), CD19 (HIB-19, Biolegend), CD25 (clone M-A251, BD Bioscience), CD69 (clone FN-50, BD Bioscience), PD-1 (EH12.2h7, Biolegend) and a viability dye (Invitrogen). For degranulation assays, co-cultures were set-up at an E:T ratio of 10:1. Anti-CD107a (clone eBioH4A3, Biolegend) was added at the start of the experiment, GolgiStop (BD Bioscience) was added after 1 hour. Five hours after the beginning of the assay, cells were collected and labeled with antibodies directed against CD4, CD8, CD19 and a viability dye. Acquisition was performed with a fluorescence-activated cell sorting (FACS) Attune (Thermo Fisher), and results were analyzed with FlowJo software.

Anna F., Bole-Richard E., LeMaoult J., Escande M., Lecomte M., Certoux J.M., Souque P., Garnache F., Adotevi O., Langlade-Demoyen P., Loustau M, & Caumartin J. (2021). First immunotherapeutic CAR-T cells against the immune checkpoint protein HLA-G. Journal for Immunotherapy of Cancer, 9(3), e001998.

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Multicolor Flow Cytometric Analysis of Immune Cell Subsets

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LIVE/DEAD™ Fixable Aqua Dead Cell Stain (L34957, Thermo Fisher) was used to exclude dead cells. CD1c (Biolegend), CD141 (Biolegend) were used for DCs analysis by flow cytometry. Fluorochrome-labeled anti-human mAbs specific for CD3 (UCHT1, Biolegend), CD4 (OKT4, Biolegend), CD45RA (HI100, Biolegend), CXCR5 (J252D4, Biolegend), CCR6 (G034E3, Biolegend), CXCR3 (G025H7, Biolegend), PD-1 (EH12.2H7, Biolegend) and ICOS (C398.4A, Biolegend) were used for analysis of Tfh cell population by flow cytometry. Fluorescein-labeled anti-human antibodies specific for CD3 (UCHT1, Biolegend), CD19 (HIB19, Biolegend), CD21 (Bu32, Biolegend), CD27 (O323, Biolegend), CD38 (HIT2, Biolegend) and IgD (IA6-2, Biolegend) were used for analysis of B cell population by flow cytometry.

Jiang Z., Lin H., Zhang H., Shi N., Zheng Z., Dong L., Yang Y, & Xia Q. (2022). An Integrative Analysis of the Immune Features of Inactivated SARS-CoV-2 Vaccine (CoronaVac). Vaccines, 10(6), 878.

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Flow Cytometric Profiling of PBMCs

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Frozen PBMCs were thawed, washed in FACS buffer (2% FBS, 1mM EDTA in PBS), and counted on TC20 (Biorad) before surface staining using two independent flow panels. For the innate panel, the following antibodies were used: CD3 (SP34, BD Pharmingen) and CD20 (2H7, BioLegend) for the exclusion of T & B lymphocytes, respectively. We further stained for CD56 (HCD56, Biolegend), CD57 (HNK-1, BioLegend), KLRG1 (SA231A2, BioLegend) CD16 (3G8, BioLegend), CD14 (M5E2, BioLegend), HLA-DR (L243, BioLegend), CD11c (3.9, ThermoFisher Scientific), CD123 (6H6, BioLegend) and CD86 (IT2.2, BioLegend). For the adaptive panel, the following antibodies were used: CD4 (OKT4, BioLegend), CD8b (2ST8.5H7, Beckman Coulter), CD45RA (HI100, TONBO Biosciences), CCR7 (G043H7, BD Biosciences), CD19 (HIB19, BioLegend), IgD (IA6–2, BioLegend), CD27 (M-T271, BioLegend), KLRG1 (SA231A2, BioLegend) and PD-1 (Eh12.2h7, BioLegend). Cells were stained with Ghost Dye viability dye (TONBO biosciences) for 30 minutes at 4C per manufacturer’s instructions, washed, surface stained with either innate or adaptive panels for 30 minutes at 4C. Samples were then washed and analyzed on Attune NxT Flow Cytometer (ThermoFisher Scientific, Waltham MA).

Sureshchandra S., Zulu M.Z., Doratt B., Jankeel A., Tifrea D., Edwards R., Rincon M., Marshall N.E, & Messaoudi I. (2021). Deep immune profiling of the maternal-fetal interface with mild SARS-CoV-2 infection. bioRxiv.

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Multiparametric Flow Cytometry Analysis

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Antibodies to CD45 (HI30), CD4 (RPA-T4), CD8 (HIT8a), CD56 (5.1h11), CD123 (6H6), CD14 (63D3), CD11c (3.9), CD45RA (HI100), CCR7 (G043H7), CD10 (HI10a), IL-2 (MQ1-17H12), IFN-γ (4S.B3), HLA-DR (L243), CD38 (HIT2), and PD-1 (EH12.2H7) were purchased from BioLegend. Antibodies to CD3 (7D6), CD19 (6D5), mouse CD45 (30-F11), and LIVE/DEAD Fixable Yellow Dead Cell Stain Kit were purchased from Invitrogen. Antibody to HIV-1 p24 (KC57) were purchased from Beckman Coulter.
Total lymphocytes were isolated prepared from peripheral blood, spleen, bone marrow (BM), and mesenteric lymph nodes (mLNs) according to standard protocols; red blood cells were lysed with ACK buffer. Intrahepatic lymphocytes were prepared as described (42 (link)). Total cell number was quantified by Guava Easycytes with Guava Express software (Guava). For surface staining, single cell suspension was stained with surface markers and analyzed on a CyAn ADP flow cytometer (Dako). For intracellular staining, cells were first stained with surface markers and then fixed and permeabilized with cytofix/cytoperm buffer (BD Bioscience), followed by intracellular staining. Data were analyzed using Summit4.3 software (Dako).

Cheng L., Ma J., Li G, & Su L. (2018). Humanized Mice Engrafted With Human HSC Only or HSC and Thymus Support Comparable HIV-1 Replication, Immunopathology, and Responses to ART and Immune Therapy. Frontiers in Immunology, 9, 817.

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