MLR assay was performed with monocyte derived dendritic cells (moDCs) co-cultured with allogeneic exhausted T cells. To generate exhausted T cells, CD4 + T cells were expanded for 7 days with Dynabeads™ Human T-Activator CD3/CD28 (1:1 bead to cell ratio; Invitrogen) replaced every second day. After 7 days, Dynabeads were removed and CD4 + T cells were rested overnight. MoDCs were generated from CD14 + monocytic cells in the presence of IL-4 and GM-CSF (Miltenyi Biotech) for 5 days and then matured for 24 h using a cocktail of Il-1β, IL-6, TNF-α (Miltenyi Biotech) and PGE2 (Merck Millipore). Mature moDCs and exhausted CD4 + T cells were co-cultured (ratio 1:10) with serially diluted Opdivo® and ATOR-1017 crosslinked with F(ab)2 anti-Ig (5:1 molar ratio to mAb) for 7 days, when IFNγ in supernatants was analyzed by ELISA. Exhausted CD4 + T cells and mature moDCs phenotypes was confirmed with flow cytometric staining: CD4-PerCpCy5.5(RPA-T4), PD-1-PE(MIH4), CD14-PerCpCy5.5(MφP9), CD86-FITC(2331FUN-1) and HLADR-BV510(G46-6) from BD Biosciences; LAG-3-FITC(11C3C65), TIM-3-BV421(F38-2E2), CTLA-4-BV421(BNI3), CD137-PECy7(4B4-1) (BioLegend); TOX-PE(REA473) (Miltenyi Biotech) and PD-1-APC(MIH4) (eBioscience).
Cd137 pecy7 4b4 1
Manufactured by BioLegend
Sourced in United States
CD137-PECy7(4B4-1) is a fluorochrome-conjugated antibody that binds to the CD137 antigen. CD137, also known as 4-1BB, is a costimulatory receptor expressed on activated T cells, natural killer cells, and other immune cell types. This product can be used for the identification and characterization of CD137-positive cells by flow cytometry.
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Lab products found in correlation
Il 1β, by Miltenyi Biotec (1 mentions)
Dynabeads human t activator cd3 cd28, by Thermo Fisher Scientific (1 mentions)
Pd 1 apc mih4, by Thermo Fisher Scientific (1 mentions)
Hladr bv510 g46 6, by BD (1 mentions)
Interleukin 6 (il 6), by Miltenyi Biotec (1 mentions)
Gm csf, by Miltenyi Biotec (1 mentions)
Interleukin 4 (il 4), by Miltenyi Biotec (1 mentions)
Tnf α, by Miltenyi Biotec (1 mentions)
D058 1283, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Cd45ra bv605, by BioLegend (1 mentions)
Foxp3 fixation permeabilization buffer kit, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Cd3 bv785 okt3, by BioLegend (1 mentions)
Cd8 v500 rpa t8, by BD (1 mentions)
3 protocols using cd137 pecy7 4b4 1
1
Exhausted T Cell Activation Assay
2
Multiparametric Flow Cytometry Panel
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Antibodies used in this study included the following: CD45RA-fluorescein isothiocyanate (FITC) (5H9, Becton Dickinson [BD], Franklin Lakes, NJ), TIGIT-peridinin chlorophyll protein (PerCP)-eFluor 710 (MBSA43, Invitrogen, Waltham, MA, USA), CD69-Alexa Fluor 647 (FN50, BioLegend, San Diego, CA, USA), Ki67-Alexa Fluor 700 (B56, BD), PD-1-allophycocyanin (APC)-eFluor 780 (J105, eBioscience), CCR7-PE (3D12, Thermo Fisher Scientific), CD154-PE-CF594 (TRAP1, BD), CD137-PE-Cy7 (4B4-1, BioLegend), CD25-V450 (4E3, eBioscience), CD4-Brilliant Violet (BV)570 (OKT4, BioLegend), CD3-BV605 (SP34-2, BD), human leukocyte antigen (HLA)-DR-BV711 (G46-6, BD), CTLA-4-BV786 (BNI3, BD), CD45-BUV395 (D058-1283, BD), and CD8-BUV737 (SK1, BD). Anti-VSV-G-PE (F-6, Santa Cruz Biotechnology, Dallas, TX, USA) was used to detect VSV-G expression. All flow cytometry was collected and analyzed on a FACSymphony using FACSDiva software (BD Biosciences). Final data analysis and plot generation were performed in FlowJo v10 (BD Biosciences).
3
Multiparametric Phenotyping of Immune Cells
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Stimulated cells were stained with fluorochrome-conjugated monoclonal antibodies to detect surface and intracellular markers. Intracellular staining was performed with Foxp3 Fixation/Permeabilization buffer kit (eBioscience, San Diego, CA, USA), and Fixable Viability eFluor 780 Dye (eBioscience) was used to exclude dead cells. The following antibodies were used: CD4-Alexa Fluor 700 (OKT4, BioLegend), CD8-V500 (RPA-T8, BD Biosciences, San Jose, CA, USA), GrB-Fitc (GB11, BioLegend), IL-2-Pe (MQ1-17H12, BioLegend), IL-4-PeDaz594 (MP4-25D2, BioLegend), CD137-Pe/Cy7 (4B4-1, BioLegend), CD154-Alexa Fluor 647 (24-31, BioLegend), TNF-α-eFl450 (MAb11, eBioscience), IFN-γ-BV650 (4S.B3, BioLegend), CD3-BV785 (OKT3, BioLegend), CD19-BV605 (HIB19, BioLegend), CD45RA-BV605 (HI100, BioLegend), CCR7-PerCP/Cyanine 5.5 (G043H7, BioLegend).
For the absolute quantification of immune cell subsets, total CD19+, CD3+, CD4+, and CD8+ T cells numbers were determined in 50 μL of the peripheral blood by flow cytometry as described below by means of CytExpress software (Beckman Coulter, USA). Absolute numbers of HBs-specific T cells were determined based on relative frequencies of HBs-specific T cells and absolute numbers of total CD4+ T cells per μL of peripheral blood.
For the absolute quantification of immune cell subsets, total CD19+, CD3+, CD4+, and CD8+ T cells numbers were determined in 50 μL of the peripheral blood by flow cytometry as described below by means of CytExpress software (Beckman Coulter, USA). Absolute numbers of HBs-specific T cells were determined based on relative frequencies of HBs-specific T cells and absolute numbers of total CD4+ T cells per μL of peripheral blood.
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