To determined transduction efficiency, surface CAR expression was evaluated by flow cytometry (FACSCanto II or Accuri, BD Biosciences) by staining transduced cells with anti-human IgG-phycoerythrin (PE) (SouthernBiotech) and CD3-allophycocyanin (APC) (Miltenyi Biotec), respectively. For phenotyping, CAR-transduced cells were stimulated with PSMA+ tumor cells (C4-2 or LNCaP) for 24 h at an E:T ratio of 1:1, before cells were harvested and evaluated. Activation was evaluated based on the expression of CD69 or CD25 (CD69-APC, clone CH4; CD25-PE, clone 3G10; both Thermo Fisher Scientific). T cell subsets were determined by staining cells with anti-human CD62L-Brilliant Violet 421 (BV421) (BD Biosciences), anti-human CD45RA-fluorescein isothiocyanate (FITC) (BioLegend), anti-human CD3-APC/H7 (BD Biosciences), and anti-human IgG-PE (SouthernBiotech). To determine exhaustion, cells were stained with anti-human CD279-FITC (PD-1, BD Biosciences), anti-human CD223-eFluor 710 (LAG-3, BD Biosciences), anti-human CD3-APC/H7, and anti-human IgG-PE.
Anti human cd3 apc h7
Manufactured by BD
Sourced in United States
The Anti-human CD3-APC-H7 is a flow cytometry reagent used for the detection and quantification of human CD3-positive T cells. It is a monoclonal antibody conjugated with the fluorescent dye APC-H7. This reagent allows for the identification and enumeration of CD3-expressing cells in biological samples.
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Lab products found in correlation
Facsaria fusion, by BD (2 mentions)
Viability dye, by Cytek Biosciences (2 mentions)
Cd19 car detection reagent, by Miltenyi Biotec (2 mentions)
Anti human cd8 bv785, by BioLegend (2 mentions)
Anti human cd62l bv421, by BioLegend (2 mentions)
Anti human cd25 viobright fitc, by Miltenyi Biotec (2 mentions)
Anti human tigit pe, by BioLegend (2 mentions)
Anti human cd27 pe cf594, by BD (2 mentions)
Hla dr fitc, by BD (2 mentions)
Cd4 pe cy7, by BD (2 mentions)
Live dead fixable dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Cd8 v500, by BD (2 mentions)
Cd38 percp cy5, by BD (2 mentions)
Facscanto 2, by BD (1 mentions)
Accuri, by BD (1 mentions)
Anti human igg pe, by BD (1 mentions)
Anti human igg pe, by Southern Biotech (1 mentions)
Anti human cd107a apc, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Lysing solution, by BD (1 mentions)
8 protocols using anti human cd3 apc h7
1
Chimeric Antigen Receptor T-Cell Phenotyping
2
Phenotypic Analysis of Activated Vγ9Vδ2 T Cells
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Vγ9Vδ2 T cells were stimulated with 5 μg/mL plate-bound anti-CD3 antibodies and 1 μg/mL soluble anti-CD28 antibodies (free mAbs) for 6 h in the presence of 5 μg/mL Brefeldin A. Then, the cells were stained with anti-human CD3-APC-H7 (BD Biosciences, clone: SK7), anti-human TCR Vδ2-PerCP (BioLegend, clone: B6), and anti-human CD107a-APC (BD Biosciences, clone: H4A3) antibodies. After staining for surface markers, the cells were fixed and permeabilized using Lysing Solution (BD Biosciences) and Permeabilizing Solution (BD Biosciences), respectively. Subsequently, the cells were stained with anti-human IFN-γ-PE-Cy7 (BD Biosciences, clone: B27), anti-human TNF-α-PE (BD Biosciences, clone: MAb11), and their corresponding isotype controls (APC-conjugated mouse IgG1, κ isotype control (clone: MOPC-21); PE-Cy™7-conjugated mouse IgG1, κ isotype control RUO (clone: MOPC-21); and PE-conjugated mouse IgG1, κ isotype control (clone: MOPC-21), all from BD Biosciences). Then, the cells were analyzed with a BD FACS Verse, and the data were analyzed with FlowJo. In addition, cell cycle, cell proliferation (Ki-67, BioLegend, clone: Ki-67), cell differentiation (CD45RA, BioLegend, clone: HI100), CD27 (BioLegend, clone: O323), mitochondrial, and costimulatory molecule (CD80, BioLegend, clone: 2D10; CD86, BioLegend, clone: BU63) analyses were performed following standard protocols.
3
CD8+ CAR T Cell Sorting and TCR Profiling
4
T Cell Activation Profiling of Healthy Volunteers
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PBMC from 20 healthy volunteers were isolated from whole peripheral blood using a density gradient (LymphoprepTM, AXIS-SHIELD) centrifugation and frozen until use. Briefly, PBMC were cultured for 3 days in RPMI 1640 media supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin and 20% fetal calf serum (GibcoTM, Life Technologies). For activation, 5 μg/ml PHA (Sigma-Aldrich) and 50 U/ml of recombinant human IL-2 (Roche, Sigma-Aldrich) were added to the media. Viability staining (Live/Dead Fixable Dead Cell Stain kit, Invitrogen) was performed to gate on viable cells and T cell activation was assessed by staining for T cell markers (antibodies: anti-human CD3-APC-H7, CD4-PE-Cy7, and CD8-V500; BD Biosciences) and markers of T cell activation (antibodies: anti-human CD25-APC, HLA-DR-FITC and CD38-PerCP-Cy5.5; BD Biosciences). Cells were collected on an LSR II instrument (Becton Dickinson) and T cell activation markers analysis was performed using the FlowJo software.
5
Comprehensive Immune Profiling of PBMCs
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Collected peripheral blood samples were analyzed using flow cytometry. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque centrifugation, then stained by the following antibodies: anti-human CD3-APC-H7 (BD biosciences, clone: SK7), anti-human TCR γδ-BV421 (BD biosciences, clone: 11F2), anti-human PD-1-BB515 (BD biosciences, clone: EH12.1), anti-human NKP46-BV510 (BD biosciences, clone: 9E2/NKP46), anti-human NKP30-Alexa Fluor®647 (BD biosciences, clone: P30-15), anti-human NKG2D-PE-Cy™7 (BD biosciences, clone: 1D11), anti-human TCR Vδ2-PE (BD biosciences, clone: B6), and anti-human TCR Vδ1-PerCP-Vio700 (Miltenyi Biotec, clone: REA173). Data was analyzed using FlowJo 10.1 software (Tree Star Inc., Ashland, OR, USA).
6
Monitoring T Cell Activation and HIV Co-Receptors
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Benzyl-2-acetamido-2-deoxy-α-d -galactopyranoside-treated and -untreated PHA-blasts were used to monitor expression of surface markers of cell activation and HIV co-receptor CCR5 and CXCR4. Briefly, PHA-blasts were prepared from 20 HIV-negative donors by stimulation with PHA for 2 days. One-half of the cells from each sample was then treated with 2 mM of BAGN overnight. The next day, cells were stained for viability markers (Live/Dead Fixable Dead Cell Stain kit, Invitrogen) and markers of T cell activation (antibodies: anti-human CD3-APC-H7, CD4-PE-Cy7, CD8-V500, CD25-APC, HLA-DR-FITC, and CD38-PerCP-Cy5.5; BD Biosciences). Cells were fixed and permeabilized (Fix and Perm, Invitrogen) to stain with Ki67-PE (BD Biosciences). Additional aliquots of BAGN-treated and -untreated PHA-blasts were used to stain for viability and T cell subsets as before and for HIV co-receptors (antibodies: anti-human CD184-APC (CXCR4), CD195-PE (CCR5); BD Biosciences). Cells were collected on an LSR II instrument (Becton Dickinson) and analysis was performed using FlowJo software.
7
Phenotyping T-cell Activation in Vaccination
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Fresh heparinized peripheral whole blood (150 µL) was stained with anti-human CD3-APC-H7, anti-human CD4-PerCP, anti-human CD8-FITC and anti-CD69-PE (BD Pharmingen, USA) at room temperature for 15 min in the dark. Red cells were lysed with 1x BD FACS lysis buffer (BD Biosciences, USA) for 10 min in the dark. After centrifugation at 450 × g for 10 min, samples were washed once with phosphate-buffered-saline (PBS) before analysis using a BD FACSCantoTM II flow cytometer (BD Bioscience, USA). Activated (CD69+) T cells were determined as shown in Supplementary Figure S1 . To account for the possibility of asymptomatic infection at CV0 (and hence elevated Th responder cell numbers), we measured the changes in CD4 T cell numbers from CV0-CV1 or from CV1-CV2. Participants were sub-grouped into non-T helper (Non-Th) responders (115/151 [76.2%]) and Th responders (36/151 [23.8%]) based on the increased numbers of helper lymphocytes between from CV0 to CV1 and/or CV2. An average of a > 1.47 fold (±SD) change in Th numbers (non-responders vs responders) was taken as a vaccine-dependent difference.
8
Sorting of Effector and Precursor CAR T Cells
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Cryopreserved GMP product cells were thawed at 37°C and washed. Cells were then stained with the CD19-CAR detection reagent (Miltenyi; cat. #130-115-965) for 10 minutes at room temperature followed by staining with an antibiotin antibody conjugated to APC (Miltenyi; cat. #130-110-952), a cocktail of antibodies (CD3, CD8, CD27, CD62L, CD25, and TIGIT), and a viability dye (Tonbo Biosciences; cat. #13-0870-T100). After staining for 10 minutes at room temperature, the cells were washed twice and resuspended in FACS buffer for sorting on the FACSAria Fusion (BD Biosciences). CD8+ CAR T cells with either the predicted effector surface profile (TIGIT+, CD62Llo, and CD27−) or the opposite, noneffector precursor profile (TIGIT−, CD62L+, and CD27+) were sorted into complete RPMI media. Cells were lysed with TRIzol for bulk TCR repertoire sequencing.
Antibodies used were anti-human CD3-APC-H7 (BD Pharmingen; cat. #560176), anti-human CD8-BV785 (BioLegend; cat. #344740), anti-human CD27-PE-CF594 (BD Horizon; cat. #562297), anti-human CD62L-BV421 (BioLegend; cat. #304828), anti-human CD25-VioBright FITC (Miltenyi Biotec; cat. #130-113-283), and anti-human TIGIT-PE (BioLegend; cat. #372703).
Antibodies used were anti-human CD3-APC-H7 (BD Pharmingen; cat. #560176), anti-human CD8-BV785 (BioLegend; cat. #344740), anti-human CD27-PE-CF594 (BD Horizon; cat. #562297), anti-human CD62L-BV421 (BioLegend; cat. #304828), anti-human CD25-VioBright FITC (Miltenyi Biotec; cat. #130-113-283), and anti-human TIGIT-PE (BioLegend; cat. #372703).
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