Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor apheresis cones obtained through the Mayo Clinic Blood Donor Center54 (link) (approved by the Division of Transfusion Medicine Research Committee at Mayo Clinic and determined to be IRB exempt). Informed consent was obtained from all donors for the use of their sample for research purposes. Cells were isolated using Lympholyte-H density separation (Cedarlane) and cultured in AimV Media (ThermoFisher) supplemented with 5% human AB serum (Sigma) and 1% PenStrep and stimulated with 100 U/mL of human IL2 and 50 ng/mL anti-CD3 OKT3 antibody (Biolegend #317326). 48 and 72 h later, cells were transduced twice with retroviral supernatant collected from the PG13-139-CD8-CD28BBZ-F10 producer cell line on RetroNectin-coated plates (Takara). Cells were split every two days and collected 4 days after the second transduction for in vitro experiments. Transduced cells were identified using a tetramer composed of biotin-PepvIII and streptavidin-AF647 and a representative gating scheme identifying murine CAR T cells is shown in Supplementary Fig. 10 . The PepvIII tetramer was generated by incubating PepvIII-biotin (Proteomics Core Mayo Clinic) with SA-AF647 (Invitrogen) at a 10:1 molar ratio and washed 4x using a 30 kDa MCWO filter.
Anti cd3 antibody okt3
Manufactured by BioLegend
Sourced in United States
The Anti-CD3 antibody (OKT3) is a laboratory tool used for immunological research. It is a monoclonal antibody that specifically binds to the CD3 complex, which is expressed on the surface of T cells. This antibody can be used to activate and stimulate T cells in in vitro experiments.
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Lab products found in correlation
Retronectin coated plate, by Takara Bio (2 mentions)
Aim 5 media, by Thermo Fisher Scientific (2 mentions)
Lympholyte h density separation, by Cedarlane (2 mentions)
Human il 2, by BioLegend (2 mentions)
Human ab serum, by Merck Group (2 mentions)
Accuri c5 flow cytometer, by BD (1 mentions)
Red blood cell lysate, by BD (1 mentions)
Anti cd28 antibody, by BioLegend (1 mentions)
Recombinant human il 21, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 6, by Thermo Fisher Scientific (1 mentions)
Anti tgf β, by BioLegend (1 mentions)
Anti il 4 antibody, by BioLegend (1 mentions)
Recombinant human il 12, by Thermo Fisher Scientific (1 mentions)
Recombinant human tgf β, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by BioLegend (1 mentions)
Anti ifn γ, by BioLegend (1 mentions)
Anti cd3 antibody, by BioLegend (1 mentions)
Lsrfortessa x 50 flow cytometer, by BD (1 mentions)
Facs staining buffer, by BioLegend (1 mentions)
Lpc 18 1, by Avanti Polar Lipids (1 mentions)
8 protocols using anti cd3 antibody okt3
1
Transduction of Murine CAR T Cells
2
Expansion and Transduction of Primary Human T Cells
3
Binding of Anti-LAG3 Antibodies to Human T Cells
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Example 13
Anti-LAG3 antibodies (Clone 2#, 8#, 13#) were tested for the ability of binding to human LAG-3 expressed on activated human T cells.
Primary T cells were isolated from peripheral blood mononuclear cells with magnetic beads and cultured in tissue culture plates coated with anti-CD3 antibody (OKT3, Biolegend). Anti-LAG-3 antibodies (Clone 2#, 8#, 13#) and negative control IgG4 were added to cells and the mixture was incubated at 4° C. for 30 minutes. The cells were washed twice. The binding activity of the anti-LAG-3 antibodies to LAG-3 expressed on T cells was detected using an R-PE-conjugated AffiniPure Goat Anti-Human IgG, Fcγ Fragment Specific (Jackson ImmunoResearch) secondary reagent, with the mixture incubated at 4° C. for 30 minutes followed by washing twice. Then, cells were resuspended in PBS buffer. Analysis of LAG-3 binding was carried out with the BD Accuri C5 flow cytometer (BD Bioscience).
Representative curves for these clones binding to LAG-3 expressed by human T cells were shown in
4
In Vitro Tfh Polarization Assay
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Regarding the protocol for the in vitro mouse Tfh polarization assay, 24‐well round‐bottom tissue culture plates were incubated with 4 μg/mL anti‐CD3 antibody (BioLegend) in PBS at 4°C overnight. Single‐cell suspensions from mouse spleens were prepared, and the red blood cells were lysed with red blood cell lysate (BD). 5 × 105 isolated splenocytes were transferred per well onto the precoated 24‐well round‐bottom tissue culture plates and additionally activated with 1 μg/mL anti‐CD28 (BioLegend, USA), 10 μg/mL anti‐IFN‐γ (BioLegend), 10 μg/mL anti‐IL‐4 antibodies (BioLegend, USA), 20 μg/mL anti‐TGF β (BioLegend), and 10 ng/mL each IL‐6 and IL‐21 (PeproTech) cytokines for 3 days. The percentage of in vitro‐polarized Tfh cells was analyzed using flow cytometry. For the induction of human Tfh cells in vitro, 24‐well plates were precoated with 4 μg/mL anti‐CD3 antibody (OKT3) (BioLegend) at 4°C overnight. The cells were then incubated on the precoated plates in presence of 1 μg/mL anti‐CD28 antibody (BioLegend), 20 μg/mL recombinant human IL‐21 (PeproTech), 5 μg/mL recombinant human IL‐12 (PeproTech), 1 μg/mL recombinant human TGF‐β (PeproTech), and 20 μg/mL recombinant human IL‐6 (PeproTech) at 37°C under 5% CO2 conditions for 3 days. Plated cells were treated with 0.1 mΜ tapinarof or 50 µM Colivelin TFA as indicated.
5
CD1a Lipid Tetramer Staining Protocol
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Biotinylated human CD1a monomers (NIH Tetramer Core Facility) were produced in HEK293-derived cell lines (36 (link), 67 (link)). CD1a (10 ug) was treated with a 100X molar excess of LPC 18:1 or LPC 18:0 (Avanti Polar Lipids) in Tris Buffer saline containing 0.25% CHAPS or vehicle alone (mock) for 16 h at 37 °C, and tetramerised with PE Streptavidin (High Concentration; BioLegend) at a molar ratio of 5:1. T cells (<1x106) were washed twice in FACS staining buffer (BioLegend) at room temperature and stained with 0.5 µl tetramer in 20 µl FACS staining buffer at 37 °C for 30 min with gentle shaking. Anti-CD3 antibody (OKT3; 0.1 µg in 10 µl; BioLegend) was added to the cells and incubated for an additional 10 min at 37 °C with gentle shaking. Tetramers and anti-CD3 antibody were removed before staining surface markers CD3 (UCHT1; BioLegend), CD4, CD8 and Zombie Fixable Viability dyes (BioLegend) for 15 min at 4°C. Cells were washed once and resuspended in FACS buffer and ready for requisition using LSRFortessa X-50 flow cytometer (BD Biosciences) and further analyzed with FlowJo (FlowJo LLC) software.
6
Trophoblast-CD8+ T Cell Interaction
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Freshly isolated trophoblasts were seeded at a density of 2 × 105 cells/ml per well in Matrigel (Coring, USA)-coated 24-well plates overnight. The cells were then washed twice with phosphate-buffered saline (PBS, HyClone, USA). Equal numbers of dCD8+ T cells or pCD8+ T cells were added to each well. In some wells, anti-HLA-C (10 μg/ml, clone W6/32; Biolegend, U.S.A), HLA-G (10 μg/ml, clone 87G; Biolegend, USA) were added. dCD8+T cells were also cultured with plate-bound anti-CD3 antibody (OKT-3; 5 μg/ml, Biolegend, USA) plus soluble anti-CD28 antibody (28.2; 1 μg/ml, Biolegend, USA), or HTR8/Svneo cells or DSCs for 48 h. In some wells, dCD8+ T cells (2 × 105 cells) were plated in the upper chamber (0.4 mm pore size cell culture inserts, Millipore, Germany), while trophoblasts were plated in the lower chamber to establish indirect cell contact. Phorbol 12-myrstate 13-acetate (PMA) (50 ng/ml, Biolegend, USA), ionomycin (1 μg/ml, Biolegend, USA) and brefeldin A (10 mg/ml, BioLegend, USA), were added 4 h before the end of the 48 h culture for intracellular cytokine analysis. The cells were then harvested for flow cytometry analysis.
7
Generating Single-Cell Suspensions from Tumor
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To generate a single-cell suspension of the tumor for cell sorting, tumors were digested in enzyme medium on a shaker for 30 min at 37°C and then successively filtered through 140, 100 and 40 μm strainers. Enzyme medium comprised RPMI-1640 (ThermoFisher Scientific) supplemented with trypsin inhibitor (Sigma-Aldrich) at 1 mg/ml, hyaluronidase (Sigma-Aldrich) at 0.3 mg/ml, collagenase (Sigma-Aldrich) at 1 mg/ml and DNase (Pulmozyme) at 10 U/ml. Fresh tumor single-cell suspensions were labeled with the fluorescent-labeled antibodies CD3-AF700 (clone: SK7), CD4-APC Fire750 (clone: SK3), CD8-BV785 (clone: SK1), PD-1-AF647 (clone: EH12.2H7), CD103-FITC (clone: Ber-ACT8) and CD39-BV421 (clone: A1) (all from BioLegend). Populations of interest were purified using a FACSAria II based on PD-1, CD39 and CD103 expression for single-cell sequencing and in vitro expansion using excess irradiated (5,000 rad) allogeneic PBMC feeder cells pooled from two different donors in 50/50 T-cell medium supplemented with 30 ng/ml anti-CD3 antibody (OKT3, Biolegend) and 3,000 IU/ml IL-2. 50/50 media consisted of CM and AIM-V media at a ratio of 1:1. After about 2 weeks, T cells were used in co-culture assays or cryopreserved for further analysis.
8
Isolation and Activation of CD8+ T Cells
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Peripheral blood was collected from three healthy drug-free adult donors after informed consent. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll separation.
The contaminating erythrocytes were removed by osmotic lysis. Briefly, 1 ml of distilled water was added to the cell pellet. After 30s, 14 ml of RPMI 1640 (Sigma-Aldrich, St.
Louis, MO, USA) was added, and the cells were washed once. Untouched CD8 + T lymphocytes were isolated by negative immunomagnetic selection, using CD8+ T Cell Isolation Kit and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer's instructions. Cell purity was checked using a FacsCanto II flow cytometer (BD, San Jose, CA, USA) and in all the cases the CD8 + T lymphocyte purity was above 90%. Enriched CD8 + T lymphocytes were resuspended in RPMI 1640 plus 10% foetal calf serum (Sigma-Aldrich, UK). Some cells were activated by culturing in the presence of 30 ng/mL anti-CD3 antibody OKT3 (BioLegend, San Diego, CA, USA) plus 600 U/mL IL-2 (Chiron, Emeryville, USA). For control samples, the non-activated CD8 + T cells were cultured in the medium for up to 4 days, and they were mechanically tested on Day 0, Day 2 and Day 4.
The contaminating erythrocytes were removed by osmotic lysis. Briefly, 1 ml of distilled water was added to the cell pellet. After 30s, 14 ml of RPMI 1640 (Sigma-Aldrich, St.
Louis, MO, USA) was added, and the cells were washed once. Untouched CD8 + T lymphocytes were isolated by negative immunomagnetic selection, using CD8+ T Cell Isolation Kit and LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer's instructions. Cell purity was checked using a FacsCanto II flow cytometer (BD, San Jose, CA, USA) and in all the cases the CD8 + T lymphocyte purity was above 90%. Enriched CD8 + T lymphocytes were resuspended in RPMI 1640 plus 10% foetal calf serum (Sigma-Aldrich, UK). Some cells were activated by culturing in the presence of 30 ng/mL anti-CD3 antibody OKT3 (BioLegend, San Diego, CA, USA) plus 600 U/mL IL-2 (Chiron, Emeryville, USA). For control samples, the non-activated CD8 + T cells were cultured in the medium for up to 4 days, and they were mechanically tested on Day 0, Day 2 and Day 4.
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