T cells induced on DLL4 proteins were seeded into 48-well plates at a density of 1–10 × 104 cells/well and stimulated with OKT3 anti-CD3 antibody (eBioscience, 500 ng/mL) in alpha-MEM supplemented with 15% FBS, 50 μg/mL PAA, 1% PSG, 10 ng/mL rhIL-7, and 10 nM dexamethasone (DEXART, Fuji Pharma) for 3 days (days 21–24).14 (link),36 (link) OKT3 and dexamethasone were removed by changing the medium (alpha-MEM supplemented with 15% FBS, 50 μg/mL PAA, 1% PSG, and 10 ng/mL rhIL-7), and cells were incubated for another 7 days (days 24–31). Anti-CD8β-PE-Cy7, anti-CD8α-APC, anti-CD45-APC-Cy7, anti-CD4-BV421, and anti-CD3-BV510 antibodies were used for FACS analysis.
Anti cd3 antibody okt3
Manufactured by Thermo Fisher Scientific
Sourced in United States, Switzerland
The Anti-CD3 antibody (OKT3) is a laboratory reagent used in immunological research. It binds to the CD3 complex on the surface of T cells, a key component of the T cell receptor complex. This antibody can be used to activate and study T cell function in various in vitro and ex vivo experimental settings.
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Lab products found in correlation
Interleukin 2 (il 2), by Merck Group (2 mentions)
Rosettesep human cd4 t cell enrichment cocktail, by STEMCELL (1 mentions)
Interleukin 2 (il 2), by Roche (1 mentions)
Anti total erk, by Cell Signaling Technology (1 mentions)
Anti phospho erk, by Cell Signaling Technology (1 mentions)
Anti total p38, by Cell Signaling Technology (1 mentions)
Anti total jnk, by Cell Signaling Technology (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Anti phospho p38, by Cell Signaling Technology (1 mentions)
Macs system, by Miltenyi Biotec (1 mentions)
Human serum, by Merck Group (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Lgm 3, by Lonza (1 mentions)
Ficoll paque, by Thermo Fisher Scientific (1 mentions)
Anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)
Protamine sulfate, by Merck Group (1 mentions)
Optmizer t cell expansion media, by Thermo Fisher Scientific (1 mentions)
Pbmcs, by Cellular Technology (1 mentions)
8 protocols using anti cd3 antibody okt3
1
T Cell Expansion and Characterization
2
ATRA-induced CD4+ T cell activation
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CD4+ T cells were isolated to a purity of 99% from healthy blood donors using RosetteSep Human CD4+ T cell Enrichment Cocktail (Stem Cell Technologies, Canada) as per the manufacturer’s protocol. Isolated cells were cultured with 10 µM all-trans retinoic acid (ATRA) (Sigma-Aldrich, MO, USA), 50 ng/ml OKT3 (anti-CD3 antibody) (eBioscience, CA, USA) and 20 U/ml IL-2 (Roche Applied Sciences, Germany) in RPMI media supplemented with 20% heat inactivated fetal bovine serum for 6 days at 37°C, 5% CO2 [3 (link)]. The purity of the CD4+ T cell population as well as the upregulation of α4β7 by ATRA was confirmed routinely by flow cytometry. Donors were designated as responders or non-responders based on whether ATRA treatment upregulated the α4β7+ population of lymphocytes as shown in Additional file 3 . Only 25% of donors responded to ATRA treatment similar to what was shown in a study by Arthos et al. [3 (link)] and only these donors were used in further experiments.
3
Regulation of T-cell Signaling Pathways
4
Expansion and Isolation of Human T and NK Cells
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Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy donors using Histopaque-1077 (Sigma-Aldrich) density gradient centrifugation. For T cell expansion, PBMCs were re-suspended at 1 × 106 cells/ml in lymphocyte growth medium 3 (LGM-3) (Lonza, Basel, Switzerland) containing 10 ng/ml anti-CD3 antibody (OKT3, eBioscience, San Diego, CA, USA), 500 IU/ml recombinant human interleukin-2 (rhIL-2 (Proleukin®), Novartis Pharmaceuticals Corp., NJ, USA), and 5% human serum (Sigma-Aldrich). On day 5, expanded T cells were transferred to a larger culture flask in LGM-3 medium containing 500 IU/ml rhIL-2 and 5% human serum. Fresh culture medium containing rhIL-2 (500 IU/ml) and 5% human serum was added to the flask every 2 to 3 days for 14 days. Purified NK cells were obtained by depleting non-NK cells with a magnetic activated cell sorting (MACS) system according to the manufacturer’s instructions (Miltenyi Biotec, Bisley, Germany). NK cell purity was assessed by flow cytometry using anti-human CD3 and CD56 mAbs. Purified NK cells were cultured for 7 days in LGM-3 containing rhIL-2 (500 IU/ml) and 5% human serum. All procedures were performed under conditions of good manufacturing practices (GMP).
5
Isolation and Expansion of T Cells
6
PBMC Cytokine Response to LY3300054
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Fresh unstimulated human PBMC isolated from six healthy donors were incubated with plate bound LY3300054 antibody or control antibodies for 24 h, pre-coated over a broad titration range from 0.003 to 100 μg/ml. Anti-CD3 antibody OKT3 (eBioscience, San Diego, CA) was used as a positive control. Using a commercially available multiplex assay based on the Luminex platform (Luminex Corporation, Austin, TX), 21 cytokines including Fractalkine, GM-CSF, IFNγ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, IL-17A, IL-21, IL-23, ITAC, MIP-1α, MIP-1β, MIP-3α, and TNF-α were measured in cell culture supernatants [27 (link)].
7
Lentiviral Vector Production and T-cell Transduction
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High-titer lentiviral vectors were produced and concentrated as previously described16 . Briefly, 20 × 106 293T cells were seeded onto 150-mm2 gelatin-coated plates 24 hours before transfection. Cells were transfected with 22.5 μg GPA7-28z vector, 7.5 μg Plp/VSVG, 15 μg Plp1 and 10 μg Plp2, using Fugene HD transfection reagent (Roche Diagnostics, IN). The viral supernatant was harvested at 48 hours post-transfection and cell debris was removed by centrifugation. Viral particles were subsequently concentrated by ultracentrifugation for 3 hours at 25,000 rpm and resuspended in PBS.
Peripheral blood obtained from healthy donors with informed consent was purified by centrifugation over Ficoll gradients. Peripheral blood mononuclear cells (PBMCs) and T-cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS and 100 IU/mL IL-2. Isolated PBMCs were activated using 5 μg/mL anti-CD3 antibody OKT3 and 2 μg/mL anti-CD28 antibody (eBioscience, San Diego, CA) on day 0. On day 2 and day 3, activated lymphocytes were transduced with concentrated lentiviral vectors at MOI 5, in the presence of 10 μg/mL protamine sulfate (Sigma-Aldrich, St. Louis, MO). Six hours after transduction, the medium was replaced with RPMI-1640 supplemented with 10% heat-inactivated FBS and 100 IU/mL IL-2.
Peripheral blood obtained from healthy donors with informed consent was purified by centrifugation over Ficoll gradients. Peripheral blood mononuclear cells (PBMCs) and T-cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated FBS and 100 IU/mL IL-2. Isolated PBMCs were activated using 5 μg/mL anti-CD3 antibody OKT3 and 2 μg/mL anti-CD28 antibody (eBioscience, San Diego, CA) on day 0. On day 2 and day 3, activated lymphocytes were transduced with concentrated lentiviral vectors at MOI 5, in the presence of 10 μg/mL protamine sulfate (Sigma-Aldrich, St. Louis, MO). Six hours after transduction, the medium was replaced with RPMI-1640 supplemented with 10% heat-inactivated FBS and 100 IU/mL IL-2.
8
IL-17 Gene Expression in PBMCs
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IL-17 gene expression was analyzed in human peripheral blood mononuclear cells (PBMCs). Briefly, PBMCs (Cellular Technologies Ltd., Shaker Heights, OH) were thawed in OpTmizer T-cell expansion media (Life Technologies) supplemented with 20 ng/ml IL-2 (Sigma-Aldrich, St. Louis, MO). Cells were pelleted by centrifugation and resuspended in media recommended by the supplier. Cells were pretreated with RVX-297 or DMSO for 1 hour, then media containing anti-CD3 antibody (OKT3; eBioscience, San Diego, CA) was added at a final concentration of 1 mg/ml. Incubation was continued for 3 hours. IL-17 gene expression was analyzed by real-time PCR. Each data point was derived from three biological replicates, and results were determined in six independent experiments. RVX-297 Ameliorates Inflammation and Autoimmune Disease 695 at ASPET Journals on September 7, 2024 molpharm.aspetjournals.org
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