Purified CD4 T cells were stimulated with 10μg/ml plate bound anti-CD3ε (clone 145-2C11) and 2μg/ml soluble anti-CD28 (37.51) in RPMI 1640 as before in flat bottom 96 well plates. For Th1 polarisation, cells were also treated with 15ng/ml recombinant mouse IL-12 and 5μg/ml anti-IL-4 (11B11), or for Th2 polarisation, 30ng/ml recombinant mouse IL-4 and 5μg/ml anti-IFNγ (XMG1.2). Phenylephrine hydrochloride (Sigma) was used at 10μM and added during both anti-CD3 dependent activation (4 days) and also during rest in 10U/ml recombinant human IL-2 (2 days). All antibodies were from Biolegend and were low endotoxin / azide free, and recombinant cytokines were from Peprotech.
Recombinant human il 2
Manufactured by Merck Group
Sourced in United States
Recombinant human IL-2 is a laboratory product that is a cytokine, specifically interleukin-2. It is produced using recombinant DNA technology. Interleukin-2 is a protein that plays a role in the immune system by stimulating the growth and function of certain types of white blood cells.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (3 mentions)
Recombinant human il 2, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Pha p, by Merck Group (2 mentions)
40 m cell strainer, by Avantor (2 mentions)
Magnetic bead positive selection, by Miltenyi Biotec (2 mentions)
Dynabeads mouse t activator cd3 cd28, by Thermo Fisher Scientific (2 mentions)
My la cd8 cells, by Merck Group (2 mentions)
Human ab serum, by Merck Group (2 mentions)
Recombinant cytokines, by Thermo Fisher Scientific (1 mentions)
Phenylephrine hydrochloride, by Merck Group (1 mentions)
Interleukin 2 (il 2), by Corning (1 mentions)
Golgi stop reagent, by BD (1 mentions)
K562 cell, by American Type Culture Collection (1 mentions)
Rosettesep, by STEMCELL (1 mentions)
Rhil 4, by R&D Systems (1 mentions)
Recombinant human gm csf rhgm csf, by R&D Systems (1 mentions)
33 protocols using recombinant human il 2
1
CD4+ T cell polarization protocol
2
NK Cell Degranulation Assay with PBMC
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On day 6 PBMC, either GAD65 AA 114–122 or FLU peptides stimulated for 4 days, additionally cultured for two days in the presence of IL-2 (100 IU/ml), were co-cultured with pulsed or unpulsed APCs at 1:3 (PBMC:APCs) ratio in 96 well round bottomed culture plates (Corning Incorporated, Corning, NY 14831–001, USA) for 2 and half hours in RPMI 10% FBS complete medium (see above) additionally supplemented with GolgiStop reagent (1:500 dilution, BD Biosciences). An experimental positive control was set-up by NK cell isolation from PBMC of a HD volunteer previously obtained with the RosetteSep method (Stem Cell Technologies, Vancouver, Canada) and FicollPaque Plus (Lympholyte, Cedarlane Laboratories, Burlington, Ontario, USA). Isolated NK cells have been routinely checked for the percentage of CD3-CD56+ cells by FACS analysis and those with purity greater than 90% were cultured with 200 IU/ml of recombinant human IL-2 (Sigma-Aldrich) at 37°C and used up to 5 days after isolation as effectors in degranulation assay. Isolated NK cells were then co-cultured with K562 cells (American Type Culture Collection, ATCC), a tumoral cell line known to induce NK cell degranulation according to standard protocols, as control target [41 (link)], and with either GAD65 AA 114–122 peptide pulsed and unpulsed APCs.
3
Peripheral Blood Mononuclear Cell Isolation and Culture
4
DAPT Modulation of Regulatory T Cells
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DAPT (CalBiochem, Merck Chemicals International, Germany) was reconstituted in DMSO to a concentration of 10 mM. CD4+ T cells were stimulated with DAPT at a final concentration of 20 μM for 24 h in the presence of anti-CD3/CD28 (eBioscience, San Diego, CA, U.S.A.; final concentration 1 mg/ml), and cells and supernatants were harvested for further experiments. Cells with DMSO stimulation were used as controls. In certain experiments, purified CD4+CD25+CD127dim/− Tregs were stimulated with DAPT or DMSO for 24 h. Cells were washed twice, and 2.5×104 of Tregs were co-cultured with autologous 105 of CD4+CD25− T cells in the presence of anti-CD3/CD28 (eBioscience) for 96 h with replacement of fresh medium containing 20 U/ml of recombinant human IL-2 (Sigma–Aldrich) 48 h post mixture.
5
Cytotoxic T-Cell Responses Evaluation
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Assays were performed according to standard protocols.60 (link) Briefly, lymphocytes were isolated from harvested spleen of 3 mice per group 5 d after the final vaccination. Cells were resuspended to 2 × 106 cells/mL and were stimulated with CEA peptide pools along with 20 IU/mL recombinant human IL-2 (Sigma). Five days later, these in vitro stimulated cells were used as CTL effector cells, and the CTL activity was determined by a standard 6 h 51Cr-release cytotoxicity assay using the indicated cell lines as targets. 51Cr labeled cells were then added to wells for 6 h at an effector to target cell ratio = 100. Specific lysis was calculated as (experimental 51Cr release − spontaneous 51Cr release)/(maximal 51Cr release − spontaneous 51Cr release) × 100.
6
IL-2 Maintenance of HepG2 and QJY-7703 Cells
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HepG2 and QJY‐7703 cells were purchased from Tongpai Biotechnology (Shanghai, China) and were maintained in Dulbecco's Modified Eagle Medium (DMEM, Wisent, CA, USA) containing 10% fetal bovine serum (FBS) (ExCell Bio, China). Recombinant human IL-2 was purchased from Sigma-Aldrich LLC.
7
Analysis of T Cell Subsets and Activation Markers
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Recombinant human TNF was purchased from Leinco Technologies (St Louis, MO, USA), and recombinant human IL-2 was purchased from Sigma-Aldrich (St Louis, MO, USA). Monoclonal antibodies against TNFR2 were produced in house or purchased from commercial vendors as previously described.8 (link) Fluorochrome-conjugated mAbs against human CD4 (RPA-T4), CD25 (M-A251), CD45RA (HI100, 2H2), CD45RO (UCHL1, 4HB), CD127 (hIL-7R-M21), HLA-DR (L243 (G46-6)) were purchased from BD Biosciences. Fluorochrome-conjugated monoclonal antibodies against CD4 (S3.5, Invitrogen, Carlsbad, CA, USA), CD120a (16803 R&D systems), CD120b (22235, R&D systems, Minneapolis, MN, USA), FOXP3 (259D, Biolegend, San Diego, CA, USA) were also used in this study.
Intracellular staining of FOXP3 and CD152 was performed using either FOXP3 Fix/Perm Buffer set (Biolegend) or Human FoxP3 Buffer set (BD Biosciences) according to the manufacturer's instructions. Flow cytometric data were obtained using FACSCalibur (BD Biosciences) flow cytometer. All the data were analyzed with Cellquest Software (BD Biosciences, San Jose, CA, USA).
Intracellular staining of FOXP3 and CD152 was performed using either FOXP3 Fix/Perm Buffer set (Biolegend) or Human FoxP3 Buffer set (BD Biosciences) according to the manufacturer's instructions. Flow cytometric data were obtained using FACSCalibur (BD Biosciences) flow cytometer. All the data were analyzed with Cellquest Software (BD Biosciences, San Jose, CA, USA).
8
Multicolor Flow Cytometry Immunophenotyping
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FITC‐labeled anti‐human CD279 (PD‐1; EH12.2H7), PE‐labeled anti‐human CD56 (HCD56), PerCP‐labeled anti‐human CD3 (SK7), and PE‐labeled Mouse IgG1κ Isotype control (MOPC‐21) antibodies were purchased from BioLegend (San Diego, CA). Anti‐human CLEC‐1 goat polyclonal, anti‐human CLEC‐2 goat polyclonal, and goat polyclonal control IgG antibodies were purchased from R&D Systems (Minneapolis, MN). Phalloidin‐Alexa Fluor 568, deoxyribonuclease I‐Alexa Fluor 488, Hoechst33342, and Calcein‐AM were obtained from Thermo Fisher Scientific (Waltham, MA). Recombinant human IL‐2 was purchased from Sigma‐Aldrich (St. Louis, MO). For NK cell incubation, IL‐2 concentrations were adjusted to 200 U/mL in RPMI‐1640 medium.
9
Transduction of Primary Human T Cells
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Cryopreserved purified peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed and resuspended in 10% FBS RPMI supplemented with 500 U/ml recombinant human IL-2 (PeproTech) and activated with plate-bound anti-CD3 (UCHT1, homemade) and anti-CD28.2 (BioLegend) (1 μg/ml). 48-72 h after activation, >99% of the cells were T cells. Activated primary human T cells were lentivirally transduced using spin infection with a virus MOI of 4 together with 5 μg/ml of protamine sulfate (Sigma) and 500 U/ml of recombinant human IL-2. 8-12 days after transduction, CAR surface levels were determined by flow cytometry. After transduction, cells were grown in 10% FBS RPMI supplemented with 100 U/ml IL-2 before being used for the indicated experiments.
10
Antigen-Specific CD4+ T-cell Isolation
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SJL/J mice (6–8 weeks old, female) were immunized subcutaneously with 100 μg of S18 emulsified in CFA. At the end of 2 weeks splenocytes were harvested and co-cultured with stimulators at a ratio of 1:1. Stimulators were prepared by loading healthy mice derived irradiated splenocytes with S18 (10 μg/ml). Co-cultures were plated (5 million cells/ml) in primary culture medium (DMEM supplemented with 10% FBS, 2 mM GlutaMax, Anti-anti, 0.5 μM β-mercaptoethanol and 10 U/ml of recombinant human IL-2 (Sigma)). Cultures were replenished with IL-2 every 3–4 days and re-stimulated weekly using stimulators. At the end of 2 weeks S18 reactive CD4+ T-cells were isolated from mixed culture by magnetic sorting (Miltenyi Biotec) and infused (20 million cells/mice) a day prior to EAE induction.
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