As a source of FVIII-specific T-cells, the murine FVIII-specific T-cell hybridoma 1G8-A2, which was generated by immunizing SureL1 mice with BDD-FVIII (see Supplementary Material ) (18 (link)), and the human FVIII-specific T-cell line D9E9 (from Dr. M. Jacquemin) (29 (link)) were used. Five day old MO-DCs from DRB1∗0101 healthy donors or mitomycin C-treated splenocytes from SureL1 mice were incubated for 24 h with the FVIII-specific T-cell hybridoma 1G8-A2 (10,000 MO-DCs or 200,000 splenocytes for 100,000 T cells) with FVIII in X-VIVO15 medium (Lonza). Levels of secreted interleukin-2 (IL-2) were assessed using BD OptEIA mouse IL-2 ELISA set (BD Biosciences). When the human FVIII-specific T-cell line D9E9 was used, 5,000 T cells were incubated with either MO-DCs from a DRB1∗1501 donor, or with the FVIII-specific human B-cell line (BO2C11) expressing the DRB1∗1501/DRB5∗0101 alleles (10,000 cells) (30 (link)) in DMEM-F12 media (Lonza) containing 10% FCS, 20 IU/ml human IL-2 (Sigma Aldrich), and FVIII fragments for 20 h at 37°C. When indicated, MO-DCs or BO2C11 were pre-incubated 30 min at 37°C with EDTA (5 mM) or mannan (1 mg/ml) prior to incubation with FVIII and T cells. The production of interferon-gamma was measured in the supernatants using the human IFN-gamma Duo Set (R&D Systems, Minneapolis, Minnesota, United States).
Human il 2
Manufactured by Merck Group
Sourced in United States, Sweden, France
Human IL-2 is a recombinant protein that serves as a laboratory reagent for research purposes. It is the human version of the interleukin-2 cytokine, which plays a crucial role in the immune system's T-cell function. This product is intended for use in various in vitro experiments and assays involving the study of T-cell biology and immunology.
Automatically generated - may contain errors
Lab products found in correlation
Lgm 3, by Lonza (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Dmem f12 media, by Lonza (1 mentions)
Opteia mouse il 2 elisa set, by BD (1 mentions)
Human ifn gamma duoset, by R&D Systems (1 mentions)
X vivo 15 medium, by Lonza (1 mentions)
T cell transact, by Miltenyi Biotec (1 mentions)
Ficoll medium, by Merck Group (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Dynal cd8 isolation kit, by Thermo Fisher Scientific (1 mentions)
Anti cd3 and anti cd28 antibody, by Thermo Fisher Scientific (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Il 15, by ProSpec (1 mentions)
X vivo 10, by Lonza (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
N acetyl l cysteine, by Merck Group (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Ficoll paque, by GE Healthcare (1 mentions)
Dnazol, by Thermo Fisher Scientific (1 mentions)
9 protocols using human il 2
1
Assessing FVIII-specific T-cell Responses
2
PBMC activation and co-culture with cancer cells
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Human peripheral blood mononuclear cells (PBMCs) were collected from blood of healthy donors by density gradient centrifugation with Ficoll medium (Sigma, F4357). Isolated PBMCs were stored in 90% FBS with 10% DMSO at −80 °C.
PBMCs were activated by 1:100 diluted T Cell TransAct (Miltenyi Biotec, 130‐111‐160) and 20 IU ml−1 human IL‐2 (Sigma, SRP3085). Activated PBMCs were cultured in RPMI 1640 medium (Gibco, 11875101) supplied with 5% FBS, 80 U ml−1 penicillin and 0.08 mg ml−1 streptomycin at 37 °C with 5% CO2.
SKBR3 and SKBR3_HR cells were seeded in 96‐well plate with DMEM for 24 h before co‐culture with PBMCs. Activated PBMCs were added to each well at a ratio of 20:1 with different trastuzumab concentrations, and PGE2 was added to the medium of SKBR3 cells to reach the same concentration as SKBR3_HR cells. After another 48 h incubation, PBMCs were removed, and the viability of cancer cells were evaluated by CCK‐8 assay.
PBMCs were activated by 1:100 diluted T Cell TransAct (Miltenyi Biotec, 130‐111‐160) and 20 IU ml−1 human IL‐2 (Sigma, SRP3085). Activated PBMCs were cultured in RPMI 1640 medium (Gibco, 11875101) supplied with 5% FBS, 80 U ml−1 penicillin and 0.08 mg ml−1 streptomycin at 37 °C with 5% CO2.
SKBR3 and SKBR3_HR cells were seeded in 96‐well plate with DMEM for 24 h before co‐culture with PBMCs. Activated PBMCs were added to each well at a ratio of 20:1 with different trastuzumab concentrations, and PGE2 was added to the medium of SKBR3 cells to reach the same concentration as SKBR3_HR cells. After another 48 h incubation, PBMCs were removed, and the viability of cancer cells were evaluated by CCK‐8 assay.
3
Isolation and Activation of CD8+ T Cells
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Four healthy males (three in their twenties and one in his fifties) and one female (in her twenties) donated 10 cc of blood in compliance with the protocol approved by the IRB of Sookmyung Women’s University (SM-IRB-08-0225). CD8+ T cells were purified using the Dynal CD8+ Isolation Kit (Invitrogen, USA) and activated by the treatment of Dynabeads conjugated with anti-CD3 and anti-CD28 antibodies (Invitrogen). The Roswell Park Memorial Institute (RPMI) medium was replaced every two days with fresh supplements of human IL-2 (60 UI/ml; Sigma-Aldrich, USA) and IL-15 (5 ng/ml; ProSpec, USA). At the start of the activation, 5 mM NAM was added. The individual donors provided written informed consent to publish these case details.
4
Cell culture protocol for various cell lines
5
Generating Anti-BCMA CAR-T Cells
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CD8+ T cells were selected from peripheral blood of healthy donors or patients. T cells were activated and expanded over time to achieve a high yield of activated CD8+ cells followed by transfection with anti-BCMA mRNA. The mRNA was a codon-optimized sequence containing the following sections (5’ to 3’); 3’UTR, human CD8 signal peptide, mouse kappa variable region, scFv linker, mouse IgH variable region, human CD8 hinge/transmembrane region, human CD28 signal domain, human CD3 signal domain, mouse alpha globin 5’UTR, and poly(A) tail (Figure 1A ). The mRNA is synthesized by in vitro transcription from a linearized DNA plasmid. Mock RNA electroporated T cells are used as controls. Upon manufacturing, cells were frozen in cryomedia containing 10% DMSO, and were stored at −80°C until the day of use. Upon thawing, cells were incubated with LGM3 (Lonza), 5% Serum, and 2 ng/ml of human IL-2 (I2644, Sigma).
6
Enhancing T Cell-Mediated Tumor Killing
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Peripheral blood mononuclear cells (PBMCs) were isolated from the fresh blood of healthy donor by density gradient centrifugation using Ficoll‐Paque (GE Healthcare, Uppsala, Sweden) The PBMCs were cultured in AIM‐V medium (Thermo Fisher Scientific) with 200 IU/mL of human IL‐2 (Sigma‐Aldrich) for 7 days. Fresh medium and IL‐2 were replenished every 3 days. After 7 days of culture, the IL‐2 activated lymphocytes including T cells, expressing PD‐1 (data not shown),29 were used for coculture experiments. The IL‐2 activated lymphocytes were cocultured with the GSK‐3 inhibitor or DMSO‐treated tumor cells at 1:1 ratio in 24‐well plates for 48 hours. After 48‐hour incubation, the proportion of apoptotic CD3‐positive cells, T cells, were analyzed with Annexin V and 7‐AAD using flow cytometry. The 3 independent coculture experiments were performed. This study was also approved by the Institutional Review Board of Fukushima Medical University.
7
Generating Anti-BCMA CAR-T Cells
8
HIV-1 Infection Kinetics in Cell Lines
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The CEM and CEM-SS cells were cultured in RPMI 1640 media (Hyclone) plus 10% heat inactivated FBS (Gibco) medium containing 1% PenStrep (Hyclone) [122] (link). For stimulation, 10 ng/mL human IL-2 (Sigma) and 10 μg/mL PHA-L (Roche) was incubated with cells for 3 days. Cells were then harvested and 8 × 105 cells/mL of cells were dispensed to tubes and infected with 0.25 MOI of HIV-1LAI. The spreading infection then progressed as described for PBMCs. Samples were taken every three days for the duration of thirty days. The final supernatant was removed and the cellular genomic DNA was harvested by DNAzol (Invitrogen) treatment, following manufacturer protocol, for provirus sequencing. The frozen virus aliquots were subsequently used for RT assay and viral RNA extraction for cDNA synthesis.
9
Cell Lines Used in Lymphoma Study
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In addition to the LCLs established in our laboratory, seven other lines were used, the characteristics of which are mentioned in Table 3 . B95-8 and the four BL lines (Jijoye, Namalwa, P3HR1, and Raji) were purchased from the ATCC (Catalog numbers CRL 1612—ECACC 85011419, CCL-87, CRL-1432, HTB-62, and CCL-86 respectively, Manassas, VA, USA). The extranodal NK/T cell lymphoma lines (MEC04 and SNK6) were kindly provided by Marion Travert (Inserm U955, Hôpital Henri Mondor, Créteil, France). All lines were grown in RPMI1640 medium with glutaMAX (ThermoFisher Scientific, Illkirch-Graffenstaden, France; catalog number 61870-010) supplemented with 10% fetal bovine serum (FBS; Eurobio Scientific, Les Ulis, France; catalog number CVFSVF00-0U) and 1% penicillin-gentamicin at 37 °C in a humidified 5% CO2 atmosphere. MEC04 and SNK6 cell lines were cultured under the same conditions and supplemented with 100 U/mL of human IL-2 (Sigma-Aldrich, Saint-Quentin Fallavier, France; catalog number I7908).
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