Recombinant human il 2

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Recombinant human IL-2 is a protein produced using recombinant DNA technology. It is a type of cytokine that plays a key role in the activation and proliferation of T cells, which are important components of the immune system.

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Recombinant human IL-2 (rhIL-2) Recombinant human interleukin (IL)-2 Recombinant human IL-2 protein Recombinant human IL-2 and IL-10 Recombinant human interleukin-2 (IL-2) Recombinant human IL-2 or mouse IL-15 15U recombinant human IL-2 Recombinant human IL (rhIL)-2 Recombinant human IL-2 (200–02) Recombinant IL-2

432 protocols using recombinant human il 2

Isolation and Activation of Human CD4+ T Cells

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CD4⁺ T cells were isolated from human PBMC or buffy coats after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) and a negative selection kit according to the manufacturer’s protocol (either #130–091-155 from Miltenyi Biotech (Bergisch Gladbach, Germany) or #17952 from Stem Cell Technologies (Vancouver, Canada)). Purity of isolated cells was typically >95%, and cells were resuspended in complete RPMI medium (10% FCS, 2mM glutamine (#25030–149, Gibco), 50U/ml Penicillin + 50μg/ml streptomycin (#15070–063, Gibco)). Purified CD4⁺ T cells were activated for the indicated time points in 96- or 48 well culture plates (Greiner, Monroe, NC) precoated with antibodies to CD3 (clone OKT3) and CD46 (clone TRA2.10)^{64 (link)} at 1.5–2 ×10⁵ cells/well (96well) or 3.5–5×10⁵/well (48 well) in media containing 50 U/ml recombinant human IL-2 (Peprotech) in an incubator at 37°C and 5% CO₂. Cells were harvested and assayed at the indicated timepoints.
For restimulation experiments, cells were harvested after 36–48 hours of activation and rested for 5 days in 5U/ml recombinant human IL-2 (Peprotech), then harvested, recounted and re-stimulated in plates precoated with CD3 and CD46 antibodies in media containing 50 U/ml recombinant human IL-2 (Peprotech) for 18 hrs.

West E.E., Merle N.S., Kamiński M.M., Palacios G., Kumar D., Wang L., Bibby J.A., Overdahl K., Jarmusch A.K., Freeley S., Lee D.Y., Thompson J.W., Yu Z.X., Taylor N., Sitbon M., Green D.R., Bohrer A., Mayer-Barber K.D., Afzali B., Kazemian M., Scholl-Buergi S., Karall D., Huemer M, & Kemper C. (2023). Loss of CD4+ T cell-intrinsic arginase 1 accelerates Th1 response kinetics and reduces lung pathology during influenza infection. Immunity, 56(9), 2036-2053.e12.

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In Vitro Induction and Stability of Regulatory T Cells

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Induction of T reg cell differentiation in vitro was conducted according to published protocols (Feng et al., 2015 (link); Yue et al., 2016 (link)). Briefly, cell culture plates or dishes were precoated with 1 µg/ml anti-CD3 (clone 145-2C11; Bio X Cell) and anti-CD28 (clone 37.51; Bio X Cell) antibodies in PBS at 37°C for 2 h and washed with PBS once before cell culture. CD4 Tn cells were grown in complete RPMI1640 supplemented with 100 U/ml recombinant human IL-2 (PeproTech) and 1 ng/ml recombinant human TGF-β (R&D Systems) with or without 0.25 mM ASC-2-phosphate (Sigma) for 4 d.
To assess the stability of Foxp3 expression in vitro, iT reg cells sorted based on GFP-Foxp3 expression after 4 d of induction were further cultured on plates coated with both anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) antibodies and either recombinant human IL-2 or 25 µg/ml IL-2 neutralization antibodies (JES6-54H and S4B6-1; Bio X Cell) for 4 d more. To culture nT reg cells, FACS-sorted nT reg cells were cultured in complete RPMI1640 supplemented with 10% FBS in the presence of Dynabeads Mouse T-Activator CD3/CD28 Beads (Thermo Fisher Scientific) with 500 U/ml recombinant human IL-2.

Zong X., Hao X., Xu B., Crawford J.C., Wright S., Li J., Zhang Y., Bai L., He M., Jiang M., Fan Y., Connelly J.P., Pruett-Miller S.M., Berns H., Janke L., Li C, & Feng Y. (2021). Foxp3 enhancers synergize to maximize regulatory T cell suppressive capacity. The Journal of Experimental Medicine, 218(8), e20202415.

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Culturing Human T-cell Leukemia Cells

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HCT‐5 cells were derived from the cerebrospinal fluid of HAM/TSP patients and were a kind gift from Dr T. Nakamura (Nagasaki International University).²² The HCT‐5 cells were maintained in RPMI‐1640 medium supplemented with 20% FBS and 0.75 μg/ml recombinant human IL‐2 (PeproTech). Blood samples were obtained with informed consent and with the approval of the Institutional Review Board of the Faculty of Medicine, University of Miyazaki. Acute T‐cell leukemia cells were collected from patients at the time of hospital admission before the start of chemotherapy. CD4⁺ lymphocytes were isolated from the blood samples of healthy volunteers by AutoMACS negative selection using a CD4⁺ T‐cell isolation kit (Miltenyi Biotech). The ATL cells were maintained in AIM‐V medium (Thermo Fisher Scientific) supplemented with 20% FBS, 10 mM 2‐mercaptoethanol, and 0.75 μg/ml recombinant human IL‐2.²³

Ichikawa T., Sugamoto K., Matsuura Y., Kunitake H., Shimoda K, & Morishita K. (2022). Inhibition of adult T‐cell leukemia cell proliferation by polymerized proanthocyanidin from blueberry leaves through JAK proteolysis. Cancer Science, 113(4), 1406-1416.

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T Cell Isolation and Expansion for Editing

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T cells for editing were isolated from whole blood via density gradient centrifugation using Biocoll (Biochrom, reference #L6115). Generally, T cells were cultured in RPMI 1640 (LifeTechnologies, reference #31870074) supplemented with 10% FCS (Sigma, reference #F7524), 50 μM 2-mercaptoethanol (Invitrogen, reference #31350-010), 1.34 mM L-glutamine (Sigma, reference #G8540), 5 mM HEPES (Roth, reference #HN77.4), 25 mg gentamycin (LifeTechnologies, reference #15750-037) and 1x penicillin/streptomycin (LifeTechnologies, reference #10378-016) and 180 IU ml^-1 recombinant human IL-2 (Peprotech, reference #200-02) (‘RPMI’ hereafter) unless indicated otherwise. Sorted cells were cultured with 1x10⁶ ml^-1 irradiated (30 Gy) allogeneic feeder peripheral blood mononuclear cells (PBMC), 1 μg ml^-1 PHA (ThermoFisher, reference #R30852801) and 180 IU ml^-1 recombinant human IL-2.
Written informed consent was obtained from the donors, and usage of the blood samples was approved according to national law by the local Institutional Review Board (Ethikkommission der Medizinischen Fakultät der Technischen Universität München). Information about age and gender of donors is not available. The study conforms to the standards of the Declaration of Helsinki.

Müller T.R., Jarosch S., Hammel M., Leube J., Grassmann S., Bernard B., Effenberger M., Andrä I., Chaudhry M.Z., Käuferle T., Malo A., Cicin-Sain L., Steinberger P., Feuchtinger T., Protzer U., Schumann K., Neuenhahn M., Schober K, & Busch D.H. (2021). Targeted T cell receptor gene editing provides predictable T cell product function for immunotherapy. Cell Reports Medicine, 2(8), 100374.

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T Cell Activation and Transduction Protocol

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T cells were thawed in warm water after removal from liquid nitrogen and then washed with T cell medium (AIM-V (Gibco) supplemented with 5% FBS, 10 mM HEPES, 1× penicillin–streptomycin–glutamate and 100 U ml⁻¹ recombinant human IL-2 (Peprotech) or RPMI (Gibco) supplemented with 10% FBS, 10 mM HEPES, 1× penicillin–streptomycin–glutamate and 100 U ml⁻¹ recombinant human IL-2). Human T-Expander αCD3/CD28 Dynabeads (Gibco) were washed and added to T cells at a volume of 30 μl resuspended beads per million T cells. T cells and beads were then resuspended at a concentration of 500,000 T cells per ml in T cell medium (day 0 for all assays). Forty-eight and 72 hours after activation, T cells were transduced (see ‘Retroviral transduction’). Ninety-six hours after activation, beads were removed by magnetic separation using a DynaMag column (Invitrogen). T cells were fed with fresh T cell medium every 48–72 h and were maintained at a density of 0.5 ×10⁶ cells per ml after feeding. For FOXO1_i experiments, T cells were provided with fresh complete T cell medium and vehicle control (dimethyl sulfoxide; DMSO) or AS1842856 (EMD Millipore) every 2–3 days from days 4 to 15 after activation.

Doan A.E., Mueller K.P., Chen A.Y., Rouin G.T., Chen Y., Daniel B., Lattin J., Markovska M., Mozarsky B., Arias-Umana J., Hapke R., Jung I.Y., Wang A., Xu P., Klysz D., Zuern G., Bashti M., Quinn P.J., Miao Z., Sandor K., Zhang W., Chen G.M., Ryu F., Logun M., Hall J., Tan K., Grupp S.A., McClory S.E., Lareau C.A., Fraietta J.A., Sotillo E., Satpathy A.T., Mackall C.L, & Weber E.W. (2024). FOXO1 is a master regulator of memory programming in CAR T cells. Nature, 629(8010), 211-218.

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Antigen-Specific T Cell Activation Assay

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Naïve CD4⁺CD25⁻ T cells were isolated from the spleens of OT-II mice using a CD4⁺ CD25⁻ T cell isolation kit (Miltenyi Botec Inc.). Both tDCs and cDCs were generated from BM cells of C57BL/6 mice, matured in the presence of 50 ng/mL IFN-γ and 50 ng/mL TNF-α for 24 h, and then pulsed with the OVA323–339 peptide (10 μg/ml) for 1 h. After washing with PBS, the DCs (2 × 10⁴ cells/well) were co-cultured with purified OT-II CD4⁺ CD25⁻ T cells (2 × 10⁵ cells/well) for 4 days in a medium containing 100 U/ml of recombinant human IL-2 (PeproTech Inc.). In allogeneic MLR, the DCs (2 × 10⁴ cells/well) generated from BM cells of C57BL/6 mice were cultured with naïve CD4⁺CD25⁻ T cells (2 × 10⁵ cells/well) isolated from the spleens of BALB/c mice for 4 days in a medium containing 10 U/ml of recombinant human IL-2 (PeproTech Inc.). In IL-10 blocking experiments, anti-IL-10 monoclonal antibody, or isotype-matched monoclonal antibody (10 μg/ml, both from BD Biosciences, San Jose, CA, USA) was added from the initiation of the culture.

Lee J.H., Park C.S., Jang S., Kim J.W., Kim S.H., Song S., Kim K, & Lee C.K. (2017). Tolerogenic dendritic cells are efficiently generated using minocycline and dexamethasone. Scientific Reports, 7, 15087.

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Optimizing IL-2 Stimulation for Flow Cytometry

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Human PBMCs or murine splenocytes (1×10⁶ cells/ml in staining buffer) were stimulated with human recombinant IL-2 (Peprotech EC ltd., London, UK) because recombinant human IL-2 has been proven to be also efficient in mouse [32 (link), 33 (link)]. For all experiments other than dose-response curves, a concentration of 10 IU/ml was used, while for dose-response curve experiments, an 8-log range of concentrations was used for human and a 9-log range was used for murine experiments. Samples were stimulated for 15 min at 37°C and were immediately washed twice with staining buffer before processing for flow cytometry staining. This time of exposure was chosen based on _phosphoSTAT5 levels observed in lymphocytes after different times of stimulation with 10 IU/ml (Supplemental Figure 5), 15 min corresponding to a time point of maximal response.

Ehx G., Hannon M., Beguin Y., Humblet-Baron S, & Baron F. (2015). Validation of a multicolor staining to monitor phosphoSTAT5 levels in regulatory T-cell subsets. Oncotarget, 6(41), 43255-43266.

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Profiling T-Cell Cytokine Response to IL-36α

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Like the previously published researches (7 (link), 18 (link)), the separated PBMCs from GD patients or controls were divided into two equal parts (1~2 × 10⁶ /ml). Both of them were seeded into culture dishes with a diameter of 35 mm. One was incubated with recombinant human IL-2 (50 ng/ml) (PeproTech, USA) and 100 U/ml penicillin/streptomycin (Hyclone, USA) in the presence of recombinant human IL-36α (100 ng/ml) (R&D system, USA) at a 37°C incubator supplemented with 5% CO2. Another was incubated only with recombinant human IL-2 (50 ng/ml) (PeproTech, USA) and served as the negative control. After 24 h, we harvested the cultured supernatants of the both and stored them at −80°C. The levels of T-cell-derived cytokines including IFN-γ, TNF-α, IL-6 and IL-17A in supernatants were determined by Human High Sensitivity T Cell Magnetic Bead Panel (Merck Millipore, Germany) according to the manufacturer's instructions.

Yao Q.M., Li L., Song Z.Y., Wang B., Qin Q., An X.F, & Zhang J.A. (2018). Elevated Interleukin-36α And CD4+IL-36α+T Cells Are Involved in the Pathogenesis of Graves' Disease. Frontiers in Endocrinology, 9, 591.

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T Cell Transduction and Expansion Protocol

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Healthy donor PBMCs were cultured in a 12-well plate in C10 medium (1 × 10⁶ cells/mL/well) for 2 days and stimulated with Dynabeads™ Human T-Activator CD3/CD28 (10 μL/mL) (GIBCO, 11161D) and recombinant human IL-2 (20 ng/mL) (Peprotech). After 2 days, Dynabeads™ were removed and cells were spin-infected with frozen-thawed Lenti/ESO-TCR, Lenti/BCAR, or Lenti/MCAR viruses supplemented with polybrene (10 μg/mL) at 660× g at 30 °C for 90 min following an established protocol. Virus-transduced T cells were expanded for another 6–8 days in C10 medium supplemented with recombinant human IL-2 (20 ng/mL) (Peprotech) and cryopreserved for future use. CAR or ESO-TCR expression levels on T cells were determined using flow cytometry.

Li Y.R., Yu Y., Kramer A., Hon R., Wilson M., Brown J, & Yang L. (2022). An Ex Vivo 3D Tumor Microenvironment-Mimicry Culture to Study TAM Modulation of Cancer Immunotherapy. Cells, 11(9), 1583.

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Human NK Cell Isolation and Culture

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Primary human NK cells were isolated from fresh peripheral blood (within 24 hours of blood donation) obtained from Hong Kong Red Cross. PBMCs were first prepared from the peripheral blood using Ficoll-Paque PLUS (GE Healthcare), from which NK cells were purified by negative selection using the EasySep Human NK Cell Enrichment Kit (Stemcell). Isolated human NK cells were either used immediately or cultured for 3 days at a density of 1 × 10⁶ cells/ml in RPMI 1640 medium containing 10 ng/ml recombinant human IL-2 (ThermoFisher), 10% heat-inactivated FCS, 100 U/ml penicillin and 100 μl streptomycin, prior to usage. The NK92-MI cell line was purchased from American Type Culture Collection (ATCC) and maintained in MyeloCult H5100 medium (Stemcell).

Zhu Y., Huang B, & Shi J. (2016). Fas ligand and lytic granule differentially control cytotoxic dynamics of natural killer cell against cancer target. Oncotarget, 7(30), 47163-47172.

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