Interleukin 2 (il 2)

Manufactured by Miltenyi Biotec

Sourced in Germany, United States, United Kingdom, Canada, France, Japan

IL-2 is a recombinant human interleukin-2 protein. Interleukin-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells, natural killer cells, and other immune cells.

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Interleukin (IL)-2 (3 citations) Interleukin 2 (IL-2) (2 citations) Recombinant human interleukin IL-2 (2 citations)

276 protocols using interleukin 2 (il 2)

Generation of CAR-T Cells Targeting B7H6

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Human peripheral blood mononuclear cells (PBMCs) were isolated from whole blood or leukapheresis of healthy donors by a ficoll density gradient and activated for 2 days with OKT-3 (Miltenyi) in the presence of IL-2 (Miltenyi). Activated cells were then transduced with a retroviral vector coding for the anti-B7H6 CAR fused to a Furin T2A cleaving site in frame with a truncated human CD19 marker (tCD19) described in [11 (link)] or a vector coding for the truncated CD19 vector described in [11 (link)]. Two days later, transduced T cells were enriched using CD19-specific magnetic beads (Miltenyi, 130-050-301), and then expanded for an additional 4 days together with IL-2. At day 8, harvested cells were evaluated for cell number and viability prior to cryopreservation. Surface expression of the CAR on T cells was validated by flow cytometry using recombinant human Fc-tagged B7H6 proteins and a secondary antibody recognizing the Fc part of human IgG. All human studies have been approved by the appropriate ethics committee.

Obiedat A., Charpak-Amikam Y., Tai-Schmiedel J., Seidel E., Mahameed M., Avril T., Stern-Ginossar N., Springuel L., Bolsée J., Gilham D.E., Dipta P., Shmuel M., Chevet E., Mandelboim O, & Tirosh B. (2019). The integrated stress response promotes B7H6 expression. Journal of Molecular Medicine (Berlin, Germany), 98(1), 135-148.

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Treg Differentiation and Polarization

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Tregs were stimulated with irradiated (40 Gy) L cells (mouse fibroblasts expressing human CD80, CD32, and CD58 [30] ) loaded with 100 ng/mL mouse a-human CD3 mAb (clone: OKT3, AbLab [Vancouver, Canada], UBC, or HIT3a, BD Biosciences [Mississauga, Canada]) at a 1:1 ratio in OpTmizer T-Cell Expansion Serum-free Medium (Life Technologies) with l-Glutamax (Life Technologies), 1% penicillin/streptomycin, 1000 IU/mL IL-2 (Miltenyi Biotec or Proleukin, San Diego, CA), and 100 ng/mL rapamycin (EMD Chemicals, Inc., Billerica, MA) at 378C/5% CO 2 . Tconv were expanded without rapamycin. On day 7, cells were restimulated with a-CD3-loaded L cells and 1000 IU/mL IL-2 (standard conditions) with or without 10 ng/mL IL-12 (Th1-polarizing conditions) or 10 ng/mL IL-1b, 10 ng/mL IL-6, 10 ng/mL IL-23, and 20 ng/mL tumor necrosis factor (TNF)-a (Th17-polarizing conditions; all Miltenyi Biotec) (30, 31) . On day 11, supernatants were stored at -308C. Expanded cells were washed and rested overnight in expansion medium with 1000 IU/mL IL-2 at 378C/5% CO 2 . Cell number and viability were analyzed using propidium iodide staining on MACSQuant Analyzer (Miltenyi Biotec) or trypan blue staining.

Dijke I.E., Hoeppli R.E., Ellis T., Pearcey J., Huang Q., McMurchy A.N., Boer K., Peeters A.M., Aubert G., Larsen I., Ross D.B., Rebeyka I., Campbell A., Baan C.C., Levings M.K, & West L.J. (2016). Discarded Human Thymus Is a Novel Source of Stable and Long-Lived Therapeutic Regulatory T Cells. American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons, 16(1).

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Isolation and Activation of NK and T Cells

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PBMCs isolated from HLA-A24⁺ and HLA-A24⁻ healthy donors were conjugated with human CD3 beads (Miltenyi Biotec). Negative selection was performed with LD columns (Miltenyi Biotec) according to the manufacturer’s protocol. NK cells were generated based on previous reports with modifications.⁵¹ (link) CD3-negative PBMCs were cultured in NS-A2 CTL medium (Nissui) supplemented with 1% PSG (Thermo Fisher Scientific) and a cocktail of 5μM zoledronic acid (Novartis International, Basel, Switzerland) and 0.01 kE/ml OK-432 (Chugai Pharmaceutical, Tokyo, Japan) in the presence of 700 IU/mL IL2 (Miltenyi Biotec) on anti-CD16 antibody (Beckman Coulter)-coated T25 flasks. Three days after culture, floating cells were harvested and transferred to a new T25 flask without coating. The cells were maintained in NS-A2 CTL medium (Nissui) supplemented with 1% PSG in the presence of 700 IU/mL IL2 (Miltenyi Biotec) for 2weeks to obtain activated NK cells for experiments. The remaining PBMCs were stimulated with TransAct (Miltenyi Biotec) following the manufacturer’s protocol and cultured in NS-A2 CTL medium (Nissui) supplemented with 1% PSG (Thermo Fisher Scientific) in the presence of 10 ng/mL IL7 and 10 ng/mL IL15 (both Miltenyi Biotec) for 2weeks to prepare activated T cells for experiments.

Furukawa Y., Ishii M., Ando J., Ikeda K., Igarashi K.J., Kinoshita S., Azusawa Y., Toyota T., Honda T., Nakanishi M., Ohshima K., Masuda A., Yoshida E., Kitade M., Porteus M., Terao Y., Nakauchi H, & Ando M. (2023). iPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer. Cell Reports Medicine, 4(12), 101327.

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Antiviral Activity of CD8+ T Cells

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Autologous CD4⁺ and CD8⁺ T cells were purified from freshly isolated PBMCs or tissue cell suspensions by positive and negative selection, respectively, using nonhuman primate CD4⁺ MicroBeads and the CD8⁺ T-Cell Isolation Kit, with the MultiMACS™ Cell24 Separator (Miltenyi Biotec). Purified CD4⁺ T cells were stimulated for 3 days with concanavalin A (5 μg/mL, Sigma-Aldrich) in the presence of IL-2 (100 IU/mL, Miltenyi Biotec). Purified CD8⁺ T cells were cultured in the absence of mitogens and cytokines (ex vivo CD8⁺ T cells). Stimulated CD4⁺ T cells (10⁵) were superinfected in U-bottom 96-well plates with SIVmac₂₅₁ (MOI = 10⁻³) in the presence (1:1 effector-to-target-cell ratio) or absence of ex vivo CD8⁺ T cells (10⁵) from the same tissue via spinoculation^{75 (link)} for 1 h (1200 × g at room temperature) and incubated for 1 h at 37 °C. Cells were then washed and cultured in R10 medium containing IL-2 (100 IU/mL, Miltenyi Biotec). Culture supernatants were assayed on day 7 using an SIV p27 Antigen ELISA Kit (XpressBio). Antiviral activity was calculated as log10 (mean p27 ng/mL in SIV-infected CD4⁺ T-cell cultures without ex vivo CD8⁺ T cells)/(mean p27 ng/mL in SIV-infected CD4⁺ T-cell cultures with ex vivo CD8⁺ T cells)^{39 (link)}.

Passaes C., Desjardins D., Chapel A., Monceaux V., Lemaitre J., Mélard A., Perdomo-Celis F., Planchais C., Gourvès M., Dimant N., David A., Dereuddre-Bosquet N., Barrail-Tran A., Gouget H., Guillaume C., Relouzat F., Lambotte O., Guedj J., Müller-Trutwin M., Mouquet H., Rouzioux C., Avettand-Fenoël V., Le Grand R, & Sáez-Cirión A. (2024). Early antiretroviral therapy favors post-treatment SIV control associated with the expansion of enhanced memory CD8+ T-cells. Nature Communications, 15, 178.

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Isolation and Culture of Primary Human Cells

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293T and MAGI cells were maintained as previously described [45 (link)]. Primary human monocytes and CD4⁺ T cells were isolated from peripheral blood buffy coats by positive selection using CD14 or CD4 beads (Miltenyi Biotec, San Diego, CA), as previously described [46 (link)], and maintained in RPMI medium with 10 % FCS and penicillin/streptomycin. CD14⁺ monocytes were matured to macrophages using 5 ng/mL human GM-CSF for 7 days (Miltenyi Biotec). CD4⁺ T cells were activated with 5 µg/mL phytohemagglutinin (Sigma-Aldrich) and 20 units/mL IL2 (Miltenyi Biotec) for 1 day, then 20 units/mL IL2 alone for 5 days.

Daly M.B., Roth M.E., Bonnac L., Maldonado J.O., Xie J., Clouser C.L., Patterson S.E., Kim B, & Mansky L.M. (2016). Dual anti-HIV mechanism of clofarabine. Retrovirology, 13, 20.

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Lentiviral Transduction of Primary Human T Cells

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Primary human T cells were isolated from healthy human donors either from fresh whole blood or from buffy coats obtained from the Australian Red Cross Lifeblood. All healthy donors provided informed consent. PBMCs were isolated by Ficoll-Paque (GE Healthcare, IL, USA)-mediated centrifugation using Leucosep tubes (Greiner Bio-One, Kremsmünster, Austria) as per the manufacturer’s instructions. PBMCs were cryopreserved prior to use. For use in transductions, PBMCs were thawed and isolated using Dynabeads human T-expander CD3/CD28 beads (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently activated for 72 h at a bead-to-cell ratio of 3:1 in the presence of 200 U/mL IL-2 (Miltenyi Biotec, Bergisch Gladbach, Germany). Following activation, bead-free T cells were incubated with lentiviral particles in RetroNectin (Takara Bio, Kusatsu, Japan)-coated plates for 48 h at a multiplicity of infection (MOI) of 50. T cell cultures were transferred to complete T cell expansion media comprising of IL-2, IL-7, IL-15, IL-21 (Miltenyi Biotec, Bergisch Gladbach, Germany), human AB (hAb) serum (Sigma-Aldrich, St. Louis, MO, USA) and Stemulate (Cook Regentec, Indianapolis, IN, USA) in TexMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) for continued expansion. Activated but non-transduced T cells (T cells [no CAR]) were maintained in parallel for all donors analyzed.

Shu R., Evtimov V.J., Hammett M.V., Nguyen N.Y., Zhuang J., Hudson P.J., Howard M.C., Pupovac A., Trounson A.O, & Boyd R.L. (2021). Engineered CAR-T cells targeting TAG-72 and CD47 in ovarian cancer. Molecular Therapy Oncolytics, 20, 325-341.

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Expansion and Isolation of Pan-T Cells

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Pan-T cells isolated from blood samples obtained from the Stanford Blood Center were thawed and activated by adding human T-cell activation and expansion MACSi Beads at 1:2 bead-to-cell ratios for 72 hours. On day 3 after activation, cells were transferred to a Grex-100 flask (Wilson Wolf). The interleukin (IL)-2 (Miltenyi Biotec) was added at a final concentration of 100 units/mL. Additional IL-2 was added every three days. Media (300 mL) was removed and replaced on Day 7 after activation. On Day 11, T cells were spun at 1500 RPM for 10 minutes and resuspended in culture media. Activation beads were removed with a magnet, and viability and the number of T cells was determined. Cells were spun again and resuspended in cold PBS for injection.

Zhu G., Foletti D., Liu X., Ding S., Melton Witt J., Hasa-Moreno A., Rickert M., Holz C., Aschenbrenner L., Yang A.H., Kraynov E., Evering W., Obert L., Lee C., Sai T., Mistry T., Lindquist K.C., Van Blarcom T., Strop P., Chaparro-Riggers J, & Liu S.H. (2019). Targeting CLDN18.2 by CD3 Bispecific and ADC Modalities for the Treatments of Gastric and Pancreatic Cancer. Scientific Reports, 9, 8420.

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Activation of NK and T-cells from Mononuclear Cells

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Mononuclear cells (LP_241; Cellular Technology, Ltd., Cleveland, OH, USA) were activated to generate NK cells by addition of 5 µl/ml/well of mixed CD2 and CD335 beads with 500 U/ml/well IL-2 (all from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) for 2 h. For T-cell activation, the mononuclear cells were stimulated with 2 µg/ml/well anti-CD3 agonistic antibodies (eBioscience; Thermo Fisher Scientific, Inc.) and 1 µg/ml/well anti-CD28 agonistic antibodies (BioLegend, San Diego, CA, USA) with 100 U/ml/well IL-2 (Miltenyi Biotec GmbH) for 2 h.

Kamei R., Yoshimura K., Yoshino S., Inoue M., Asao T., Fuse M., Wada S., Kuramasu A., Furuya-Kondo T., Oga A., Iizuka N., Suzuki N., Maeda N., Watanabe Y., Matsukuma S., Iida M., Takeda S., Ueno T., Yamamoto N., Fukagawa T., Katai H., Sasaki H., Hazama S., Oka M, & Nagano H. (2017). Expression levels of UL16 binding protein 1 and natural killer group 2 member D affect overall survival in patients with gastric cancer following gastrectomy. Oncology Letters, 15(1), 747-754.

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PBMC Expansion and TCR Redirection

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Following isolation from buffy coats, PBMCs were either frozen in 90% FBS + 10% DMSO or underwent MAIT cell expansion as previously described.³³ On Day 12/14 of the MAIT cell expansion, paired donor PBMCs were thawed and cultured in AIM-V (Thermo Fisher) + 2% AB serum and activated for 7 days in the presence of 50 ng/ml anti-CD3 (eBioscience) and 600 IU/ml IL-2 (Miltenyi) to generate ConT cells as previously reported.¹⁷ (link) On Day 7, the concentration of IL-2 was increased to 1,000 IU/ml. All cells underwent TCR redirection by electroporation the following day.

Healy K., Pavesi A., Parrot T., Sobkowiak M.J., Reinsbach S.E., Davanian H., Tan A.T., Aleman S., Sandberg J.K., Bertoletti A, & Sällberg Chen M. (2021). Human MAIT cells endowed with HBV specificity are cytotoxic and migrate towards HBV-HCC while retaining antimicrobial functions. JHEP Reports, 3(4), 100318.

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Expansion of NK, CIK, and gamma-delta T cells

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NK cells were expanded as described [33 (link)]. Briefly, PBMCs were resuspended in AIM-V (Invitrogen) medium with 5 % auto-plasma, 500 U/mL IL-2, 2 ng/mL IL-15 (both from Miltenyi Biotec, Germany), and 1 μg/mL OK432 (Shandong Luya Pharmaceutical Co., China) at a concentration of 1 × 10⁶ cells/mL. PBMCs were cultured in flasks coated with anti-CD16 (Beckman, USA) for 24 h at 39 °C in a humidified 5 % CO₂ atmosphere. The cells were cultured in AIM-V medium supplemented with 5 % auto-plasma, 1000 U/mL IL-2, and 2 ng/mL IL-15 at 37 °C for the next 13 days.
To generate CIK cells, PBMCs were cultured in AIM-V medium with 5 % auto-plasma at 37 °C with 1000 U/mL IFN-γ (Miltenyi Biotec). After 24 h, 100 ng/mL mouse anti-human CD3 monoclonal antibody (Peprotech, USA), 1000 U/mL IL-2, and 1000 U/mL IL-1α (Miltenyi Biotec) were added. Fresh complete medium and IL-2 supplement (1000 U/mL) were added every three days.
To amplify γδ T cells, PBMCs were cultured in complete medium with 1 μM zoledronate (Zoledronic Acid, Jilin Province Xidian Pharmaceutical Sci-Tech Development Co., China) and 400 U/mL human IL-2. Fresh complete medium and IL-2 supplement (400 U/mL) were added every 2 or 3 days.

Niu C., Jin H., Li M., Xu J., Xu D., Hu J., He H., Li W, & Cui J. (2015). In vitro analysis of the proliferative capacity and cytotoxic effects of ex vivo induced natural killer cells, cytokine-induced killer cells, and gamma-delta T cells. BMC Immunology, 16, 61.

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