Interleukin 2 (il 2)

Manufactured by Novartis

Sourced in Switzerland, United Kingdom, Germany, United States, Canada

IL-2 is a laboratory equipment product that functions as an interleukin-2 protein. Interleukin-2 is a cytokine that plays a crucial role in the regulation of the immune system.

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

181 protocols using interleukin 2 (il 2)

Isolation and Activation of Human CD4+ T Cells

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Blood (120 ml) was collected by venepuncture from two times four donors for RNA-IC and OOPS. Peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll gradient (Pancoll R ) centrifugation and CD4 + T cells were isolated from 1x10 8 cells using CD4 + Microbeads (Miltenyi) to arrive at 2-3x10 7 cells. These were resuspended at 2x10 6 /ml in T cell medium ((AIM-V (Invitrogen), 10% heat-inactivated human serum, 2mM Lglutamine, 10 mM HEPES and 1,25 µg/ml Fungizone), supplemented with 500 ng/ml PHA (Murex) and 100 IU IL-2/ml (Novartis). Cells were dispensed in 24-well plates at 2 ml per well and on day 3 old medium was replaced by fresh T cell medium and cell were harvested and counted at day 4,5. For generating iTreg, naïve CD4 + T cells were isolated from PBMCs using Microbeads (Naive CD4+ T Cell Isolation Kit II, Miltenyi) and resuspended at 1x10 6 cells/ml in T cell medium supplemented with 500 ng/ml PHA (Murex), 500 IU IL-2/ml (Novartis), 500 nM Rapamycin (Selleckchem), and 5 ng/ml TGF-ß1 (Miltenyi). Cells were cultured in 24-well plates at 2 ml per well together with 1x10 6 irradiated (40 Gy) PBMCs derived from three donors. On day 3, old medium was replaced by fresh T cell medium including supplements and the cells harvested and counted at day 5.

Hoefig K.P., Reim A., Gallus C., Wong E.H., Behrens G., Conrad C., Xu M., Ito-Kureha T., Defourny K.A.Y., Geerlof A., Mautner J., Hauck S.M., Baumjohann D., Feederle R., Mann M., Wierer M., Glasmacher E., & Heissmeyer V. (2020). Defining the RBPome of T helper cells to study higher order post-transcriptional gene regulation.

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Suppressive Activity of CD8+ CD45RC low/- Tregs

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CD8⁺CD45RC^low/− Tregs suppressive activity was assessed on syngeneic responder CD4⁺CD25⁻ T cells stimulated with allogeneic APCs, cDCs, or pDCs, at 1:1 ratio. CD8⁺CD45RC^low/− Tregs were stimulated overnight with coated anti-CD3 and soluble anti-CD28 MAbs (1 µg/ml each) in medium supplemented with 250U/ml IL-2 (Proleukin^®, Novartis) when indicated. 1,000 U/ml IL-2 (Proleukin^®, Novartis) or 50 µg/ml blocking mAbs or isotypic control mAbs were added at day 0 (Table 1). Transwell membrane (0.4 µM pores) (ThermoFisher Scientific) was used. Proliferation of CFSE-labeled (ThermoFisher Scientific) CD4⁺ responder cells was analyzed by flow cytometry after 5 days of coculture in complete RPMI1640 medium supplemented with 5% AB serum, by gating on CD3⁺CD4⁺ living cells (DAPI negative) and excluding CPD-V450 (ThermoFisher Scientific) labeled CD4⁺Tregs.

Bézie S., Meistermann D., Boucault L., Kilens S., Zoppi J., Autrusseau E., Donnart A., Nerrière-Daguin V., Bellier-Waast F., Charpentier E., Duteille F., David L., Anegon I, & Guillonneau C. (2018). Ex Vivo Expanded Human Non-Cytotoxic CD8+CD45RClow/− Tregs Efficiently Delay Skin Graft Rejection and GVHD in Humanized Mice. Frontiers in Immunology, 8, 2014.

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Measuring NK and CTL Degranulation

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Degranulation of resting and activated NK cells was measured by surface staining of CD107a without (medium-cultured cells) and 3 h after stimulation with K562 cells at a ratio of 1:1 (ref. 41 (link)). The erythroleukemic cell line K562 (ATCC, CCL-243) was used as target cell line. NK cells were cultured in medium containing 600 U ml⁻¹ IL-2 (Novartis) for 48 h to assess degranulation of activated NK cells. CTL (cytotoxic T lymphoblasts) degranulation was evaluated in T lymphoblasts 48 h after stimulation with 1.25 μg ml⁻¹ phytohemagglutinin-L (PHA-L, Sigma) and 200 U ml⁻¹ IL-2 (Novartis). CTL degranulation was calculated by the difference in median fluorescence intensity of CD107a of CTLs stimulated with CD3/CD28 coated microbeads (ThermoFisher Scientific) at a ratio of 1:10 for 3 h and medium-cultured cells.

Schober T., Magg T., Laschinger M., Rohlfs M., Linhares N.D., Puchalka J., Weisser T., Fehlner K., Mautner J., Walz C., Hussein K., Jaeger G., Kammer B., Schmid I., Bahia M., Pena S.D., Behrends U., Belohradsky B.H., Klein C, & Hauck F. (2017). A human immunodeficiency syndrome caused by mutations in CARMIL2. Nature Communications, 8, 14209.

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Lentiviral Transduction of Fabrack-CAR in T Cells

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T cells were stimulated with Dynabeads Human T-Expander CD3/CD28 (Invitrogen) at a 1:3 ratio (T cell:bead) overnight in X-VIVO-15 (Lonza) supplemented with 10% FBS, 2 mM L-glutamine, and IL2/IL15 [50 U/mL IL2 (Novartis), 0.5 ng/mL IL15 (CellGenix)]. Stimulated T cells were then transduced with lentiviral vector (M.O.I. of 1.0) encoding the Fabrack-CAR. Mock and CAR transduced T cells were cultured with indicated cytokines three times a week for 18 days before subsequent analyses. Efficiency of CAR transduction in human T cells was 40%–60% (online supplemental figure S6).

Kuo Y.C., Kuo C.F., Jenkins K., Hung A.F., Chang W.C., Park M., Aguilar B., Starr R., Hibbard J., Brown C, & Williams J.C. (2022). Antibody-based redirection of universal Fabrack-CAR T cells selectively kill antigen bearing tumor cells. Journal for Immunotherapy of Cancer, 10(6), e003752.

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Neoepitope-specific CD8+ T cell expansion

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Putative neoepitope HLA class I multimers were generated using conditional MHC class I ligands and peptide exchange technology [12 (link)]. Healthy donor PBMCs isolated from anonymised National Blood Service apheresis cones were incubated with phycoerythrin (PE)-labelled neoepitope multimers, before enrichment by magnetic-activated cell sorting (MACS) using anti-PE microbeads [Miltenyi Biotec]. Enriched cells were expanded using anti-CD3 mAb (30 ng/ml) [ebioscience] and IL-2 (3000 U/ml) [Novartis]. Neoepitope multimer⁺ CD8⁺ T cell populations were identified by combinatorial-encoded multimer staining [13 (link)]. To establish T cell clones, neoepitope multimer⁺ CD8⁺ cells were single-cell sorted and expanded using anti-CD3 mAb (30 ng/ml) [ebioscience] and IL-2 (3000 U/ml) [Novartis]. T cell clones were confirmed by neoepitope multimer and anti-CD8-APC (1:50) [BD] staining.

Tubb V.M., Schrikkema D.S., Croft N.P., Purcell A.W., Linnemann C., Freriks M.R., Chen F., Long H.M., Lee S.P, & Bendle G.M. (2018). Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies. Journal for Immunotherapy of Cancer, 6, 70.

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PBMC Expansion and T-Cell Activation

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PBMC were isolated as described above. Cells were cultured in RPMI-1640
medium supplemented with L-glutamine (2 mM, Sigma-Aldrich),
penicillin/streptomycin (100 IU/ml penicillin, 100 μg/ml streptomycin
(Sigma-Aldrich)) and 10% FCS (v/v, (Gibco, Massachusetts, USA)). T-cell
expansion was induced by addition of anti-CD3 (OKT3) and anti-CD28 (clone
CD28.2). 100 IU/ml IL-2 (Aldesleukin, Novartis, Frimley, UK) was added to PBMC
suspension after PBMC isolation (day 1) and was replenished every 2–3
days by removing half of the media from the well and replacing with fresh media
containing 200 IU/ml IL-2.

Fisher J., Sharma R., Don D.W., Barisa M., Hurtado M.O., Abramowski P., Porter L., Day W., Borea R., Inglott S., Anderson J, & Pe’er D. (2019). Engineering γδT cells limits tonic signaling associated with chimeric antigen receptors. Science signaling, 12(598), eaax1872.

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CFSE-based T cell proliferation assay

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Total PBMCs or sorted naive T cells (defined as CD3⁺, CD45RA⁺, CCR7⁺ cells) were stained with carboxy-fluorescein diacetate succinimidyl ester (CFSE, Thermo Fisher Scientific) at a final concentration of 2.5 µM for 5 min at room temperature in the dark. Following two washes to remove residual CFSE, 10⁵ cells were stimulated in a 96-well U-bottom plate with anti-CD3–coupled beads (Bio-anti-CD3, OKT3 from eBioscience coupled with anti-Biotin MACSiBeads from Miltenyi Biotec) at a ratio of 4:1, 500 ng/ml soluble anti-CD28 mAb (CD28.2, eBioscience), or with 0.5 ng/ml PMA (Sigma-Aldrich) and 1 µM ionomycin (Sigma-Aldrich). Titration of the anti-CD3–coupled bead-to-cell ratio had been performed earlier (Magg et al., 2021 (link)). For IL-2 (Novartis) rescue experiments, we added 500 IU/ml IL-2. After 4 or 5 d of incubation at 37°C under an atmosphere containing 5% CO₂, the cells were washed and stained with Abs targeting CD3 (SK7), CD4 (SK3), CD8 (RPA-T8), and CD25 (M-A251, all from BD). Naive T cells were sorted with a BD FACSAria II cell sorter.

Lévy R., Gothe F., Momenilandi M., Magg T., Materna M., Peters P., Raedler J., Philippot Q., Rack-Hoch A.L., Langlais D., Bourgey M., Lanz A.L., Ogishi M., Rosain J., Martin E., Latour S., Vladikine N., Distefano M., Khan T., Rapaport F., Schulz M.S., Holzer U., Fasth A., Sogkas G., Speckmann C., Troilo A., Bigley V., Roppelt A., Dinur-Schejter Y., Toker O., Bronken Martinsen K.H., Sherkat R., Somekh I., Somech R., Shouval D.S., Kühl J.S., Ip W., McDermott E.M., Cliffe L., Ozen A., Baris S., Rangarajan H.G., Jouanguy E., Puel A., Bustamante J., Alyanakian M.A., Fusaro M., Wang Y., Kong X.F., Cobat A., Boutboul D., Castelle M., Aguilar C., Hermine O., Cheminant M., Suarez F., Yildiran A., Bousfiha A., Al-Mousa H., Alsohime F., Cagdas D., Abraham R.S., Knutsen A.P., Fevang B., Bhattad S., Kiykim A., Erman B., Arikoglu T., Unal E., Kumar A., Geier C.B., Baumann U., Neven B., Rohlfs M., Walz C., Abel L., Malissen B., Marr N., Klein C., Casanova J.L., Hauck F, & Béziat V. (2022). Human CARMIL2 deficiency underlies a broader immunological and clinical phenotype than CD28 deficiency. The Journal of Experimental Medicine, 220(2), e20220275.

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Expansion and Culture of NK Cell Lines and Primary NK Cells

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The HL cell lines KM-H2, L-428, and L-540, the multiple myeloma cell lines RPMI-8226 and U266, and the AML cell line K562 were cultured in RPMI-1640 (Sigma-Aldrich; St. Louis, MO) supplemented with 10% fetal bovine serum (Gibco; Gaitherburg, MD) at 37 °C in a humidified atmosphere with 5% CO₂. The NK cell lines, NK-92 and haNK (kindly provided by NantKwest, Inc.), were cultured in X-VIVO 10 (Lonza; Anaheim, CA) plus 5% heat-inactivated human AB serum at 37 °C in a humidified atmosphere with 5% CO₂. NK-92 cell culture media was supplemented with 450 U/ml of IL-2 (Proleukin; Novartis), while haNK cells were cultured without IL-2 because of self-production capacity. Primary NK cells were obtained from two different healthy donors. Peripheral blood mononuclear cells were isolated by Lymphoprep (Stemcell Technologies; Vancouver, BC) density gradient centrifugation. Subsequently, NK cells were negatively enriched using a NK Cell Isolation Kit (Stemcell Technologies) according to the manufacturer’s instruction. Isolated NK cells were incubated in AIM-V medium supplemented with 10% human AB serum (Sigma-Aldrich) and 10 ng/mL recombinant human IL-15 (Peprotech; Rocky Hill, NJ) for 2 weeks. Half volume of the medium was replaced twice a week. All experiments using cell lines were performed within 12 weeks of cell culture.

Ernst D., Williams B.A., Wang X.H., Yoon N., Kim K.P., Chiu J., Luo Z.J., Hermans K.G., Krueger J, & Keating A. (2019). Humanized anti-CD123 antibody facilitates NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of Hodgkin lymphoma targets via ARF6/PLD-1. Blood Cancer Journal, 9(2), 6.

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CFSE-based Immune Cell Activation Assay

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Freshly isolated PBMCs and NK cells were stained with 5 µM FITC-conjugated carboxyfluorescein succinimidyl ester (CFSE, BioLegend) in phosphate-buffered saline (PBS) (Life Technologies) at room temperature for 5 min. CSFE stained cells were washed with flow cytometer buffer three times. CSFE-labeled healthy PBMCs and NK cells were cultured in 96-well plates with X-VIVO20% and 1% PS for 3 and 6 days, respectively. Healthy PBMCs were incubated with Human T-activator CD3/CD28 beads at 1:4 ratio (Thermo Fisher Scientific) and IL-2 (Novartis) at 100 IU/mL. Healthy donor NK cells were incubated with 1000 IU/mL IL-2 only. 2×10⁵ PBMCs and 5×10⁴ NK cells were counted for treatment with ADO (Sigma-Aldrich) and 12 μM of A_2BAR antagonist at the same day.

Tay A.H., Prieto-Díaz R., Neo S., Tong L., Chen X., Carannante V., Önfelt B., Hartman J., Haglund F., Majellaro M., Azuaje J., Garcia-Mera X., Brea J.M., Loza M.I., Jespers W., Gutierrez-de-Teran H., Sotelo E, & Lundqvist A. (2022). A2B adenosine receptor antagonists rescue lymphocyte activity in adenosine-producing patient-derived cancer models. Journal for Immunotherapy of Cancer, 10(5), e004592.

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Retroviral Transduction of CD8+ T Cells

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MART-1 (1D3) TCR retrovirus was produced in a packaging cell line^{28 (link)}, and supernatant was either directly used or snapfrozen for later use. NY-ESO and CDK4 TCR retrovirus was produced in Fly cells using polyethylenimine. Peripheral blood mononuclear cells were isolated from healthy donor buffycoats (Sanquin, Amsterdam, the Netherlands) by density gradient centrifugation using Lymphoprep (Stem Cell Technologies). CD8⁺ T cells were purified using CD8 Dynabeads (Thermo Fisher Scientific), activated for 48 h on a non-tissue culture treated 24-well plate that was pre-coated overnight with αCD3 and αCD28 antibodies (eBioscience, 16-0037-85 and 16-0289-85) at 2 × 10⁶ per well. Activated CD8 T cells were harvested and mixed with TCR retrovirus and spinfected on a Retronectin coated (Takara, 25 μg per well) non-tissue culture treated 24-well plate for 2 h at 2000 g. After 24 h, T cells were harvested and maintained in RPMI (Gibco) containing 10% human serum (One Lamda), 100 units per ml of penicillin, 100 μg per ml of streptomycin, 100 units per ml IL-2 (Proleukin, Novartis), 10 ng per ml IL-7 (ImmunoTools), and 10 ng per ml IL-15 (ImmunoTools).

Boshuizen J., Vredevoogd D.W., Krijgsman O., Ligtenberg M.A., Blankenstein S., de Bruijn B., Frederick D.T., Kenski J.C., Parren M., Brüggemann M., Madu M.F., Rozeman E.A., Song J.Y., Horlings H.M., Blank C.U., van Akkooi A.C., Flaherty K.T., Boland G.M, & Peeper D.S. (2020). Reversal of pre-existing NGFR-driven tumor and immune therapy resistance. Nature Communications, 11, 3946.

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