Interleukin 2 (il 2)

Manufactured by InvivoGen

Sourced in United States

IL-2 is a recombinant protein that corresponds to the human interleukin-2 cytokine. Interleukin-2 is a key regulator of immune cell function and proliferation.

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7 protocols using interleukin 2 (il 2)

Isolation and Activation of T Cells

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Spleen and lymph nodes from WT, Cpeb4^−/−, and CPEB4‐TKO mice were mechanically processed into a single‐cell suspension. CD8 or CD4 T cells were purified using Dynabeads™ FlowComp™ Mouse CD8 Kit (Themo Fisher) or Dynabeads™ FlowComp™ Mouse CD4 Kit (ThemoFisher), respectively. Isolated cell purity was confirmed by flow cytometry (> 95%). T cells were cultured in RPMI medium (Gibco) supplemented with 10% Fetal Bovine Serum, 1% penicillin/streptomycin, 2 mM L‐glutamine (Gibco), 1 mM sodium pyruvate (Gibco), and 55 μM β‐mercaptoethanol at 37°C, 5% CO₂, and 5% O₂. Purified T cells were activated with 1 μg/ml anti‐CD3 antibody (clone 145‐2C11, #MA5‐17655, Thermo Fisher), 1 μg/ml anti‐CD28 antibody (clone CD28.6, #16‐0288‐85, eBiosciences), and 20 ng/ml IL‐2 (212‐12, PeproTech) for the indicated periods. OT‐I WT and CPEB4‐TKO splenocytes were processed into a single‐cell suspension and plated into T‐cell media supplemented as described above. For OT‐I activation, 1 μg/ml OVA_257‐264 peptide (vac‐sin, InvivoGen) and 20 ng/ml IL‐2 were added to the media. Activated OT‐I CD8 cells were purified at the indicated time points for downstream analysis by either Dynabeads™ or FlowComp™ Mouse CD8 Kit.

Fernández‐Alfara M., Sibilio A., Martin J., Tusquets Uxó E., Malumbres M., Alcalde V., Chanes V., Cañellas‐Socias A., Palomo‐Ponce S., Batlle E, & Méndez R. (2023). Antitumor T‐cell function requires CPEB4‐mediated adaptation to chronic endoplasmic reticulum stress. The EMBO Journal, 42(9), e111494.

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Expansion of Natural Killer Cells

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PBMC (5 × 10⁶ cells) were stimulated with 1 × 10⁷ γ-irradiated K562 Clone 9.mbIL21 cells [28 (link)] in a volume of 40 mL of NKEM: RPMI 1640 (Invitrogen) supplemented with 10% FBS (Invitrogen), glutamine (Invitrogen), Primocin (InvivoGen), and 50 U/mL IL-2 (NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH, contributed by Dr. Maurice Gately, Hoffmann—La Roche Inc.) [51 (link)]. On days 3 and 5 after culture, cells were resuspended in fresh medium. The expanded NK cells were re-stimulated on day 7, and weekly thereafter, with additional γ-irradiated K562 Clone 9.mbIL21 cells at a 1:1 ratio. From day 7 onward, expanded cells were resuspended in fresh medium at 4 × 10⁵ cells/mL 2–3 times weekly.

Schafer J.L., Ries M., Guha N., Connole M., Colantonio A.D., Wiertz E.J., Wilson N.A., Kaur A, & Evans D.T. (2015). Suppression of a Natural Killer Cell Response by Simian Immunodeficiency Virus Peptides. PLoS Pathogens, 11(9), e1005145.

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Multiparametric flow cytometry analysis of immune cell signaling

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The SCNP assay was performed as described previously [7 (link)]. Modulators and concentrations were as follows: 1000 IU/ml IFN-α (PBL); 50 ng/ml IL-4, 5 μg/ml anti-IgD (BD Biosciences); 50 ng/ml IL-2, 50 ng/ml IL-6, 50 ng/ml IL-27, 5 μg/ml R848 (Invivogen); 40 nM PMA (Sigma Aldrich), 3 μg/ml anti-CD3 (eBioscience), 10 μg/ml anti-mouse (Santa Cruz Biotechnology), 10 μg/ml anti-IgM (Southern Biotech). For TCR stimulation, cells were exposed to anti-CD3 for 12 min, with anti-mouse added for the last 2 min; the anti-IgM modulation time was 10 min; all other modulation times were 15 min. Staining was performed using Ab cocktails with each cocktail consisting of 5 Abs to detect phenotypic markers and 2 Abs to detect intracellular protein readouts. Abs used include anti-CD3, -CD4, -CD45RA, -CD20, -p-NF-κB, -c-poly(ADP-ribose) polymerase, -p-Stat1, -p-Stat3, -p-Stat5, -p-Stat6, -p-Erk, -p-ZAP70/Syk (BD Biosciences); -p-Akt, -p-S6 (CST); and -CD14 (Beckman Coulter). Flow cytometry data was acquired on FACS Canto II Flow Cytometers (BD Biosciences). All flow cytometry data were analyzed with WinList (Verity House Software). PBMC subpopulations were delineated according to the immunophenotypic gating scheme described previously [7 (link)].

Longo D.M., Louie B., Ptacek J., Friedland G., Evensen E., Putta S., Atallah M., Spellmeyer D., Wang E., Pos Z., Marincola F.M., Schaeffer A., Lukac S., Railkar R., Beals C.R., Cesano A., Carayannopoulos L.N, & Hawtin R.E. (2014). High-dimensional analysis of the aging immune system: Verification of age-associated differences in immune signaling responses in healthy donors. Journal of Translational Medicine, 12, 178.

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OT-1 T Cell Polarization and Expansion

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OT-1 polarizations were carried out as previously described (Carrio et al, 2004). Briefly, splenocytes from OT-1 mice were cultured at 1×10⁶ cells/mL in 24 well-plates of complete RPMI (UCSF Media Core facility) supplemented with 10% FBS (Omega Scientific, Tarzana, California), 100U/mL penicillin-streptomycin (Fisher Scientific, Hampton, New Hampshire), 2mM L-glutamine (Sigma-Aldrich, St. Louis, Missouri) and 50μM β-mecaptoethanol (Thermo Fisher Scientific, Waltham, Massachusetts ) and 10mM HEPES (UCSF Media Core Facility) in the presence of OVA_257–264 peptide (0.1nM) (Invivogen, San Diego, California) and IL-2 (100U/ml) (Teceleukin) kindly provided by NCI Frederick. After 3 days in culture, activated cells were washed 3 times with RPMI 1640 and recultured in T25 culture flasks at 1×10⁵ cells/mL in the presence of either IL-7, IL-15 (Biolegend, San Diego, California) or IL-2 (Teceleukin) kindly provided by NCI Frederick. (all cytokines 10ng/ml) After 2 additional days in culture, cells were passaged and recultured under the same conditions without peptide for an additional two days for total of 7 days in culture. Viability was confirmed by trypan blue exclusion (Thermo Fisher, Waltham, Massachusetts) or mass cytometry as described below.

Levine L.S., Hiam-Galvez K.J., Marquez D.M., Tenvooren I., Madden M.Z., Contreras D.C., Dahunsi D.O., Irish J.M., Oluwole O.O., Rathmell J.C, & Spitzer M.H. (2021). Single-cell analysis by mass cytometry reveals metabolic states of early activated CD8+ T cells during the primary immune response. Immunity, 54(4), 829-844.e5.

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B Cell Differentiation and Antibody Production

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B cell subsets were sorted using FACS Aria III (BD Biosciences). The gating strategy of cell sorting was shown in Supplementary Fig. 4. Sorted cells (5 × 10⁴) were cultured in RPMI media supplemented with 3 conditions of stimuli; Stimuli 1 (S1): 1 μg/ml R848 (Invivogen, USA), 10 ng/ml IL-2 (Peprotech, USA), 10 ng/ml BAFF (Peprotech); S2: 1 μg/ml R848, 10 ng/ml IL-2, 10 ng/ml BAFF and 100 ng/ml IL-21 (Peprotech); S3: 1 μg/ml R848, 10 ng/ml IL-2, 10 ng/ml BAFF, 100 ng/ml IL-21 and 20 ng/ml IFN-γ (Invivogen), at 37 °C, 5% CO₂ for 11 days. Culture supernatants were tested by IgG ELISA and the cells were harvested and stained with CD19, CD27 and CD38 antibodies before acquisition on a flow cytometry.

Kochayoo P., Thawornpan P., Wangriatisak K., Changrob S., Leepiyasakulchai C., Khowawisetsut L., Adams J.H, & Chootong P. (2022). Interferon-γ signal drives differentiation of T-bethi atypical memory B cells into plasma cells following Plasmodium vivax infection. Scientific Reports, 12, 4842.

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Optimized Human IgG Production from PBMCs

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PBMCs were thawed, seeded at 1x10⁶ per ml in complete media (RPMI containing 25mM Hepes and L-glutamine + 10% FCS +1% Pen/Strep) and stimulated with 1000 U/ml recombinant human IL-2 (StemCell Technologies) and 2.5 μg/ml R848 (Invivogen) for 7 days at 37^oC. On day 4, half the media was removed and replaced with fresh media containing 1X IL-2/R848. Day 7 culture supernatants were collected, spun down to remove cellular debris, assayed for total IgG concentration, and stored at -80^oC. Total IgG was measured by cytometric bead array (CBA) assay (BD Biosciences) using a FACS Lyric instrument (BD Biosciences), according to the manufacturer’s instructions. Data were analyzed using BD FACSuite software v1.2.1.5657 (BD Biosciences).

Buckner C.M., Kardava L., El Merhebi O., Narpala S.R., Serebryannyy L., Lin B.C., Wang W., Zhang X., Lopes de Assis F., Kelly S.E., Teng I.T., McCormack G.E., Praiss L.H., Seamon C.A., Rai M.A., Kalish H., Kwong P.D., Proschan M.A., McDermott A.B., Fauci A.S., Chun T.W, & Moir S. (2022). Interval between prior SARS-CoV-2 infection and booster vaccination impacts magnitude and quality of antibody and B cell responses. Cell, 185(23), 4333-4346.e14.

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OT-1 T Cell Activation and Expansion

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OT-1 polarizations were carried out as previously described (Carrio et al, 2004) . Briefly, splenocytes from OT-1 mice were cultured at 1*10 6 cells/mL in 24 well-plates of complete RPMI (UCSF Media Core facility) supplemented with 10% FBS (Omega Scientific, Tarzana, California), 100U/mL penicillin-streptomycin (Fisher Scientific, Hampton, New Hampshire), 2mM L-glutamine (Sigma-Aldrich, St. Louis, Missouri) and 50µM b-mecaptoethanol (Thermo Fisher Scientific, Waltham, Massachusetts ) and 10mM HEPES (UCSF Media Core Facility) in the presence of OVA257-264 peptide (0.1nM) (Invivogen, San Diego, California) and IL-2 (100U/ml) (Teceleukin) kindly provided by NCI Frederick. After 3 days in culture, activated cells were washed 3 times with RPMI 1640 and recultured in T25 culture flasks at 1*10 5 cells/mL in the presence of either San Diego, California) or IL-2 (Teceleukin) kindly provided by NCI Frederick. (all cytokines 10ng/ml) After 2 additional days in culture, cells were passaged and recultured under the same conditions without peptide for an additional two days for total of 7 days in culture. Viability was confirmed by trypan blue exclusion (Thermo Fisher, Waltham, Massachusetts) or mass cytometry as described below.

Levine L.P., Hiam K., Marquez D.M., Tenvooren I., Contreras D.C., Rathmell J.C., & Spitzer M.H. (2020). Single-cell metabolic dynamics of early activated CD8 T cells during the primary immune response to infection.

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