Recombinant murine il 2

Manufactured by Thermo Fisher Scientific

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Recombinant murine IL-2 is a protein expressed in Escherichia coli. It is a cytokine that plays a key role in the immune system by promoting the growth and differentiation of T cells.

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Recombinant murine IL-2 peptide (2 citations) Murine IL-2 (56 citations) Recombinant IL-2 (98 citations)

75 protocols using recombinant murine il 2

T Cell Activation and Expansion

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T cells were cultured in RPMI-1640 Medium (Sigma-Aldrich) containing 10% Fetal Bovine Serum (Sigma-Aldrich), 2% penicillin-streptomycin (Sigma-Aldrich) and 2 mM l-Glutamine (Sigma-Aldrich). For antigen-induced activation, total splenocytes isolated from OT-II mouse spleens were cultured with OVA 323–339 peptide (1 mg/ml, InvivoGen) and recombinant murine IL-2 (20 ng/ml, PeproTech). For anti-CD3/CD28-induced activation, CD4+ T cells were stimulated with Dynabeads Mouse T-Activator CD3/CD28 (Gibco) and recombinant murine IL-2 (20 ng/ml, PeproTech), following manufacturer’s instructions.

Zhou D., Borsa M., Puleston D.J., Zellner S., Capera J., Sanderson S., Schifferer M., Hester S.S., Ge X., Fischer R., Jostins L., Behrends C., Alsaleh G, & Simon A.K. (2022). Mapping autophagosome contents identifies interleukin-7 receptor-α as a key cargo modulating CD4+ T cell proliferation. Nature Communications, 13, 5174.

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Regulation of CD4+ T Cell Activation by EV

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Naive splenic CD4⁺ T lymphocytes were captured using negative selection magnetic beads (Miltenyi Biotec, Germany). These cells were stimulated with αCD3/αCD28 beads (Thermo Fisher, USA) and 5 ng/ml of recombinant murine IL‐2 (Peprotech, USA) for 48 h while also being co‐cultured with 2×10⁹ particles/mL of EKO‐BMDM‐EV, or WT‐BMDM‐EV, or PBS. Measurements of T lymphocyte activation were assessed using flow cytometric detection of CD69 and CD25. For detection of IFN‐γ, naive splenic CD4⁺ T lymphocytes were stimulated with αCD3/αCD28 beads (Thermo Fisher, USA) and 5 ng/ml of recombinant murine IL‐2 (Peprotech, USA) for 12 h while also being co‐cultured with 2×10⁹ particles/mL of EKO‐BMDM‐EV, or WT‐BMDM‐EV, or PBS. Cells were cultured in the presence of the Protein Transport Inhibitor cocktail (Invitrogen, USA). They were then permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, USA) and stained with anti‐IFNγ (clone XMG1.2) at 1:100 concentration for 60 min in room temperature according to the manufacturer's protocol.

Phu T.A., Ng M., Vu N.K., Gao A.S, & Raffai R.L. (2023). ApoE expression in macrophages communicates immunometabolic signaling that controls hyperlipidemia‐driven hematopoiesis & inflammation via extracellular vesicles. Journal of Extracellular Vesicles, 12(8), 12345.

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T Cell Differentiation Under Polarizing Conditions

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Naïve CD4⁺ T cells (CD4⁺TCRβ⁺CD1d-αGC⁻NK1.1⁻CD44⁻CD62L^hi) were FACS sorted from the pooled spleens of IRE1α^F/F (n = 5) and CD4^cre;IRE1α^Δ/Δ mice (n = 5), resuspended in complete RPMI 1640 media containing polarizing cytokines and plated at 1 × 10⁵ cells/well in 96-well plates coated with anti-CD3ε/α-CD28 (3 μg/ml, 5 μg/ml). Plated CD4⁺ T cells were differentiated under TH1-polarizing conditions using recombinant murine IL-12 (10 ng/ml, eBioscience), recombinant murine IL-2 (10 ng/ml, eBioscience), and anti-IL-4 (10 μg/ml, eBioscience); TH2-polarizing conditions using recombinant murine IL-4 (10 ng/ml, eBioscience), recombinant murine IL-2 (10 ng/ml) and anti-IFNγ (10 μg/ml, eBioscience); and TH17-polarizing conditions using recombinant murine IL-6 (20 ng/ml, eBioscience), recombinant murine IL-23 (10 ng/ml),⁵⁵, recombinant murine IL-1β (10 ng/ml, PSF, VIB), recombinant human TGFβ1 (2 ng/ml, eBioscience), anti-IL-4 (10 μg/ml), anti-IFNγ (10 μg/ml) and anti-IL-2 (10 μg/ml, eBioscience), respectively. Cells were then removed from TCR stimuli after 3 days, resuspended at 1 × 10⁶/ml in fresh media and cultured in a new 96-well plate without TCR stimuli for 2 days. 1 × 10⁶ polarized T cells were then resuspended in complete RPMI 1640 medium and restimulated using anti-CD3ε/CD28 (3 μg/ml, 5 μg/ml; eBioscience).

Govindarajan S., Gaublomme D., Van der Cruyssen R., Verheugen E., Van Gassen S., Saeys Y., Tavernier S., Iwawaki T., Bloch Y., Savvides S.N., Lambrecht B.N., Janssens S., Elewaut D, & Drennan M.B. (2018). Stabilization of cytokine mRNAs in iNKT cells requires the serine-threonine kinase IRE1alpha. Nature Communications, 9, 5340.

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Murine and Human CD8+ T Cell Culture

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Murine CD8⁺ T cells were isolated (STEMCELL, 19853) and activated with 1 μg/mL αCD3 (eBioscience, 16-0031-86) and 2.5 μg/mL αCD28 (eBioscience,16-0281-82) for 2 days prior to subculture in the presence of 20 U/mL recombinant murine IL-2 (Peprotech, 50-813-288).
For human T cell culture, frozen negatively isolated CD8⁺ T cells (Hemacare, PB08NC1) were activated with CD3-CD28 Dynabeads (Gibco, 11131D) for 72 hours in the presence of 20 U/mL IL-2 (Roche, 11011456001) and 5 ng/mL IL-7 (Peprotech, 200-07) and IL-15 (Peprotech, 200-15). Cells were subcultured with fresh media and cytokines.

Stairiker C.J., Pfister S.X., Hendrickson E., Yang W., Xie T., Lee C., Zhang H., Dillon C., Thomas G.D, & Salek-Ardakani S. (2021). EZH2 Inhibition Compromises α4-1BB-Mediated Antitumor Efficacy by Reducing the Survival and Effector Programming of CD8+ T Cells. Frontiers in Immunology, 12, 770080.

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Cytokine Regulation of Immune Cells

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The inhibitors PD98059, SP600125, SB203580, LY294002, BAY11–7082, and Stattic were purchased from MCE (Shanghai, China). Recombinant murine IL-2 and IL-12 were purchased from PeproTech (Rocky Hill, USA). CFSE were purchased from Invitrogen/Thermo Fisher Scientific. IL-6, IL-10 and IL-21 neutralizing antibodies were purchased from R&D Systems. Lymphocyte separation media were purchased from MultiSciences (Hangzhou, China). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO).

Chen Y.Q., Li P.C., Pan N., Gao R., Wen Z.F., Zhang T.Y., Huang F., Wu F.Y., Ou X.L., Zhang J.P., Zhu X.J., Hu H.M., Chen K., Cai Y.L, & Wang L.X. (2019). Tumor-released autophagosomes induces CD4+ T cell-mediated immunosuppression via a TLR2–IL-6 cascade. Journal for Immunotherapy of Cancer, 7, 178.

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PDAC Organoids Potentiate CD8+ T Cell Killing

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Mouse PDAC cells stably expressing OVA and carrying doxycycline (Dox)-inducible mTurquoise2-Atg4B^C74A were grown as organoids, treated with or without Dox (1 μg/mL) for 96 hrs. Organoids were dissociated into single cells, which were then incubated with either anti-H-2K^b-SIINFEKL antibody (clone 25-D1.16, BioXCell, BE0207) or isotype control (clone MOPC-21, BioXCell, BE0083) at 100 μg/mL for 30 min at 4 °C. Total splenocytes were harvested from OT-I mice and CD8⁺ T cells were enriched using Dynabeads® Untouched™ Mouse CD8 Cells (Invitrogen, 11417D) following manufacturer’s instructions. Isolated CD8⁺ T cells were labelled with 10 μM CFSE (BioLegend) for 10 min at RT in the dark, washed 3x with RPMI-1640 supplemented with 10% FBS. Ten thousand PDAC cells and forty thousand CD8⁺ cells were seeded in 96-well plates and cultured in 100 μL 50% DMEM and 50% RPMI-1640 supplemented with 10% FBS, 10 ng/mL recombinant murine IL-2 (Peprotech), 27.5 μM 2-Mercaptoehanol (Gibco), and 100 μg/mL of respective antibodies. After 48 hrs, CD8⁺ T cells were harvested and stained with anti-CD8a antibody (AF647, clone 53–6.7, BioLegend) and DAPI, and proliferation was analyzed by CFSE dilution using flow cytometry. After removal of CD8⁺ T cells, the viability of remaining PDAC cells were measured by CellTiter-Glo (Promega).

Yamamoto K., Venida A., Yano J., Biancur D.E., Kakiuchi M., Gupta S., Sohn A.S., Mukhopadhyay S., Lin E.Y., Parker S.J., Banh R.S., Paulo J.A., Wen K.W., Debnath J., Kim G.E., Mancias J.D., Fearon D.T., Perera R.M, & Kimmelman A.C. (2020). Autophagy promotes immune evasion of pancreatic cancer by degrading MHC-I. Nature, 581(7806), 100-105.

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Molecular Inhibitors for Cell Signaling

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Chemical inhibitors used included 1uM MG132 (S2619; Selleckchem), 1.3 uM Chir911, 1 uM Rapamycin (S1039, Selleckchem), 1uM Torin2 (S2817; Selleckchem), 1 ug/mL DON (D2141, Sigma-Aldrich), 1 ug/mL alpha keto-glutarate (K2000; Sigma-Aldrich), 0.1 mM actinomycin D (114666; Calbiochem), 100 ug/mL cyclohexamide (C7698; Sigma-Aldrich), 1uM 2DG (D6134; Sigma-Aldrich), and 1 or 5 uM JQ1 (A1910; Apexbio). Cytokines included recombinant murine IL-7 (5 ng/mL) (217-17; Peprotech), recombinant murine IL-2 (5 ng/mL) (212-12; Peprotech), and recombinant murine GMCSF for BMDC generation (1000 U/mL) (31503; Peprotech).

Verbist K.C., Guy C.S., Milasta S., Liedmann S., Kamiński M.M., Wang R, & Green D.R. (2016). Metabolic Maintenance of Cell Asymmetry following Division in Activated T Lymphocytes. Nature, 532(7599), 389-393.

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IL-2/anti-IL-2 and N-803 treatment for bm12 graft

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For each mouse, 1 μg of recombinant murine IL-2 (PeproTech) was combined with 10 μg anti-mouse IL-2 (clone S4B6; BioXCell) in 200 μL Hank’s Balanced Salt Solution (HBSS), then incubated overnight at 4°C while mixing. For each control mouse, 1 μg of recombinant murine IL-2 was incubated overnight with 10 μg rat IgG2a (BioXCell) in 200 μL HBSS. As a pre-disease treatment, each mixture was intraperitoneally injected every other day for a total of 3 injections, after which a bm12 splenocyte graft was injected the following day (see above). As a post-disease treatment, injections were administered once daily for three days beginning one month after graft injection [29 (link)].
Experimental mice received 200 μg/kg of N-803 (provided via collaboration with Altor Biosciences, now part of NantWorks LLC) in PBS injected subcutaneously, while control mice received a molar equivalent of recombinant human IL-15 (Peprotech) in PBS. As a pre-disease treatment, two injections were administered, once every other day, finishing one day prior to bm12 graft injection. As a post-disease treatment, injections were administered once weekly over the course of 3 months, beginning 1 week after bm12 graft injection (for a total of 11 treatments).

Reighard S.D., Krishnamurthy D., Cevik H., Ochayon D.E., Ali A., Seelamneni H., Brunner H.I, & Waggoner S.N. (2020). Immunomodulatory effects of cytokine-induced expansion of cytotoxic lymphocytes in a mouse model of lupus-like disease. Cytotherapy, 23(1), 37-45.

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Single Cell Isolation from Lymphoid Tissues

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Single cell suspensions from LNs, spleens and thymus were generated by passing them through a 40 μm cell strainer. BM cells were isolated by flushing out femur and tibia. TILs were isolated by dissociating tumor tissue in the presence of collagenase D (1 mg/ml, Roche) and DNAse I (0.25 mg/ml, Sigma) for 45 min before passing through a 40 μm cell strainer. For LN cells activation, 4 × 10⁵ total tdLN cells were cultured with Dynabeads™ mouse T-activator CD3/CD28 (Gibco) in a ratio 1:1 for 48 h. Recombinant murine IL-2 (1000 U/ml, Peprotech) and recombinant murine IL-33 (20 ng/ml, Immunotools) were added where indicated.

Hatzioannou A., Banos A., Sakelaropoulos T., Fedonidis C., Vidali M.S., Köhne M., Händler K., Boon L., Henriques A., Koliaraki V., Georgiadis P., Zoidakis J., Termentzi A., Beyer M., Chavakis T., Boumpas D., Tsirigos A, & Verginis P. (2019). An intrinsic role of IL-33 in Treg cell-mediated tumor immunoevasion. Nature immunology, 21(1), 75-85.

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Cytokine and Antibody Staining Protocol

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Recombinant murine IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-21, IL-23, IFN-γ, and TGF-β were purchased from PeproTech. Mouse IFN-β was purchased from PBL Biomedical Laboratories. LCMV GP33 peptide (KAVYNFATM; specific for P14 TCR) was purchased from Bioneer. Cell suspensions of spleen, LN, or thymus were prepared and stained for FACS analysis with the fluorochrome-conjugated antibodies. Detailed information of the antibodies used is depicted in Supplementary Table 2. To stain intracellular Nur77, Ki-67, CD107a, and Granzyme B, splenocytes were stained for cell surface markers, fixed and permeabilized using eBioscience Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and then stained with fluorochrome-conjugated intracellular antibodies. Flow cytometry samples were run using a LSRII or FACSCanto II (BD Biosciences) using FACSDiva software (BD Biosciences) and analyzed by FlowJo software (Tree Star). The gating strategies are depicted in Supplementary Fig. 8.

Ju Y.J., Lee S.W., Kye Y.C., Lee G.W., Kim H.O., Yun C.H, & Cho J.H. (2021). Self-reactivity controls functional diversity of naive CD8+ T cells by co-opting tonic type I interferon. Nature Communications, 12, 6059.

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