Recombinant mouse il 15

Manufactured by Thermo Fisher Scientific

Sourced in United States

Recombinant mouse IL-15 is a cytokine that promotes the proliferation and activation of natural killer cells and memory CD8+ T cells. It is produced in E. coli and provided as a lyophilized powder.

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18 protocols using recombinant mouse il 15

Metabolic profiling of Ncr1Ki Ptpn11 NK cells

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NK cells were enriched from control and Ncr1^KiPtpn11^fl/fl mice and stimulated overnight with recombinant mouse IL-15 (50 ng/mL PeproTech) at 37 °C. The following day, 2.5 × 10⁵ enriched NK cells were seeded in a Seahorse Bioscience culture plate in DMEM (Sigma) with 10 mM glucose (Sigma), 2 mM glutamine (Life Technology) in a non-CO₂ incubator for at least 30 min. Basal OCR and ECAR were then measured by an XF96 Seahorse Extracellular Flux Analyzer following the manufacturer’s instruction. Basal OCR and ECAR were calculated as the mean of the first three time points (basal) for each replicate in Ncr1Ki Ptpn11^wt/wt and Ncr1Ki Ptpn11^fl/fl mice. For the calculation of the SRC, the mean of basal OCR was subtracted to the mean of the three time points at maximal OCR for each replicate in Ncr1Ki Ptpn11^wt/wt and Ncr1Ki Ptpn11^fl/fl mice.

Niogret C., Miah S.M., Rota G., Fonta N.P., Wang H., Held W., Birchmeier W., Sexl V., Yang W., Vivier E., Ho P.C., Brossay L, & Guarda G. (2019). Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells. Nature Communications, 10, 1444.

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In Vitro and In Vivo Characterization of Rfx7-Deficient NK Cells

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Congenically (CD45.1) marked Rfx7-deficient and control NK cells were purified (STEMCELL technologies Cat. No. 19855).
NK cells were then co-cultured in vitro with recombinant mouse IL-15 or IL-2 (Peprotech) for 3 or 4 days, as indicated. IL-15 was used at 10 ng/ml (“high”), 3 ng/ml (“medium”), or 1 ng/ml (low) dose and IL-2 at 100 ng/ml (“high”), 30 ng/ml (“medium”), or 10 ng/ml (low). The medium used for in vitro experiments was previously described55 (link).
For in vivo transfer, congenically marked NK cells from Vav Rfx7^fl/fl or Vav Rfx7^wt/wt mice were CTV-labeled and approximately 1.8 x 10⁶ NK cells were adoptively co-transferred into C57BL/6 hosts.

Castro W., Chelbi S.T., Niogret C., Ramon-Barros C., Welten S.P., Osterheld K., Wang H., Rota G., Morgado L., Vivier E., Raeber M.E., Boyman O., Delorenzi M., Barras D., Ho P.C., Oxenius A, & Guarda G. (2018). The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity. Nature immunology, 19(8), 809-820.

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Splenocyte Coculture Assay with Malaria Parasites

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In coculture experiments, isolated splenocytes of WT, IL‐12p35^−/−, and IL‐18R^−/−mice were cultured in RPMI1640 supplemented with 10% FCS, 100 U/mL penicillin/streptomycin, and 2 mM l‐glutamine (all Gibco Life Technologies) containing 2 ng/mL recombinant mouse IL‐15 (Peprotech, Rocky Hill, NJ, USA) with uRBC or Py17XNL iRBC at a ratio of 5 uRBC/iRBC per splenocyte. iRBCs were purified from blood obtained from Py17XNL‐infected WT animals by passage over a magnetic column (Miltenyi Biotech, Bergisch‐Gladbach, Germany). Blocking antibodies to IL‐12p35 (eBioscience) and IL‐18 (MBL) or control rat IgG1 or IgG2a (both eBioscience) were added at a concentration of 2.5 μg/mL. Cells were cultured at 37°C at 5% CO₂ for 19 h with Brefeldin A present for the last 3 h of culture. Stimulated cells were characterized by flow cytometry as described above.

Stegmann K.A., De Souza J.B, & Riley E.M. (2015). IL‐18‐induced expression of high‐affinity IL‐2R on murine NK cells is essential for NK‐cell IFN‐γ production during murine Plasmodium yoelii infection. European Journal of Immunology, 45(12), 3431-3440.

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Comprehensive Immune Cell Phenotyping

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Immunostaining was performed using antibodies against the following markers: CD45R (RA3-6B2), CD45RB (C363-16A), CD45.1 (A20), CD45.2 (104), TCRβ (H57-597), CD3ϵ (145-2C11), CD4 (RM4-5 or GK1.5), CD8α (53-6.7), CD16/32 (2.4G2), CD25 (PC61), CD28 (37.51), CD44 (IM7), CD62L (MEL-14), CD69 (H1.2F3), CD122 (TM-β1), CD103 (2E7), CXCR3 (CXCR3-173), CTLA4 (UC10-4F10-11), PD1 (J43), KLRG1 (2F1), Lag3 (C9B7W), Tim3 (B8.2C12), Ly49A (JR9.318), Ly49C/I (5E6), Ly49C/I/F/H (14B11), Ly49G (4D11), Ly49F (HBF-719), Ly49D (4E5), Ly49H (3D10), IFNγ (XMG1.2), IL2 (JES6-5H4), TGFβ (TW-716B4), Granzyme B (GB11), TNFα (MP6-XT22), IL10 (JES5-16E3).
Dead cells were excluded by the use of live/dead staining with either 7-AAD, Zombie Aqua™ fixable viability dye (Biolegend), or Fixable Viability Dye eFluor™ 450 (Invitrogen). Anti-CD16/32 (2.4G2, BD Bioscience) antibodies were used to block Fc receptors during sample preparation. Recombinant mouse IL-2 (eBioscience), recombinant mouse IL-15 (PeproTech), CellTrace™ Violet (CTV, Invitrogen), Brefeldin A (Biolegend), phorbol 12-myristate 13-acetate (PMA) and Ionomycin (Beyotime), Percoll solution (GE Healthcare) were used in experiments.

Shytikov D., Rohila D., Li D., Wang P., Jiang M., Zhang M., Xu Q, & Lu L. (2021). Functional Characterization of Ly49+CD8 T-Cells in Both Normal Condition and During Anti-Viral Response. Frontiers in Immunology, 11, 602783.

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Reagents for Immune Cell Culture

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α-GalCer (KRN7000) was purchased from Funakoshi (Tokyo, Japan). RPMI 1640, DMEM, newborn calf serum (NBCS), and red blood cell (RBC) lysis buffer were purchased from Gibco™ (Waltham, MA, USA). Fetal bovine serum (FBS) was purchased from Biological Industries (Beit-Haemek, Israel). Recombinant mouse IL-2 and GM-CSF were purchased from BioLegend (San Diego, CA, USA). Recombinant mouse IL-15, human IL-4, and GM-CSF were purchased from PeproTech (Rocky Hill, NJ, USA). Recombinant human IL-2 was purchased from eBioscience (San Diego, CA, USA).

Lai A.C., Chi P.Y., Thio C.L., Han Y.C., Kao H.N., Hsieh H.W., Gervay-Hague J, & Chang Y.J. (2019). α-Lactosylceramide Protects Against iNKT-Mediated Murine Airway Hyperreactivity and Liver Injury Through Competitive Inhibition of Cd1d Binding. Frontiers in Chemistry, 7, 811.

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Selectin Ligand Synthesis Assay

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To detect synthesis of P- and E-selectin ligands, WT and CD62L^-/- memory P14 CD8⁺ T cells from spleens were stimulated for 3 days with 250 ng/ml of recombinant mouse IL-15 (Peprotech). Recombinant E-selectin (5 μg/ml) and P-selectin (1.5 μg/ml) human IgG Fc chimeric proteins (R & D Systems) were incubated with cells for 30 minutes in 1% FBS/DPBS containing Ca²⁺ and Mg²⁺ (Gibco) at room temperature. Binding of selectins was detected using anti-human IgG-Fc PE (eBioscience). Cells were then stained with fluorescent antibodies as described in Flow Cytometry and Antibodies.

Osborn J.F., Hobbs S.J., Mooster J.L., Khan T.N., Kilgore A.M., Harbour J.C, & Nolz J.C. (2019). Central memory CD8+ T cells become CD69+ tissue-residents during viral skin infection independent of CD62L-mediated lymph node surveillance. PLoS Pathogens, 15(3), e1007633.

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Intracellular Signaling Analysis in Ncr1cre Myc Mice

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For signaling analysis, splenocytes from control and Ncr1cre Myc^fl/fl mice were left unstimulated or stimulated with 50 ng/ml recombinant mouse IL-15 (PeproTech) for 40 min at 37°C and analyzed for intracellular protein phosphorylation by flow cytometry. The cells were then surface stained on ice, fixed with 2% paraformaldehyde at 4°C for 20 min, and permeabilized with 90% methanol for 30 min at 4°C. Intracellular phosphostainings were performed 1 h at room temperature in the dark with antibodies against phospho-STAT5 (Tyr694; D47E7 XP rabbit mAb #4322; Cell Signaling, 1:150), phospho-S6 ribosomal protein (Ser235/236; D57.2.2E; XP rabbit mAb #4858; Cell Signaling, 1:200), phospho-ERK1/2 (Thr202/Tyr204; D13.14.4E; XP rabbit mAb #4370; Cell Signaling, 1:150).

Khameneh H.J., Fonta N., Zenobi A., Niogret C., Ventura P., Guerra C., Kwee I., Rinaldi A., Pecoraro M., Geiger R., Cavalli A., Bertoni F., Vivier E., Trumpp A, & Guarda G. (2023). Myc controls NK cell development, IL-15-driven expansion, and translational machinery. Life Science Alliance, 6(7), e202302069.

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Precomplexing IL-15 with IL-15Rα

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Precomplexing of IL-15/IL-15Rα was performed with recombinant mouse IL-15 (Peprotech) and mouse IL-15Rα Fc chimera (R&D) by incubating 2.5 µg of IL-15 and 12 µg of IL-15Rα at room temperature for 30 minutes as previously described (15 (link)).

Nguyen R., Moustaki A., Norrie J.L., Brown S., Akers W.J., Shirinifard A, & Dyer M.A. (2019). Interleukin-15 Enhances Anti-GD2 Antibody-Mediated Cytotoxicity in an Orthotopic PDX Model of Neuroblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 25(24), 7554-7564.

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Sustained IL-15 Delivery in Mice

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Recombinant mouse IL-15 (Peprotech) dissolved in PBS was administered into IL-2Rβ^Tg BALB/c mice using Alzet osmotic pumps (Durect) following the manufacturer’s instruction. The pumps (model 1002) were set to release IL-15 at a rate of 3 µg of recombinant IL-15 per 24 hours.

Won H.Y., Kim H.K., Crossman A., Awasthi P., Gress R.E, & Park J.H. (2021). The Timing and Abundance of IL-2Rβ (CD122) Expression Control Thymic iNKT Cell Generation and NKT1 Subset Differentiation. Frontiers in Immunology, 12, 642856.

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Cytokine-Induced Lymphocyte Proliferation

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After CTV labeling, live cells were counted and adjusted to the concentration of 1*10⁶/ml. 100 μl of the cell suspension was seeded into a 96 well plate. For IL-15 stimulation, 100 μL of media with recombinant mouse IL-15 (Peprotech, New Jersey, USA) at the concentration of 2 μg/ml was added to the cells. The cells were incubated at 37°C for 5 days and then analyzed for proliferation by the CTV dilution on a flow cytometer. For IL-7/12/18 (Peprotech, New Jersey, USA) stimulation, 100 μl media with either IL-7 or a combination of either IL-7 and IL-12 or IL-7 and IL-18 (all at 100ng/ml) was added to the cells. The cells were incubated at 37°C for 7 days and then analyzed for proliferation by the CTV dilution on a flow cytometer.

Prasad M., Wojciech L., Brzostek J., Hu J., Chua Y.L., Tung D.W., Yap J., Rybakin V, & Gascoigne N.R. (2021). Expansion of an Unusual Virtual Memory CD8+ Subpopulation Bearing Vα3.2 TCR in Themis-Deficient Mice. Frontiers in Immunology, 12, 644483.

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