Mouse il 7

Manufactured by Thermo Fisher Scientific

Mouse IL-7 is a recombinant protein that represents the interleukin-7 cytokine from the mouse. Interleukin-7 is a key regulator of T-cell and B-cell development and homeostasis.

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10 protocols using mouse il 7

Investigating Cytokine-Mediated Signaling

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Recombinant human IL-2 (200–02) and mouse IL-7 (217–17) were from Peprotech. IAA (I2515), CoCl₂ (232696), sodium citrate (PHR1416), and UK5099 (PZ0160) were from Sigma-Aldrich. PX-478 2HCl (px478, S7612) was from Selleck.

Zhu Y., Zhao Y., Zou L., Zhang D., Aki D, & Liu Y.C. (2019). The E3 ligase VHL promotes follicular helper T cell differentiation via glycolytic-epigenetic control. The Journal of Experimental Medicine, 216(7), 1664-1681.

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Intestinal Organoid-Intraepithelial Lymphocyte Coculture

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We followed a modified protocol from a previous report (27 (link)). Briefly, intestinal organoids were cultured for 2 days prior to the co-culture with IELs. On the first day of co-culture, SI-IELs collected from the WT mice aged 9-weeks old or 89 weeks old were stained with anti-mouse CD3e-APC-Cy7, anti-mouse CD4-PE-Cy7, and the CD3⁺ (CD3⁺) and CD4⁺ (CD3⁺ CD4⁺) -IELs were sorted using FACSMelody (BD Bioscience). Cultured organoids released from Matrigel were washed and counted. We combined 200 organoids and 2.0 × 10⁵ IELs and centrifuged the samples for 3 min at 200 g. In the control group, the same number of organoids as the co-culture group was centrifuged. The pellet was suspended in 20 μL of Matrigel and placed in 24-well plates. After Matrigel polymerization, 500 μL of the complete medium with 100 U/mL recombinant human IL-2 (Roche), 10 ng/mL mouse IL-7 (Peprotech) and 10 ng/mL mouse IL-15 (Peprotech) were supplemented. The medium was refreshed every 1–2 days. Images of organoids were taken with a BZ-X710 microscope (Keyence).

Yonemoto Y., Nemoto Y., Morikawa R., Shibayama N., Oshima S., Nagaishi T., Mizutani T., Ito G., Fujii S, & Okamoto R. (2024). Single cell analysis revealed that two distinct, unique CD4+ T cell subsets were increased in the small intestinal intraepithelial lymphocytes of aged mice. Frontiers in Immunology, 15, 1340048.

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Reconstitution and Preparation of Immunotherapeutic Agents

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ALT-803 was generously provided by Altor BioScience Corporation and was diluted in sterile PBS for in vitro and in vivo studies. Recombinant human IL-15 (Novoprotein), human IL-2, human IL-21, mouse IL-7 (Peprotech), human IL-27 (eBioscience), and Concanavalin A (Type VI, Sigma-Aldrich) were reconstituted in sterile PBS and stored at −20°C. Trametinib (GSK-1120212) was purchased from LC Laboratories and suspended in vehicle solution of 10% PEG-300 (Sigma Aldrich) and 10% Cremophor EL (EMD Millipore) in sterile dH₂0 for in vivo oral gavage experiments. For in vitro assays, all inhibitors were dissolved in sterile DMSO and diluted in the assays 1:1000, so that the final concentration of DMSO was 0.1%.

Allegrezza M.J., Rutkowski M.R., Stephen T.L., Svoronos N., Tesone A.J., Perales-Puchalt A., Nguyen J.M., Sarmin F., Sheen M.R., Jeng E.K., Tchou J., Wong H.C., Fiering S, & Conejo-Garcia J.R. (2016). IL-15 AGONISTS OVERCOME THE IMMUNOSUPPRESSIVE EFFECTS OF MEK INHIBITORS. Cancer research, 76(9), 2561-2572.

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Cytokine Production by Cultured ILC2s

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Fresh lung ILC2s (CD45⁺Lin⁻CD127⁺CD90.2⁺ST2⁺) sorted from IL33-treated mice were cultured in 96-well round-bottom plates at a density of 5 × 10³ cells per well in RPMI-1640 medium containing 10% FCS, 100 U/ml penicillin and 100 mg/ml streptomycin in the presence of 10 ng/ml human IL-2 (PeproTech), 20 ng/ml mouse IL-7 (PeproTech, 217-17-100), 1 ng/ml mouse IL-33 (BioLegend, 580506), 20 ng/ml human IL-7 (R&D Systems, 207-IL-010) and 50 ng/ml human IL-33 (R&D Systems, 3625-IL-010) with or without α-MSH (10 ng/ml) or β-endorphin (MCE, HY-P1502, 10 ng/ml). Then, the levels of cytokines in the culture supernatants were measured by ELISA (mouse IL-5 ELISA kit, Invitrogen, 88-7054-76; mouse IL-13 ELISA kit, Invitrogen, 88-7137-76; human IL-5 ELISA kit, Invitrogen, 88-7056-88; and human IL-13 ELISA kit, Invitrogen, 88-7439-88). For intracellular cytokine staining, 50 ng/ml PMA, 1 µg/ml ionomycin, and 1 µg/ml BFA were added to the cultures 2–4 h before staining.

Huang Y., Zhu L., Cheng S., Dai R., Huang C., Song Y., Peng B., Li X., Wen J., Gong Y., Hu Y., Qian L., Zhu L., Zhang F., Yu L., Yi C., Gu W., Ling Z., Ma L., Tang W., Peng L., Shi G., Zhang Y, & Sun B. (2023). Solar ultraviolet B radiation promotes α-MSH secretion to attenuate the function of ILC2s via the pituitary–lung axis. Nature Communications, 14, 5601.

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NK Cell Differentiation from Hematopoietic Progenitors

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NK cell differentiation from HPCs was performed as previously described. In brief, to isolate HPCs, Lin⁻ (B cell (B220), T/NK cells (CD2), granulocytes (Gr-1), monocytes (CD11b), NK/NKT cells (NK1.1), and erythrocytes (TER-119)) cells were purified using the MACS Cell Seperation kit (Miltenyi Biotec). c-Kit⁺ cells from the Lin⁻ cells were positively selected using the CD117 (c-Kit) microbeads (Miltenyi Biotec). The purified HPCs were plated into a 24-well plate (BD) at 1 × 10⁶ cells/well and cultured for 7 d in complete RPMI1640 medium supplemented with a mixture of mouse Flt3L (50 ng/ml, Peprotech), mouse SCF (30 ng/ml, Peprotech), mouse IL-7 (0.5 ng/ml, Peprotech), Indometacin (2 ug/ml, Sigma), and gentamycin (20 ug/ml, Sigma). The cell were refreshed with the same media on day 3. To generate the mNK cells, 7 d-HPCs were maintained with IL-15 (30 ng/ml, Peprotech) for 6~7 days.

Kim W.S., Kim M.J., Kim D.O., Byun J.E., Huy H., Song H.Y., Park Y.J., Kim T.D., Yoon S.R., Choi E.J., Jung H, & Choi I. (2017). Suppressor of Cytokine Signaling 2 Negatively Regulates NK Cell Differentiation by Inhibiting JAK2 Activity. Scientific Reports, 7, 46153.

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LCMV-Specific Tfh and Th1 Cell Isolation

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Lentivirus was produced by co-transfecting HEK293T cells with the lentiviral metabolic library plasmids, psPAX2 (Addgene plasmid # 12260) and pCAG4-Eco. At 48 h after transfection, virus was harvested and frozen at −80 °C. Naïve Cas9-expressing SMARTA cells were isolated from 6 Cas9-SMARTA mice and transduced at a MOI of 0.3 to achieve ~20% transduction efficiency. After viral transduction, cells were cultured with human IL-2 (140 IU/ml; PeproTech) for 3 days, then transferred to medium containing 2 ng/ml mouse IL-7 (PeproTech) for 1 day. Transduced cells expressing Ametrine were sorted using a Reflection sorter (i-Cyt), and an aliquot of 5 × 10⁶ transduced SMARTA cells was saved as “input” (~500× cell coverage per sgRNA). Transduced SMARTA cells (1 × 10⁶ cells per recipient) were i.v. transferred into recipient mice followed by LCMV infection 24 h later. Forty-five recipients were randomly divided into 3 groups as biological replicates in each sub-library screening. At 7 days after infection, donor-derived Tfh (CXCR5⁺SLAM⁻) and Th1 (CXCR5⁻SLAM⁺) cells were sorted using a Reflection sorter. On average, 5 × 10⁶ SMARTA cells per sample (~500× cell coverage per sgRNA) were recovered for further analysis.

Fu G., Guy C.S., Chapman N.M., Palacios G., Wei J., Zhou P., Long L., Wang Y.D., Qian C., Dhungana Y., Huang H., KC A., Shi H., Rankin S., Brown S.A., Johnson A., Wakefield R., Robinson C.G., Liu X., Sheyn A., Yu J., Jackowski S, & Chi H. (2021). Metabolic control of Tfh cells and humoral immunity by phosphatidylethanolamine. Nature, 595(7869), 724-729.

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Notch Signaling Activation in Mouse and Human T Cells

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Mouse and human T cells were activated using the methods mentioned above. To activate Notch signalling, activated T cells were cocultured with Notch ligand-expressing OP9 feeder cells, OP9-DL1 cells for mouse T cells and OP9-hDL1 cells for human T cells. We cultured mouse T cells and OP9-DL1 cells with mouse IL-7 (PeproTech; 10 ng ml⁻¹), an anti-mouse IFN-γ antibody (R4-6A2; 2 μg ml⁻¹), in alpha MEM for 11–12 days. Human T cells and OP9-hDL1 cells were cocultured with human IL-7 (PeproTech; 10 ng ml⁻¹), an anti-human IFN-γ antibody (Bio Legend, B27; 2 μg ml⁻¹), in alpha MEM for 11 days.

Kondo T., Morita R., Okuzono Y., Nakatsukasa H., Sekiya T., Chikuma S., Shichita T., Kanamori M., Kubo M., Koga K., Miyazaki T., Kassai Y, & Yoshimura A. (2017). Notch-mediated conversion of activated T cells into stem cell memory-like T cells for adoptive immunotherapy. Nature Communications, 8, 15338.

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Retroviral Transduction of OT-I Cells

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pRV-luc plasmid was co-transfected in Plate-E with the packaging plasmid, pCL-ECO, using lipofectamine 2000 (Invitrogen, Carlsbad, CA). Supernatants were collected 48 and 72 h later and were combined to generate stocks, which were then used to transduce OT-I cells. Briefly, single-cell suspensions of OT-I cells were activated with Concanavalin A (2 μg/ml; Sigma, St. Louis, MO) and mouse IL7 (1ng/ml; PeproTech, Rocky Hill, NJ) for 24 h. Cells were then transduced with pRV-luc retrovirus in non–tissue culture 24-well plates precoated with RetroNectin (Takara Bio. Inc., Shiga, Japan). The transduced OT-I cells were then cultured for 48 hours in fresh medium supplemented with hIL-2 (300 U/ml; NIH AIDS Reagent Program, Germantown, MD) to allow the cells to recover. They were then used directly for adoptive transfer.

Fu X., Rivera A., Tao L, & Zhang X. (2014). An HSV-2 based oncolytic virus can function as an attractant to guide migration of adoptively transferred T cells to tumor sites. Oncotarget, 6(2), 902-914.

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In vitro Lymphoid Progenitor Culture

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Ebf1-deficient lymphoid progenitor cell lines (31 ) were cultured in IMDM (Wako) with 10% fetal bovine serum (Sigma–Aldrich), penicillin–streptomycin–glutamine, 50 μM β-mercaptoethanol (Sigma–Aldrich), 10 ng/ml mouse SCF (PeproTech), 10 ng/ml human Flt3L (PeproTech), 10 ng/ml mouse IL-7 (PeproTech) with mitomycin C (Wako) treated OP9. For T-cell induction, lymphoid progenitors were cocultured on OP9-DLL4 for 2 days under the same conditions as the maintenance of the lymphoid progenitors.

Koizumi M., Kama Y., Hirano K.I., Endo Y., Tanaka T., Hozumi K, & Hosokawa H. (2022). Transcription factor Zbtb1 interacts with bridging factor Lmo2 and maintains the T-lineage differentiation capacity of lymphoid progenitor cells. The Journal of Biological Chemistry, 298(11), 102506.

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Proliferation Assay of IEL and SP CD4+ Cells

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1 x 10⁵ of sort-purified IEL- or SP CD4⁺ cells were labelled with 2.5μM CFSE (CellTrace CFSE Cell Proliferation Kit, ThermoFisher SCIENTIFIC), and then stimulated with PMA+ionomycin PMA+ionomycin (PMA 50ng/ml, ionomycin 500ng/ml) in the complete medium with 100 U/mL recombinant human IL2 (Roche), 50 ng/mL mouse IL7 (Peprotech), and 50 ng/mL mouse IL15 (Peprotech) under the condition of 37°C, 5% CO2. After 72 hours, cells were harvested and stained with appropriate antibodies and analyzed by FACS Canto II (BD Bioscience).

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