Interleukin 7 (il 7)

Manufactured by Miltenyi Biotec

Sourced in Germany, Canada, United States

IL-7 is a cytokine that plays a crucial role in the development and homeostasis of T cells. It is involved in the survival, proliferation, and differentiation of T cell progenitors and mature T cells.

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Lab products found in correlation

101 protocols using interleukin 7 (il 7)

T Cell Transduction with RetroCAR

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Isolated TRBC1⁺ or TRBC2⁺ T cells were resuspended at 1 × 10⁶ cells/mL in R10 and stimulated with TransAct (Miltenyi Biotec; 130-111-160), 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). 24 h after, cells were collected, plated at a density of 1 × 10⁶ cells per well (1 mL) on retronectin-coated (Takara, T100B) 6-well plates in the presence of retroviral supernatant at an MOI of 1. Total volume was adjusted to 3 mL using R10 supplemented with 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). The plate were centrifuged at 1000 g for 40 min. 24 h post spinoculation, the T cells were harvested and re-plated in complete R10 media supplemented with 10 ng/ml IL-7 (Miltenyi Biotec; 130-095-367) and 10 ng/ml IL-15 (Miltenyi Biotec; 130-095-760). Transduction efficiency was determined on day 5 after transduction, and further experiments were commenced on days 5–9 after transduction. CAR expression was assessed by staining with aCD3-PE/Cy7 (317334, Biolegend) and QBend10 APC (FAB7227A, R&D System).

Ferrari M., Righi M., Baldan V., Wawrzyniecka P., Bulek A., Kinna A., Ma B., Bughda R., Akbar Z., Srivastava S., Gannon I., Robson M., Sillibourne J., Jha R., El-Kholy M., Amin O.M., Kokalaki E., Banani M.A., Hussain R., Day W., Lim W.C., Ghongane P., Hopkins J.R., Jungherz D., Herling M., Welin M., Surade S., Dyson M., McCafferty J., Logan D., Cordoba S., Thomas S., Sewell A., Maciocia P., Onuoha S, & Pule M. (2024). Structure-guided engineering of immunotherapies targeting TRBC1 and TRBC2 in T cell malignancies. Nature Communications, 15, 1583.

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Cell Culture and PBMC Isolation

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HEK-293T and HT1080 cells were cultivated in DMEM (Biowest, Nuaille, France) (10% FCS (Biochrom, Berlin, Germany)). Raji and SupT1 cells were cultured in RPMI (Biowest, Nuaille, France) supplemented with 10% FCS and 2 mM L-glutamine (Biowest, Nuaille, France). PBMCs were isolated from buffy coat derived from healthy donors via density centrifugation using Ficoll (Pan Biotech, Aidenbach, Germany) and cultured in TexMACS™ (Miltenyi Biotec, Bergisch Gladbach, Germany) supplemented with 12.5 ng/mL IL-7 and 12.5 ng/mL IL-15 (both Miltenyi Biotec, Bergisch Gladbach, Germany). For the selectivity assay, PBMCs were cultivated in TexMACS™ supplemented with 50 IU/mL IL-4, 12.5 ng/mL IL-7, and 12.5 ng/mL IL-15 (all Miltenyi Biotec, Bergisch Gladbach, Germany) and 1 µg/mL CD40-ligand (RD Systems, Minneapolis, MN, USA).

Cordes N., Winter N., Kolbe C., Kotter B., Mittelstaet J., Assenmacher M., Cathomen T., Kaiser A, & Schaser T. (2022). Adapter-Mediated Transduction with Lentiviral Vectors: A Novel Tool for Cell-Type-Specific Gene Transfer. Viruses, 14(10), 2157.

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Stimulating CD4+ T cell activation

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Resting CD4+ T cells were treated with 20 ng/mL IL-7 (Miltenyi Biotec) and 10 IU/mL IL-2 (CFAR) in complete RPMI for 4 d. Cells were then infected by spinoculation as described above and cultured for 48 h in medium containing IL-7 and IL-2. After 48 h, the cells were activated by plate-bound anti-CD3 and soluble anti-CD28 in IL-2 supplemented RPMI medium. Cells were harvested 24 h postactivation and analyzed by flow cytometry.

Mesner D., Hotter D., Kirchhoff F, & Jolly C. (2020). Loss of Nef-mediated CD3 down-regulation in the HIV-1 lineage increases viral infectivity and spread. Proceedings of the National Academy of Sciences of the United States of America, 117(13), 7382-7391.

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Differentiation of iPSCs into T Cells

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GD2-2840z-CAR-iPSCs were cultivated on C3H10T1/2 feeder cells in IMDM supplemented with 15% FBS (catalog no. SH30070, HyClone, GE Healthcare UK) and a cocktail of 10 mg/mL insulin, 5.5 mg/mL transferrin, 5 ng/mL sodium selenite, 2 mmol/L l-glutamine (catalog no. 41400045; all Thermo Fisher Scientific), 0.45 mmol/L a-monothioglycerol (catalog no. 155723, Sigma-Aldrich), and 50 mg/mL ascorbic acid (catalog no. 87314, Takeda Pharmaceuticals) in the presence of 20 ng/mL VEGF (catalog no. 130-109-386, Miltenyi Biotec). On day 14 of coculture, sac-like structures containing hematopoietic progenitor cells were extracted and transferred onto C3H10T1/2 feeder cells expressing Delta-like ligands 1 and 4 in MEMα supplemented with FBS (HyClone) in the presence of 20 ng/mL human stem cell factor, 10 ng/mL human Fms-related tyrosine kinase 3 ligand (catalog no. 130-096-474), and 10 ng/mL IL7 (all Miltenyi Biotec). On day 28 of coculture, T-lineage cells were harvested and stimulated with irradiated PBMCs in NS-A2 in the presence of 5 mg/mL phytohemagglutinin (catalog no. 11249738001, Sigma-Aldrich) and 10 ng/mL IL7 and IL15 (both Miltenyi Biotec).

Kinoshita S., Ishii M., Ando J., Kimura T., Yamaguchi T., Harada S., Takahashi F., Nakashima K., Nakazawa Y., Yamazaki S., Ohshima K., Takahashi K., Nakauchi H, & Ando M. (2024). Rejuvenated iPSC-derived GD2-directed CART Cells Harbor Robust Cytotoxicity Against Small Cell Lung Cancer. Cancer Research Communications, 4(3), 723-737.

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OP9 Co-Culture for T-Cell Differentiation

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LOUCY (ACC-394) and HEK293T (ACC-305) cells were purchased at the DSMZ. OP9-GFP and OP9-DL1 cell lines were provided by JC Zúñiga-Pflücker (University of Toronto, Toronto, Canada). The identity of these and the cell lines LOUCY (ACC-394) and HEK293T (ACC-305, DSMZ) were confirmed by DNA fingerprinting, and cells were regularly tested negative for mycoplasma contamination. OP9 cocultures were performed according to the original protocol (86 (link)). Recombinant human SCF (10 ng/mL, R&D Systems), FLT3L (5 ng/mL, Miltenyi Biotec), and IL-7 (2 ng/mL, Miltenyi Biotec) were added at the initiation and every 2–3 days upon splitting the human cocultures. Murine recombinant cytokines (Miltenyi Biotec) for coculture were: IL-7 (5 ng/mL) and FLT3L (5 ng/mL). Overnight recovery of BM lineage-depleted cells prior to the start of the coculture was done in Stemspan (Stemcell Technologies) supplemented with recombinant murine TPO (100 ng/mL), SCF (100 ng/mL), and FLT3L (50 ng/mL).

Canté-Barrett K., Meijer M.T., Cordo’ V., Hagelaar R., Yang W., Yu J., Smits W.K., Nulle M.E., Jansen J.P., Pieters R., Yang J.J., Haigh J.J., Goossens S, & Meijerink J.P. (2022). MEF2C opposes Notch in lymphoid lineage decision and drives leukemia in the thymus. JCI Insight, 7(13), e150363.

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Expansion of Genetically Modified PBMCs

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PBMCs were separated from buffy coats obtained from healthy donors with Ficoll density gradient centrifugation (Ficoll-Paque, GE Healthcare). All donors gave written informed consent in accordance with Helsinki ethical guidelines. Cells were cultivated at 37°C in 5% CO₂ in CellGenix GMP DC medium (CellGenix) supplemented with heat-inactivated 10% fetal bovine serum (Thermo Fisher Scientific), penicillin/streptomycin 100 U/mL (Thermo Fisher Scientific), and cytokines IL-4 (20 ng/mL), IL-7 (10 ng/mL), and IL-21 (40 ng/ml), all from Miltenyi Biotec. For large-scale expansion, we used G-Rex10 flasks (Wilson Wolf). For electroporations, 1 × 107 PBMCs were resuspended in 100 μL of buffer T containing plasmid DNA or linear transposon+ mRNA. Cells were then electroporated with the Neon Electroporation System (Thermo Fisher Scientific, USA) by a single 20-ms/2,300-V pulse and then were transferred to complete cell media. Next day, cells were polyclonally stimulated with TransAct reagent (anti-CD3/CD28, Miltenyi Biotec, Germany) and expanded for 21 days in the presence of IL-4, IL-7, and IL-21, identically as already described.¹³

Kaštánková I., Štach M., Žižková H., Ptáčková P., Šmilauerová K., Mucha M., Šroller V, & Otáhal P. (2021). Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells. Molecular Therapy. Methods & Clinical Development, 23, 119-127.

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Transcriptomics Analysis of T-cell Subsets

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The TCRγδ⁺ population and the CD10⁺ PD-1⁺ population were cultured in cIMDM supplemented with IL-15 (10 ng/mL; Miltenyi, 130-095-765) and the CD10⁻ PD-1⁻ population in cIMDM supplemented with IL-7 (10 ng/mL; Miltenyi, 130-095-362) for 11 days before the cells were harvested for transcriptomics analysis. RNA extraction was performed using the miRNeasy Mini Kit (Qiagen, Venlo, The Netherlands; 217004). For poly(A) RNA-seq, the QuantSeq 3′ mRNA FWD kit (Lexogen, Vienna, Austria) was used, followed by single-ended sequencing on the NextSeq500 Sequencing System (Illumina, San Diego, CA, USA) with a read length of 75bp. RNA-seq reads were aligned to hg38-noalt using STAR v2.6.0c and quantified on Ensembl v93.

Billiet L., Goetgeluk G., Bonte S., De Munter S., De Cock L., Pille M., Ingels J., Jansen H., Weening K., Van Nieuwerburgh F., Kerre T., Taghon T., Leclercq G, & Vandekerckhove B. (2020). Human Thymic CD10+ PD-1+ Intraepithelial Lymphocyte Precursors Acquire Interleukin-15 Responsiveness at the CD1a– CD95+ CD28– CCR7– Developmental Stage. International Journal of Molecular Sciences, 21(22), 8785.

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Generating T Cell Precursors from Murine Bone Marrow

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OP9-DL1, a mouse BM stromal cell line of (C57BL/6 x C3H)F₂-op/op origin transduced to express the Notch 1 ligand DLL1, was obtained under an MTA from J.-C. Zúñiga-Pflücker (University of Toronto, Toronto, Canada). Cell culture medium consisted of αMEM (Invitrogen) supplemented with 20% heat-inactivated FBS, 100 U/ml penicillin (Invitrogen), and 100 µg/ml streptomycin (Invitrogen). T cell precursors were generated in vitro as described previously with modifications (Min et al., 2002 (link)). In brief, LSK cells were sorted as described above and added to tissue culture–treated polystyrene 24-well tissue culture plates that were seeded with 4,000 OP9-DL1 cells per cm² the day before. The tissue culture media was supplemented with 10 ng/ml IL-7 (Miltenyi Biotec) and 10 ng/ml FLT3-ligand (Miltenyi Biotec) and different concentrations of Degarelix or vehicle (mannitol). Cultures were passaged every 4 d.

Velardi E., Tsai J.J., Holland A.M., Wertheimer T., Yu V.W., Zakrzewski J.L., Tuckett A.Z., Singer N.V., West M.L., Smith O.M., Young L.F., Kreines F.M., Levy E.R., Boyd R.L., Scadden D.T., Dudakov J.A, & van den Brink M.R. (2014). Sex steroid blockade enhances thymopoiesis by modulating Notch signaling. The Journal of Experimental Medicine, 211(12), 2341-2349.

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Automated CAR T Cell Manufacturing with CliniMACS Prodigy

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CAR T cell manufacture was automated with the use of the CliniMACS Prodigy® device using the TCT software program and TS520 tubing set (Miltenyi Biotec, Bergisch Gladbach, Germany). The instrument setup and technical protocol were described by Zhu et al.(59 (link)). The clinical-grade reagents applied in this process were CliniMACS Buffer, TexMACS Media, CliniMACS CD4 reagent, CliniMACS CD8 reagent, TransAct, and the cytokines IL-7 and IL-15 (Miltenyi Biotec, Bergisch Gladbach, Germany). Peripheral blood apheresis products were loaded into the machine and CD4 and CD8 T cells were isolated using CliniMACS CD4 reagent and CliniMACS CD8 reagent according to the manufacturer’s instructions. The isolated T cells were then stimulated with IL-7 and IL-15 (Miltenyi Biotec, Bergisch Gladbach, Germany) at a concentration of 25μg/2L bag of TexMACS Media with 3% human AB serum (Innovative Research, Novi, MI, USA). human AB serum was removed after the 6^th day of culture. The viability, purity, and potency of the products were confirmed as previously described(23 (link)). This process was performed at the Cellular Therapy Lab of University Hospitals Cleveland Medical Center Seidman Cancer Center/Case Western Reserve University Center for Regenerative Medicine.

Jackson Z., Hong C., Schauner R., Dropulic B., Caimi P.F., de Lima M., Giraudo M.F., Gupta K., Reese J.S., Hwang T.H, & Wald D.N. (2022). Sequential single cell transcriptional and protein marker profiling reveals TIGIT as a marker of CD19 CAR-T cell dysfunction in patients with non-Hodgkin’s lymphoma. Cancer discovery, 12(8), 1886-1903.

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Cell Culture and T-cell Activation Protocol

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U2-OS (ATCC) and HEK 293 cells (ATCC) were cultured in Advanced DMEM (Life Technologies), supplemented with 10% FBS (Thermo Fisher Scientific), 2 mM GlutaMax (Thermo Fisher Scientific) and penicillin-streptomycin (50 U/ml) (Thermo Fisher Scientific) at 37°C with 5% CO₂. Human primary CD4⁺/CD8⁺ T-cells were cultured in X-Vivo 15 media (Lonza) supplemented with 5% human heat-inactivated serum (Fisher), 10 ng/ml IL-7 (Miltenyi), and 10 ng/ml IL-15 (Miltenyi). T-cells were stimulated with MACS GMP T-cell TransAct polymeric nanomatrix (Miltenyi) for 3 days according to the manufacturer’s instructions prior to transfection.

Lazzarotto C.R., Malinin N.L., Li Y., Zhang R., Yang Y., Lee G., Cowley E., He Y., Lan X., Jividen K., Katta V., Kolmakova N.G., Petersen C.T., Qi Q., Strelcov E., Maragh S., Krenciute G., Ma J., Cheng Y, & Tsai S.Q. (2020). CHANGE-seq reveals genetic and epigenetic effects on CRISPR-Cas9 genome-wide activity. Nature biotechnology, 38(11), 1317-1327.

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