Il 7 and il 15

Manufactured by Miltenyi Biotec

Sourced in Germany

IL-7 and IL-15 are cytokines that play important roles in the immune system. IL-7 is essential for the development and survival of T cells, while IL-15 is involved in the activation and proliferation of natural killer (NK) cells and memory CD8+ T cells. These cytokines have potential applications in various areas of immunology and cell biology research.

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IL-15 (145 citations) Recombinant human IL-7 and IL-15 (3 citations)

9 protocols using il 7 and il 15

Expansion and scRNA-Seq Analysis of CAR-T cells

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Leukapheresis samples were thawed, washed and seeded at a concentration of 2 × 10⁶ cells/ml in TexMacs (Miltenyi) supplemented with human AB serum (Seralab) and IL-7 and IL-15 (Miltenyi) in 24 well plates.
Similarly, product samples were thawed, washed, and resuspended at a concentration of 1 × 10⁵ CAR⁺ cells/ml in 0.5 ml of culture media and cultured for 24 hours in 48 well plates. For the antigen-exposure experiments, product samples were co-cultured in the presence of MM1.S cells in a 1:1 effector (CAR⁺ cells)/target (MM1.S) ratio.
For single-cell RNA-Seq analysis, following 20 hours of incubation, wells from the same conditions were pooled and all samples were FACS sorted, selecting live cells by using DAPI staining. Cells were washed and counted before entering the droplet-based scRNA-Seq workflow.

Wang X., Peticone C., Kotsopoulou E., Göttgens B, & Calero-Nieto F.J. (2021). Single-cell transcriptome analysis of CAR T-cell products reveals subpopulations, stimulation, and exhaustion signatures. Oncoimmunology, 10(1), 1866287.

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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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Culturing Human Immune Cells for Research

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Human embryo kidney-293T (HEK-293T) cells and its derivatives were propagated in Iscove’s modified Dulbecco’s modified Eagle medium (IMDM) supplemented with 10% fetal calf serum (FCS) and Penicillin-Streptomycin-Glutamine (PSG) (Lonza, Basel, Switzerland). CEM A3.01 T cells were grown in RPMI 1640 supplemented with 10% FCS and PSG.
Human peripheral blood mononuclear cells were isolated from healthy donors after centrifugation on a Ficoll-Hypaque gradient (Lymphoprep; Stemcell Technologies, Vancouver, Canada) and then frozen in aliquots. Peripheral blood mononuclear cells were subsequently thawed and cultured in RPMI 1640 supplemented with 10% FCS, PSG, and IL-7 and IL-15 (5 ng/ml, each) (Miltenyi Biotec, Bergisch Gladbach, Germany) after preactivation with CD3/CD28 Dynabeads (Life Technologies Italia, Monza, Italy) for 48 hours as described in Kaneko et al.^{30 (link)}

Marin V., Stornaiuolo A., Piovan C., Corna S., Bossi S., Pema M., Giuliani E., Scavullo C., Zucchelli E., Bordignon C., Rizzardi G.P, & Bovolenta C. (2016). RD-MolPack technology for the constitutive production of self-inactivating lentiviral vectors pseudotyped with the nontoxic RD114-TR envelope. Molecular Therapy. Methods & Clinical Development, 3, 16033-.

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Expansion of CMV-specific CTLs

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For 50 × 10⁶ non-adherent PBMCs, 3.75 μg of Streptamer (ST) Magnetic Beads and 5 μg of ST MHC class I (HLA-A*02:01/CMVpp65-NLVPMVATV Streptamer; both from IBA GmbH, Göttigen, Germany) were incubated overnight at 4 °C in the dark to generate the ST-magnetic bead complex. This complex was added to the cell pellet and incubated for 45 min at 4 °C in the dark. ST+ cells were isolated using a Possel_ds selection program on the AutoMACS Pro separator (Miltenyi Biotec, Bergisch Gladbach, Germany). ST was dissociated from the eluted cells with 1 mM d-biotin (IBA GmbH), or left bound to the cell in order to compare the effect of constant binding of the multimer to the TCR.
Following magnetic enrichment, up to 100.000 ST+ CMV-specific CTLs were co-cultured with 8 × 10⁶ γ-irradiated (30Gy) autologous PBMCs that were pre-loaded with 10 μg/ml CMVpp65_495–503 peptide (NLVPMVATV) overnight (Proimmune, Oxford, UK). The expansion was carried out in round bottom tissue culture tubes (Falcon BD Biosciences) in RPMI 1640 supplemented with 10 % human AB serum, 1 % penicillin/streptomycin (Lonza) and 10 ng/ml of IL-7 and IL-15 (Miltenyi Biotec). Cells were expanded over 21 days, culture medium was changed every 2 or 3 days and cells split when necessary. Viable cell counts were performed every 2–3 days using 0.4 % trypan blue staining.

Beloki L., Ciaurriz M., Mansilla C., Zabalza A., Perez-Valderrama E., Samuel E.R., Lowdell M.W., Ramirez N, & Olavarria E. (2015). Assessment of the effector function of CMV-specific CTLs isolated using MHC-multimers from granulocyte-colony stimulating factor mobilized peripheral blood. Journal of Translational Medicine, 13, 165.

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Large-Scale GMP-Like CAR-T Cell Manufacturing

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Large-scale manufacturing of CAR-T cells on CliniMACs Prodigy was carried out under GMP-like conditions into Gene-Cell Therapy clean rooms of the Cell Therapy Unit of Hospital Universitario Reina Sofía (Córdoba, Spain). Two different aphereses from a healthy donor were thawed, and around 100 × 10⁶ (link) T cells were inoculated into the CliniMACs prodigy bioreactor (Miltenyi Biotec). CD4 and CD8 cells were selected with CD8 and CD4 Reagent (Miltenyi Biotec), cultured with IL-7 and IL-15 (Miltenyi Biotec), and activated with αCD3 andαCD28 GMP T cell TransAct (Miltenyi Biotec). On day 2 of the process, these cells were transduced with AWARI-LVs (MOI = 5). Cells were cultured in TexMACs GMP medium containing GMP-grade IL-15 and IL-7 (Miltenyi Biotec) for 9 or 10 days. Final product was collected with 100 mL of NaCl 0.9% + 0.5% human serum albumin (HSA).
Cells were stained with CD3-APC, CD4-FITC, CD8-APC Vio770, CD14-PE Vio770, and CD45-Vioblue (Miltenyi Biotec). To assess the efficiency of transduction, CAR-T cells were stained with CD-19 Biotin and anti-Biotin PE (Miltenyi Biotec). Viability was tested by using 7-AAD (Miltenyi Biotec). Phenotype was determined with CD45RA-APC (HI100) and CCR7-BV421 (2-L1-A RUO) from BD Pharmingen. Cells were acquired on a MACsQuant cytometer (Miltenyi Biotec) and analyzed with MACsQuantify Analysis Software (Miltenyi Biotec).

Tristán-Manzano M., Maldonado-Pérez N., Justicia-Lirio P., Muñoz P., Cortijo-Gutiérrez M., Pavlovic K., Jiménez-Moreno R., Nogueras S., Carmona M.D., Sánchez-Hernández S., Aguilar-González A., Castella M., Juan M., Marañón C., Marchal J.A., Benabdellah K., Herrera C, & Martin F. (2022). Physiological lentiviral vectors for the generation of improved CAR-T cells. Molecular Therapy Oncolytics, 25, 335-349.

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Primary T Cell Isolation and Activation

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Primary T cells were isolated from fresh or frozen apheresis products obtained from healthy donors at the Hematology Department of the Hospital Universitario Reina Sofía (Córdoba, Spain). All donors gave their written informed consent, and the study was performed according to the guidelines of the local ethics committee and complies with the requirements regarding quality and safety for donation, obtaining, storage, distribution, and preservation of human cells and tissues under the Spanish-specific regulation (RD-L 9/2014). Pan-T cells were isolated by negative selection using immunomagnetic beads (Pan T cell Isolation Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) and following MACSExpress Separator (Miltenyi Biotec) or AutoMACs Pro Separator’s (Miltenyi Biotec) protocol and cultured in TexMACS (Miltenyi Biotec) supplemented with 20 UI/mL of IL-2 (Miltenyi Biotec) for EGFP experiments or with 10 ng/mL of IL-7 and IL-15 (Miltenyi Biotec) for CAR experiments. T cells were activated with T cell TransAct (Miltenyi Biotec) during 48 h as recommended by the manufacturer.

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Efficient T Cell Transduction via PTD-Mediated Viral Delivery

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T cells were activated with TransAct (Miltenyi Biotec) as previously described for 48 h and then washed with PBS prior to transduction. Protranduzin (PTD, JPT Peptide Technologies) was prepared following manufacturer’s instructions to obtain a concentration of 10 mg/mL. Then, PTD was gently dissolved in PBS (1 mg/mL) and allowed for the fibril’s formation for 10 min at RT. After that, different concentrations of PTD (12.5, 25, 50 μg/mL) were added to the viral supernatant and incubated for 5 min at RT. 50 μL of a cell suspension at 1 × 107 cells/ml was inoculated to obtain a final volume of 250 μL for a 48-well-plate. T cells were spinoculated at 800 g for 1 h at 32°C. After 5 h, cells were washed and seeded at 1 × 10⁶ cells/ml and cultured with TexMACs supplemented with 10 ng/mL of IL-7 and IL-15 (Miltenyi Biotec) and 1% P/S (Biowest)

Tristán-Manzano M., Maldonado-Pérez N., Justicia-Lirio P., Cortijo-Gutierréz M., Tristán-Ramos P., Blanco-Benítez C., Pavlovic K., Aguilar-González A., Muñoz P., Molina-Estevez F.J., Griesche V., Marchal J.A., Heras S.R., Benabdellah K, & Martin F. (2023). Lentiviral vectors for inducible, transactivator-free advanced therapy medicinal products: Application to CAR-T cells. Molecular Therapy. Nucleic Acids, 32, 322-339.

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CRISPR-Cas9 Mediated Gene Editing of T Cells

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CD4 and CD8 T cells were positively enriched from healthy donor leukapheresis products by magnetic selection (Miltenyi) and activated for 48 h with CD3 and CD28 stimulation (TransACT, 1:17.5 by volume; Miltenyi) in T cell medium (TexMACS medium, Miltenyi, supplemented with 12.5 ng ml^–1 IL-7 and IL-15, Miltenyi, and 3% human AB serum, Valley Biomedical). After activation, the T cells were centrifuged and resuspended in P3 buffer (Lonza). CRISPR–Cas ribonucleoproteins (RNPs) were formulated by complexing guide RNAs targeting TRAC and TRBC (Synthego) to spCas9 protein (Aldevron) in a 6:1 molar ratio. The patient-specific HR template and RNPs were mixed with the cell suspension, electroporated (Lonza, X-unit, EO-115) and transferred into T cell medium in a 24-well G-rex (Wilson Wolf) for 4–5 days with changes of the medium as needed.

Foy S.P., Jacoby K., Bota D.A., Hunter T., Pan Z., Stawiski E., Ma Y., Lu W., Peng S., Wang C.L., Yuen B., Dalmas O., Heeringa K., Sennino B., Conroy A., Bethune M.T., Mende I., White W., Kukreja M., Gunturu S., Humphrey E., Hussaini A., An D., Litterman A.J., Quach B.B., Ng A.H., Lu Y., Smith C., Campbell K.M., Anaya D., Skrdlant L., Huang E.Y., Mendoza V., Mathur J., Dengler L., Purandare B., Moot R., Yi M.C., Funke R., Sibley A., Stallings-Schmitt T., Oh D.Y., Chmielowski B., Abedi M., Yuan Y., Sosman J.A., Lee S.M., Schoenfeld A.J., Baltimore D., Heath J.R., Franzusoff A., Ribas A., Rao A.V, & Mandl S.J. (2022). Non-viral precision T cell receptor replacement for personalized cell therapy. Nature, 615(7953), 687-696.

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Isolation and Expansion of Primary T Cells

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For the isolation of primary T cells, peripheral blood mono-nucleated cells (PBMCs) were isolated from fresh buffy coats obtained from healthy volunteer donors (Service de transfusion, Epalinges, Switzerland). PBMCs were separated by density centrifugation using Lymphoprep (Axonlab, Cat#1114545). Pan-T cells were subsequently extracted by magnetic separation using a human pan-T cell isolation kit (Miltenyi Biotec, Cat#130-096-535) and stimulated with human T cell activator CD3/CD28 beads (Life Technologies, Cat#11161D) and 50 RU IL-2 (Peprotech, Cat#200-02-50UG) for 5 days. After the removal of the beads, primary T cells were further expanded with IL-7 and IL-15 (Miltenyi Biotec, Cat#130-095-367 and Cat#130-095-765) for a further 5–10 days.

Fierle J.K., Brioschi M., de Tiani M., Wetterwald L., Atsaves V., Abram-Saliba J., Petrova T.V., Coukos G, & Dunn S.M. (2021). Soluble trivalent engagers redirect cytolytic T cell activity toward tumor endothelial marker 1. Cell Reports Medicine, 2(8), 100362.

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