Rhil 15

Manufactured by CellGenix

RhIL-15 is a recombinant human interleukin-15 product manufactured by CellGenix. Interleukin-15 is a cytokine that plays a key role in the activation and proliferation of natural killer cells and memory T cells. The product is intended for use in cell culture and research applications.

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Lab products found in correlation

Rhil 7, by CellGenix (3 mentions) Clinimacs device, by Miltenyi Biotec (2 mentions) Knockout sr, by Thermo Fisher Scientific (2 mentions) Rhil 21, by Miltenyi Biotec (2 mentions) Cts dynabeads cd3 cd28, by Thermo Fisher Scientific (2 mentions) Sepax 2rm device, by Cytiva (2 mentions) Cryostor cs5, by BioLife Solutions (2 mentions) X vivo 15, by Lonza (2 mentions) Rhil 2, by Novartis (2 mentions) Dynabeads human t expander cd3 cd28, by Thermo Fisher Scientific (2 mentions) Cd3 cd28 dynabead, by Thermo Fisher Scientific (1 mentions) Dynabeads m 270 epoxy beads, by Thermo Fisher Scientific (1 mentions) Recombinant human cd19 protein, by Abcam (1 mentions) Streptomycin, by Harvard Bioscience (1 mentions) Ifnγ secretion assay cell enrichment and detection kit, by Miltenyi Biotec (1 mentions) Vle rpmi 1640, by PAN Biotech (1 mentions) Rituximab, by Genentech (1 mentions) Rhil 12, by Thermo Fisher Scientific (1 mentions) Cd45 j33, by Beckman Coulter (1 mentions) Pstat5, by Thermo Fisher Scientific (1 mentions)

8 protocols using rhil 15

Generation of CD19 CAR T Cells

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PBMC from three healthy donors were activated with CD3/CD28 Dynabeads (Invitrogen) at 1:3 (cells:beads ratio). CD19 CAR T cells were generated by transducing activated PBMCs the next day with a lentivirus encoding CD19R (EQ):CD28:ζ/EGFRt at an MOI of 3 and expanded in the presence of 50 U/mL rhIL-2 (CellGenix) and 0.5 ng/ml rhIL-15 (CellGenix) as previously described (PMID: 23090078, PMID: 28331616). Cultures were then supplemented with 50 U/mL rhIL-2 and 0.5 ng/mL IL-15 every 48 h up to day 14. CD19 antigen stimulation of CD19CAR T cells was carried out by Dynabeads M-270 Epoxy beads (ThermoFisher Scientific) coated with recombinant human CD19 protein (Abcam). 100 μg CD19 protein was used to coat 2.6x10^8 beads. Beads were washed following the coupling protocol from ThermoFisher Scientific (14311D). CD19 protein was added to the beads and incubated overnight (16–24 hours) at 37°C with constant mixing. Coupled beads were further washed and separated using DynaMag following wash protocol. 10x10^6 coupled beads were washed once with 3 ml 1% human serum albumin (HAS in PBS) and 3 ml RPMI media supplemented with 10% FBS. 10x10^6 CD19-coupled beads were introduced to CD19CAR T cells on day 14 at a 1:1 ratio and was supplemented with 50 U/mL rhIL-2 and 0.5 ng/mL IL-15 every 48 h for 7 more days (up to day 21).

Goldberg L., Haas E.R., Vyas V., Urak R., Forman S.J, & Wang X. (2022). Single-cell analysis by mass cytometry reveals CD19 CAR T cell spatiotemporal plasticity in patients. Oncoimmunology, 11(1), 2040772.

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Anti-B7-H3 CAR-T Cell Production

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CD4+ and CD8+ T cells were isolated from patient apheresis products using the CliniMACS device (Miltenyi Biotec). A 1:1 mixture of CD4-enriched and CD8-enriched cell fractions were then pooled, suspended in X-Vivo 15 (Lonza) media supplemented with 2% KnockOut SR (Life Technologies), 5 ng/mL rhIL-7 (CellGenix), 0.5 ng/mL rhIL-15 (CellGenix) and 10 ng/mL rhIL-21 (Miltenyi Biotec), initiated into culture in a G-Rex 100MCS vessel (Wilson-Wolf), and stimulated using CD3/CD28 CTS® Dynabeads (Life Technologies). Cell products were then transduced with a GMP-grade SIN (self-inactivating) lentivirus encoding the anti-B7-H3CAR, the methotrexate-resistant human DHFR mutein huDHFRdm, and the cell-surface marker EGFRt. On day 3 of culture, 50nM methotrexate (Mylan) was added to the culture vessel to select for cells possessing DHFRdm. On day 7 of culture, CD3/CD28 CTS® beads were removed from the cell suspension using the Dynamag CTS device. Following 10–13 days in culture, patient cells were harvested and washed using the Sepax 2RM device (Cytiva) and resuspended in CryoStor-CS5 (Biolife Solutions) for cryopreservation in CellSeal closed-system vials (Sexton).

Vitanza N.A., Wilson A.L., Huang W., Seidel K., Brown C., Gustafson J.A., Yokoyama J.K., Johnson A.J., Baxter B.A., Koning R.W., Reid A.N., Meechan M., Biery M.C., Myers C., Rawlings-Rhea S.D., Albert C.M., Browd S.R., Hauptman J.S., Lee A., Ojemann J.G., Berens M.E., Dun M.D., Foster J.B., Crotty E.E., Leary S.E., Cole B.L., Perez F.A., Wright J.N., Orentas R.J., Chour T., Newell E.W., Whiteaker J.R., Zhao L., Paulovich A.G., Pinto N., Gust J., Gardner R.A., Jensen M.C, & Park J.R. (2022). Intraventricular B7-H3 CAR T Cells for Diffuse Intrinsic Pontine Glioma: Preliminary First-in-Human Bioactivity and Safety. Cancer Discovery, 13(1), 114-131.

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Engineered Antigen-Specific T Cell Therapy

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CD4⁺ and CD8⁺ T cells were isolated from patient apheresis products using the CliniMACS device (Miltenyi Biotec). A 1:1 mixture of CD4-enriched and CD8-enriched cell fractions were then pooled, suspended in X-Vivo 15 (Lonza) media supplemented with 2% KnockOut SR (Life Technologies), 5 ng/mL rhIL7 (CellGenix), 0.5 ng/mL rhIL15 (CellGenix), and 10 ng/mL rhIL-21 (Miltenyi Biotec), initiated into culture in a G-Rex 100MCS vessel (Wilson-Wolf), and stimulated using CD3/CD28 CTS Dynabeads (Life Technologies). Cell products were then transduced with a GMP-grade SIN (self-inactivating) lentivirus encoding the anti-B7-H3 CAR, the MTX-resistant human DHFR mutein huDHFRdm, and the cell-surface marker EGFRt. On day 3 of culture, 50 nmol/L MTX (Mylan) was added to the culture vessel to select for cells possessing DHFRdm. On day 7 of culture, CD3/CD28 CTS beads were removed from the cell suspension using the Dynamag CTS device. Following 10 to 13 days in culture, patient cells were harvested and washed using the Sepax 2RM device (Cytiva) and resuspended in CryoStor-CS5 (Biolife Solutions) for cryopreservation in CellSeal closed-system vials (Sexton).

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Isolation and Culture of SARS-CoV-2-Specific T Cells

Cited 1 time

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Virus-specific T cells were isolated from PBMCs derived from 100 mL of peripheral blood from convalescent donors following a 6-h stimulation with overlapping SARS-CoV-2-specific peptide pools (JPT Peptide Technologies; 1 μg/mL each) using an IFN-γ Secretion Assay—Cell Enrichment and Detection Kit according to the manufacturer’s instructions (Miltenyi Biotec). Isolated virus-specific T cells were cultured in complete medium (VLE RPMI 1640 [PAN-Biotech] supplemented with penicillin [100 IU/mL], streptomycin [Biochrom], 10% fetal calf serum [FCS, PAA], 10 ng/mL recombinant human IL-7 [rhIL-7] and rhIL-15 [CellGenix]) in 24-well plates, in humidified incubators at 37°C and 5% CO₂ as described previously.⁸⁸ (link)^,¹²² (link) Cells were split 1:1 upon reaching 100% confluency.

Peter L., Wendering D.J., Schlickeiser S., Hoffmann H., Noster R., Wagner D.L., Zarrinrad G., Münch S., Picht S., Schulenberg S., Moradian H., Mashreghi M.F., Klein O., Gossen M., Roch T., Babel N., Reinke P., Volk H.D., Amini L, & Schmueck-Henneresse M. (2022). Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients. Molecular Therapy. Methods & Clinical Development, 25, 52-73.

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Characterization of NK Cell Function

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The following anti-human monoclonal antibodies (mAbs) were used: CD56(N901), CD3(UCHT1), CD159a(Z199), CD158a,h(EB6B), and CD45(J.33; all Beckman Coulter); CD16(3G8), CD158b(CH-L), IFNγ(B27), CD107a(H4A3), Syk(4D10), ZAP-70(1E7.2), pZAP70(pY319)/pSYK(pY352), pERK1/2(pT202/pY204), pAkt(pS473; all BD); CD3z(6B10.2; eBioscience); CD158e1(DX9), CD107a(H4A3), goat anti-mouse IgG(Poly4053; all Biolegend); FcεR1γ (Milli-Mark); and rituximab (Genentech). The following endotoxin-free recombinant human (rh) cytokines were used: rhIL-12, rhIL-18 (Peprotech), rhIL-15 (CellGenix and Miltenyi). The following cell lines were used: K562 (ATCC, CCL-243) and Raji (ATCC, CCL-86). Cells were obtained from ATCC in 2008, viably cryopreserved and stored in LN2, thawed for use in these studies, and maintained for <2 months at a time in continuous culture as per ATCC instructions. K562 cells were authenticated in 2015 by confirming cell growth morphology (lymphoblast), short tandem repeat profile, growth characteristics, and functionally as NK cell sensitive targets. Raji cells were authenticated in 2015 by confirming cell growth morphology (lymphoblast), short tandem repeat profile, growth characteristics, phenotype of uniform expression of human CD20, and functionally as anti-CD20 mAb opsonized targets for ADCC.

Wagner J.A., Berrien-Elliott M.M., Rosario M., Leong J.W., Jewell B.A., Schappe T., Abdel-Latif S, & Fehniger T.A. (2016). Cytokine-induced memory-like differentiation enhances unlicensed NK cell anti-leukemia and FcγRIIIa-triggered responses. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 23(3), 398-404.

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Cytokine-Activated NK Cell Expansion

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The following anti-human mAbs were used, Beckman Coulter: CD56(N901), CD3(UCHT1); BD: CD16(3G8), IFN-γ(B27), CD25(M-A251), pSTAT5(pY694); purified anti-CD25 (B-B10, eBioscience) or isotype IgG1k (Biolegend). The following endotoxin-free cytokines were used: rhIL-2 (Chiron); rhIL-15 (CellGenix); rhIL-12, rhIL-18 (Peprotech). The K562 (ATCC) cell line was maintained in RPMI-1640 plus 10% FCS and supplements.^{20 (link)}

Leong J.W., Chase J.M., Romee R., Schneider S.E., Sullivan R.P., Cooper M.A, & Fehniger T.A. (2014). Pre-activation with IL-12, IL-15, and IL-18 induces CD25 and a functional high affinity IL-2 receptor on human cytokine-induced memory-like NK cells. Biology of blood and marrow transplantation : journal of the American Society for Blood and Marrow Transplantation, 20(4), 463-473.

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Expansion and Transduction of T Cells

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Tn/mem cells were stimulated with Dynabeads Human T-Expander CD3/CD28 (catalog 11141D, Invitrogen) at a 1:3 cell-to-bead ratio and transduced with lentivirus at a multiplicity of infection of 1.5 to 3 in X-Vivo15 containing 10% FBS and 100 μg/mL protamine sulfate (catalog NDC 63323-0229-15, APP Pharmaceuticals), 50 U/mL rhIL2 (catalog NDC 76310-0022-01, Clinigen), and 0.5 ng/mL rhIL15 (catalog 1013-050, CellGenix). Cultures were then maintained at 37 °C and 5% CO₂, with addition of X-Vivo15 media containing 10% FBS as required to keep cell density around 6 × 10⁵ cells/mL, with cytokine supplementation 3 times a week. On day 7 of culture, the CD3/CD28 Dynabeads were removed from cultures using a DynaMag 5 magnet (catalog 12303D, Invitrogen). T cell lines were enriched with EasySep CD19 selection kit II (catalog 17854, Stemcell) around day 14 and propagated for 19 to 24 days prior to cryopreservation.

Stern L.A., Gholamin S., Moraga I., Yang X., Saravanakumar S., Cohen J.R., Starr R., Aguilar B., Salvary V., Hibbard J.C., Kalbasi A., Shepphird J.K., O’Hearn J., Garcia K.C, & Brown C.E. (2022). Engineered IL13 variants direct specificity of IL13Rα2-targeted CAR T cell therapy. Proceedings of the National Academy of Sciences of the United States of America, 119(33), e2112006119.

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Episomal Generation of Integration-free iPSCs

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Tn/mem cells were reprogrammed into pluripotent stem cells (iPSCs) by an integration-free method modified from a published protocol (Okita et al., 2013 (link)). In brief, one million Tn/mem cells were electroporated with 3 μg of plasmid mixture using the Human T Cell Nucleofector Kit and the Nucleofector 4D electroporation device (Lonza). The plasmid mixture was composed of episomal plasmids encoding OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for TP53 (Okita et al., 2013 (link)). The transfected cells were cultured in X-VIVO 15 medium (Lonza) supplemented with 10% FBS (HyClone), 50 U/mL rhIL-2 (Novartis Oncology), 0.5 ng/mL rhIL-15 (CellGenix) and Dynabeads Human T-Expander CD3/CD28 (ThermoFisher Scientific) (bead to cell ratio of 1:1). Two days after the transfection, an equal volume of pluripotent stem cell (PSC) medium containing rhFGF-basic and 10 μM Y27632 was added (Okita et al., 2013 (link)). The medium was then completely changed to PSC medium 4 days after transfection. iPSC colonies were visible at day 20–30 and individual colonies were picked under a microscope for further culture/expansion in cGMP-grade mTeSR1 medium (StemCell Technologies) in Matrigel-coated (Corning) plates.

Wang Z., McWilliams-Koeppen H., Reza H., Ostberg J., Chen W., Wang X., Huynh C., Vyas V., Chang W.C., Starr R., Wagner J., Aguilar B., Yang X., Wu X., Wang J., Chen W., Koelker-Wolfe E., Seet C., Montel-Hagen A., Crooks G.M., Forman S.J, & Brown C. (2022). 3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR T cells. Cell stem cell, 29(4), 515-527.e8.

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