Rhil 21

CAR-T cells were generated from donors’ peripheral blood mononuclear cells (PBMCs). For T cells activation, 1 μg/ml anti-CD3 monoclonal antibody (OKT3, Takara) was pre-coated overnight at 4 °C and 50 ng/ml anti-CD28 monocolonal antibody (CD28.2, Biolegend) was added into the medium. The PBMCs were activated for 2 days before infection. Infection was carried out by centrifugation at 850 g in a 24-well plate at 31 °C for 2 hours, and an agent called Envirus™-LV (Engreen Biosystem) was applied to promote the infection efficiency. After infection, the CAR-T cells were cultured in GT-T551 medium (Takara) with 0.5% fetal bovine serum (FBS, Gibco) and 300 U/ml recombinant human IL-2 (rhIL-2, PeproTech).
In another culture protocol, 10 ng/ml recombinant human IL-7 (rhIL-7, PeproTech), 10 ng/ml recombinant human IL-15 (rhIL-15, PeproTech) and 10 ng/ml recombinant human IL-21 (rhIL-21, PeproTech) were used instead of IL-2.

Antigen specific T cells were generated by stimulation of the lymphocyte population with peptide pulsed moDCs at a T cell to antigen presenting cell (APC) ratio of 5:1. Where indicated, starting lymphocyte populations were enriched for CD4⁺ T cells by immunomagnetic negative selection (EasySep Stem Cell Technologies). Cells were co-cultured in cell growth medium supplemented with either standard T cell growth cytokines of 10 ng/ml rhIL-7 and rhIL-15 (Peprotech) or enhanced cytokine conditions of 20 ng/ml rhIL-1β, 20 ng/ml rhIL-6, 10 ng/ml rhIL-7, 10 ng/ml rhIL-15, 50 ng/ml rhIL-21, 25 ng/ml rhIL-23, and 5 ng/ml rhTGFβ (Peprotech). After 72 h of culture, 30 IU/ml rhIL-2 was added to the culture. Cultures were fed or split every 2–3 days as needed. Cultures were re-stimulated up to 2 times with peptide pulsed moDCs every 10–14 days.

The anti-human monoclonal antibodies (mAbs) for flow cytometry were fluorescein iso-thiocyanate (FITC)-conjugated CD3, phycoerythrin-cyanine 5-conjugated CD56, phycoerythrin (PE)-conjugated CD11a, PE-conjugated CD16, PE-conjugated CD107a, PE-conjugated CD279, PE-conjugated CD335, PE-conjugated CD337, PE-conjugated CD314 and IgG1 isotype control, purchased from BD Biosciences (San Jose, CA, USA), and anti-human Abs PE-conjugated CD159c, PE-conjugated CD159a, PE-conjugated IgG1 and PE-conjugated IG2A, purchased from R&D systems (Minneapolis, MN, USA). The following recombinant human interleukins, rhIL-2, rhIL-15, and rhIL-21 (PeproTech, Rocky Hill, NJ, USA), were used to expand the NK cells. Vita-Orange Cell Viability Reagent (WST-8; Biotool, Houston, TX, USA) was used for the cytotoxicity assay. Matrigel (BD Biosciences, San Jose, CA, USA), the reconstituted basement membrane matrix, was used for inducing MDA-MB-231 tumor growth in NSG mice. The use of animals for this study was approved by the Institutional Animal Care and Use Committee of Chonnam National University.

Fresh whole blood (approximately 100 mL per donor) was obtained from healthy adult volunteer donors under approval by Purdue University’s Institutional Review Board (IRB). NK cells were isolated by immunomagnetic negative selection using the EasySep Direct Human NK Cell Isolation Kit (StemCell Technologies). The cells were expanded at 37 °C and 5% CO₂ in either CTS OpTmizer™ T Cell Expansion medium (ThermoFisher, A1048501), henceforth referred to as OpTmizer™, or the in-house RPMIf medium. The complete OpTmizer™ medium was supplemented with 2.6% OpTmizer™ supplement, 5% human AB (hAB) serum (Akron Biotech, AK9905), 1% penicillin/streptomycin (Pen/strep) (Gibco, 15140122), 0.2 mM l-glutamine (Gibco, 25030081), 10 ng/mL rhIL-15 (Shenandoah, 100-86), 500 IU/mL rhIL-2 (Akron Biotech, AK8227), and 25 ng/mL rhIL-21 (Gold Biotechnology, 1110-21-2). The RPMIf medium consisted of RPMI 1640 (Gibco, 11875085), 10% fetal bovine serum (FBS) (Corning, 35-016-CV), 1% Pen/strep, 50 ng/mL 4-1BBL (Peprotech, 310-11), 500 IU/mL rhIL-2, 50 ng/mL rhIL-21. The RPMIf expansion medium is commonly used for the expansion of NK cells ex vivo, largely due to the high rates of expansion that such a medium is able to achieve when feeder cells are present. Feeder cells were K562, chemically treated with 50 µg/mL mitomycin C (Cayman Chemical, 11435) for three hours.

Beginning with culture Step 2 (d2 of overall culture), rhIL-7 (Miltenyi Biotec premium grade, Auburn CA, #130-095-364) was added at an optimized final concentration of 50 ng/ml to fresh medium added at culture splits. Other agents tested in Step 2 but absent from the optimized formulation included rhIL-2 (aldesleukin, Prometheus Labs, San Diego, CA, final concentration 24 IU/ml); rhIL-15 (Peprotech #200-15, final concentration 5 ng/ml) and rhIL-21 (Peprotech #200-21, final concentration 3 ng/ml).