Accu prep

Murine T cells were isolated from splenocytes as previously described (Pegram et al., 2012 (link)). Murine CAR T cells were maintained in culture with RPMI in the presence of 100 IU/mL recombinant human IL-2 (Proleukin; Novartis, Basel, Switzerland), selected with Nylon Wool Fiber (Polysciences, Warrington, PA), and activatedwith CD3/CD28 beads, accordingto manufacturer’s instructions (Gibco, Thermo Fisher, Waltham, MA). Human T cells were derived from fresh blood-derived leukocyte concentrate (Leukopack) obtained from the New York Blood Center. Mononuclear cells were separated using density gradient centrifugation with Accu-prep (axis-Shield PoC AS, Oslo, Norway). T cells were isolated, activated, and expanded with 2 × 10⁶/mL PHA (Sigma Aldrich, St. Louis, MO). T cells were cultured in RPMI 1640 in the presence of 100 IU/mL recombinant human IL-2 (Proleukin). Viable cells were enumerated using flow cytometry and counting beads (Ebioscience), following manufacturer’s protocol. Activated human and mouseT cells were retrovirallytransduced as previously described (Pegram et al., 2012 (link), 2015 (link)). CAR expression was detected using Armenian hamster 12D11 antibody (anti-CD19 CAR) or an Alexa Fluor 647-conjugated hamster antibody that specifically binds the 4H1128z CAR (Monoclonal Antibody Facility, Memorial Sloan Kettering Cancer Center).

Fresh UCB units not meeting cell dose criteria for public banking were obtained from the Cleveland Cord Blood Center (Cleveland, OH, USA), and mononuclear cells were separated using density gradient centrifugation with Accu-prep (axis-Shield PoC AS, Oslo, Norway). T cells were isolated, activated and expanded with Dynabeads ClinExVivo CD3/CD28 magnetic beads (Invitrogen), according to the manufacturer's instructions. UCB-derived T cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin and streptomycin, and 2 mm L-glutamine, in the presence of 100 IU/ml recombinant human IL-2 (Proleukin, Novartis, Basel, Switzerland), 10 ng/ml recombinant human IL-12, 10 ng/ml recombinant human IL-7 or 10 ng/ml recombinant human IL-15 (all from R&D Systems, Minneapolis, MN, USA). Viable cells were enumerated using Trypan blue (Invitrogen) exclusion. Secondary stimulation was achieved with ClinExVivo CD3/CD28 beads at a bead to T-cell ratio of 1:2.