Human cell lines and primary human cells

The Raji lymphoblast-like cell line was used as a CD19-expressing cell line for target in cytotoxicity assays and in vivo tumor challenges (ATCC CCL­86). Peripheral blood from anonymous donors was used to isolate peripheral blood mononuclear cells (PBMC) through Ficoll-Paque Plus (GE Healthcare Life Sciences 17–1440-02) density gradient separation. Dynabeads® Human T-Activator CD3/CD28 beads (ThermoFisher Scientific 11132D) were used to activate T lymphocytes through incubation for 72 hours. After 72 hours, the T cells were harvested and beads removed through a magnetic column system. Once the beads were removed, 5 × 10⁵ cells were transduced in R10 at 4 × 10⁷ TU/mL. These cells were kept in culture in RPMI plus 10% FBS (R10) with rhuIL-2 (R&D Systems 202-IL-500) and reactivated with the anti-CD3/CD28 beads every 7–10 d.

Umbilical cord blood units were obtained through anonymous collection from the delivery rooms at UCLA. Isolation of human CD34+ cells were obtained through immunomagnetic beads (MACS CD34 MicroBead Separation Kit, Miltenyi 130–046-702) and stored at −170°C. After thawing, these cells were prestimulated for 14 hours in X-Vivo15 medium (Lonza 04–744Q) with human stem cell factor (SCF), Flt-3 ligand, and thrombopoietin (R&D Systems 255-SC-200, 308-FK and 288-TP). 1 × 10⁶ cells in 1 mL with wells coated with recombinant human fibronectin fragment RetroNectin (Clontech T100B) were transduced at a vector concentration of 5 × 10⁷ TU/mL for 24 hours.¹⁸ Myeloid differentiation conditions were created by culture for 12–15 d in Iscove's Modified Dulbecco's medium (IMDM) (Corning 10–016-CM) with 10% FBS, plus SCF and IL-3.¹⁸