HF-LVs are highly superior for transduction of cancer B cells as compared with VSV-G-LVs. (A) Isolated chronic lymphocytic leukemia B cells (B-CLL) or peripheral marginal zone lymphoma B cells (MZL) were transduced with 100 μL of concentrated VSV-G-LV (MOI 300) or H/F-LV (MOI 3) viral supernatant. Both vectors were titrated on 293T cells. Vectors were concentrated by ultrafiltration-centrifugation as described in Hazan-Halevy et al.¹  For the B-CLL donor cells, transductions were performed in parallel in the presence of polybrene (Pb; 10 ng/mL). At 48 hours upon transduction, cells were evaluated for green fluorescence by fluorescence-activated cell sorting (FACS) analysis without washing (48 hours) or followed by 2 subsequent washes with phosphate-buffered saline before FACS analysis (48 hours, 2× wash). Additionally, at 48 hours of transduction, part of the B cells were washed and were maintained on MS5 stromal cells in RPMI medium supplemented with 10% fetal calf serum, IL-2, and IL-15 to confirm stable transduction. At 7 days upon culture, the cells were harvested, passed through a mesh, and stained with anti-CD19APC. Cells were then evaluated for GFP expression by FACS analysis (MS5, 7 days). Data are representative of 3 different donors. (B) Transduction of MZL cells was performed with concentrated H/F- or VSV-G-LVs as mentioned. Two different concentration methods were used: (1) low-speed concentration (3000g, 4°C overnight, 100× concentration) or (2) ultrafiltration-centrifugation (UltraF) as performed by Hazan-Halevy et al.¹  For VSV-G- and HF-LV transductions, cells were harvested at 48 hours and total levels of reverse transcribed proviral DNA (total LTR/cell) were determined by quantitative polymerase chain reaction (see “Methods” in Frecha et al³ ). Freshly isolated, nonstimulated B cells of different B-CLL (C) and MZL (D) donors were transduced with 2 different preparations of H/F-LVs (MOI 5, HF-LV1 and HF-LV2, low-speed concentration) or with VSV-G-LVs (MOI 50, VSV-G-LV1, VSV-G-LV2, low-speed concentration) for 48 hours. Cells were washed subsequently and maintained in culture on MS5 stromal cells in RPMI supplemented with 10% fetal calf serum, IL-2, and IL-15 for 5 more days before FACS analysis was performed as in panel A. (E-F) The same transduction protocols as described for panels C and D were applied on B-CLL and MZL cells prestimulated for 48 hours with Staphylococcus aureus Cowen and IL-2 (10 ng/mL). Note that for non- and BCR-stimulated MZL-3 cells, transduction was performed with only the H/F-LV1 vector preparation. Similarly, prestimulated B-CLL-4 and B-CLL-10 cells were transduced with only H/F-LV1. FACS analysis was performed 2 days after transduction followed by 5 days of culture on MS5 cells to allow evaluation of stable transduction.