Figure 2.

Optimization of measles pseudotyped lentiviral vector transduction of primary human B cells

(A) Schematic of the workflow and timeline of the B cell studies performed. Primary B cells were isolated from PBMCs of donors 2 and 3. See materials and methods for culture conditions, assays, flow cytometry, and vector copy number per cell (VCN) information. (B) Comparison of B cell transduction efficiencies of MV-M-eGFP-eCD4-Ig, MV-EB-eGFP-eCD4-Ig, VSV-G-M-eGFP-eCD4-Ig, and VSV-G-EB-eGFP-eCD4-Ig LVs. LV transduction efficiency was assessed by flow cytometry for eGFP 5 days post transduction (n = 3, mean and SD are shown; ns, not significant; ∗∗p < 0.01, ∗∗∗∗p < 0.0001). Donors 2 and 3 are shown. (C) Representative flow-cytometric analysis of B cells transduced with MV-M-eGFP-eCD4-Ig, MV-EB-eGFP-eCD4-Ig, and mock (no LV). B cells were assessed by flow cytometry for eGFP, CD19, IgM, and IgG expression 5 days post LV transduction. Donor 1 is shown. (D) Genomic DNA was extracted 11 days post LV transduction from both B cell donor cells to assess the VCN. (E) Supernatants from LV transduced primary B cells were harvested 5 days post LV transduction (expansion period day 7) and assessed for eCD4-Ig production. eCD4-Ig production was normalized to the total number of live cells per day post transduction. Data presented are mean ± SD of triplicates, with the ranges shown.