Figure 3. Stable transduction of unstimulated primary human B lymphocytes by CD20-LV and CD19ds-LV.

(A) Freshly isolated primary human B lymphocytes from six different donors were transduced with CD20-LV, CD19ds-LV or MV-LV at an MOI of 2, or VSVG-LV at an MOI of 100. Forty-eight hours later, B lymphocytes were identified by their CD20 expression and the percentage of EGFP⁺ cells in the CD20-gated cells was determined by FACS analysis. Results are expressed as mean ± SEM. *, ** and *** indicate P<0.05, P<0.01 and P<0.001 versus transduction with CD20-LV, respectively. Data were analyzed by one-way ANOVA. (B–C) To verify stable integration of the egfp gene into the B lymphocyte genome, freshly isolated primary human B lymphocytes (purity was ∼99.8%) from three different donors were transduced with the indicated vectors. Transduced cells were cultivated in presence of 10 ng/ml rhIL-15 and 10 ng/ml rhIL-2 for 10 days on MS-5 feeder cells. The percentage of CD19⁺/EGFP⁺ cells in the CD20-gated cells was determined by FACS analysis at the indicated time points. (B) FACS blots of one representative experiment are shown (at least 2000 B lymphocytes were gated for analysis). (C) Diagram showing a summary of all three independent experiments. Dark gray column: CD20-LV; light gray column: CD19ds-LV; white column: VSVG-LV. Results are expressed as mean ± SEM. * and ** indicate P<0.05 and P<0.01 for CD20-LV and CD19ds-LV versus VSVG-LV, respectively. Data were analyzed by Student’s t-test without adjustment of multiple comparisons.