Fig. 5
Impact of VLP-delivered Vpr on CSR efficiency in primary B-cells. a Primary B-cells were transduced with VLPs HA-Vpr or ΔVpr at a M.O.I. of 10. 24 h later CSR was induced by IL-4/α-CD40/TGF-β stimulation for 3 days. IgM-to-IgG1 isotype class switch efficiency was evaluated by flow cytometry by measuring the % IgG1+ B220+. DNA from corresponding cells was extracted and uracil content (dUrd per 105 bp) was determined by LC/MS/MS. b Primary B-cells isolated from either wt C57BL/6 or AID−/− mice, were transduced with increasing MOI of VLP HA-Vpr. 24 h later, isotype switching was induced by cytokine stimulation for 3 days and evaluated by flow cytometry. Values are the mean of duplicate experiments ± SD. Ut, untransduced, non stim: non stimulated. c Primary B-cells were transduced with VLP HA-Vpr (wt or mutants R90K, Q65R, W54R) or ΔVpr. 24 h later CSR was induced as stated above. Class switch efficiency was evaluated by flow cytometry by measuring % IgG1+ B220+ cells. For VLP-transduced cells, % switched cells was determined from GFP+-gated populations. Values are derived from three independent experiments with n corresponding to the number of replicates