B-cell development and Ig isotype switching following ZAP-70 gene therapy.

(A) The percentages of splenic B cells present in WT mice and ZAP-70^−/− mice undergoing transplantation with WT BM or ZAP-70^−/− progenitor cells transduced with either EGFP or ZAP-70 retroviral vectors were assessed by staining with a PE-conjugated αB220 mAb and a Cy-conjugated αIgM mAb. The percentages of B220⁺/IgM⁺ cells are noted. (B) To assess IgG3 and IgG1 isotype switching, splenocytes were cultured in the presence of LPS and LPS plus IL-4, respectively. After 6 days of culture, cells were collected and stained with αIgG1 and αIgG3 mAbs. The percentages of B220⁺/IgG3⁺ and B220⁺/IgG1⁺ cells within the EGFP⁻and EGFP⁺ splenocyte populations of ZAP-70^−/−mice undergoing transplantation with control EGFP or ZAP-70/EGFP retroviral vectors are indicated. Data are representative of results obtained using B cells isolated from 3 individual mice in each group.