Flow cytometry and Cμ-deletion analysis of circulating sIgD⁺ B cells. (A) After FACS sorting of sIgD⁺ and sIgD^- subsets (left), the PCR products obtained (right) from extracted DNA showed Cμ deletion in the fraction of large sIgD⁺ (R2) but not sIgD^- (R3) cells (lane 1, 1000 R2 cells [∼ 2.5 kb]; lane 2, 3000 R3 cells; lane 3, 3000 unsorted mononuclear cells; lane 4, 30 sIgD⁺IgM^- tonsillar cells [∼ 0.7 kb]; and lane 5, H₂O control). The fraction of naive sIgD⁺IgM⁺ cells (R1) was not tested. Sample equivalent to 2 million unsorted mononuclear cells was positive (not shown). (B) Top row shows the gating performed to select large sIgA⁺ and sIgD⁺ cells (encircled) with relatively low levels of CD19 and sIg for further analysis (CD14⁺ monocytes excluded). CCR10 was variably expressed by both categories of blasts, while CCR4 and particularly CCR7 were mainly expressed by the large sIgD⁺ cells. One of 3 representative experiments is displayed with mean ± SD. (C) Whereas large sIgA⁺ cells were strongly positive for α4β7, large sIgD⁺ cells expressed mainly intermediate to low levels of this integrin, like naive sIgD⁺ small B lymphocytes. Both large sIgD⁺ and large sIgA⁺ cells expressed abundant levels of the postgerminal center marker CD27 (right). One of 3 representative experiments is displayed. Cursors were set on the basis of isotype- and concentration-matched control Abs, and in Figure 4C (left) to discriminate 3 levels of α4β7 expression.