The characteristics and amino acid sequences of V_H5 genes in plasma cells generated from naive B cells.

(A) Highly purified CB B cells were cultured with SAC (0.01%), IL-2 (50 U/mL), IL-10 (50 ng/mL), and anti-CD40 mAb (1 μg/mL) with CD32T for 8 days. The cells were then stained with anti-CD38–FITC and anti-CD20–PE and the antibody-coated cells gated on living cells by cell size and granularity and finally counted by means of flow cytometric analysis. Expressions of CD38 and CD20 on B cells are shown on a log scale. The cells were separated into CD20⁺CD38⁻ B cells (Ai) and CD20⁻CD38⁺ plasma cells (Aii) by sorting, cytospun, and stained with May-Giemsa staining. Original magnification, × 400. (B) Total RNA was isolated from the CD20⁺CD38⁻ B cells (Bi), CD20⁻CD38⁺ plasma cells (Bii), and resting CD27⁺ memory B cells (Biii) and then reverse transcribed into cDNA. V_H5 genes were amplified by using primers corresponding to the 5′ region of the V_H5 leader sequence and to the 3′ Cμ constant region. PCR products were ligated and transformed. The DNA sequencing of individual clones was performed with a dideoxy termination technique. Each dash represents identity with the germline sequence; amino acid differences are indicated. Complementarity determining regions are boxed. These data represent each of 3 different experiments. The same results were obtained when sort-pure adult CD27⁻ B cells were stimulated with SAC + IL-2 + IL-10 + CD40/CD32T.