Induction of mature γ1 and γ2 transcripts from naive B cells.

Highly purified adult CD20⁺CD27⁺, CD20⁺CD27⁻, or CB B cells at the cell numbers of 0.5 × 10⁶ cells were cultured with medium alone (lanes 1, 4, 7), 0.01% SAC, 50 U/mL IL-2, 50 ng/mL IL-10, and 1 μg/mL anti-CD40 cross-linked with CD32T for 1 day (lanes 2, 5, 8) or 4 days (lanes 3, 6, 9). After extraction of total RNA, RT-PCR was performed as described in “Materials and methods.” PCR primers of germline Cγ transcripts were prepared in the initiation region (Iγ1 or Iγ2) and the hinge of constant region (Cγ1 or Cγ2). PCR primers of mature Cγ transcripts were prepared in the regions of J_H of V region and of the hinge of constant region. Each template contained the same of cDNA from RNA extracted from highly purified B cells. The C lanes represent control of contamination-free reaction, in which all PCR reagents are present, but there is no cDNA. The β₂-microglobulin (β₂-MG) was used as a positive control. Data are representative of the results of 3 experiments using different donors.