Switch recombination and germ-line transcription are division-regulated events in B lymphocytes

Treatment of resting murine B lymphocytes with CD40 ligand (CD40L) and IL-4 induces proliferation and a switch in immunoglobulin (Ig) isotype surface expression from IgM and IgD to IgG1 and IgE. Using a fluorescent dye to ena ble cell sorting according to cell division cycle number, we have examined molecular events associated with B cell differentiation, namely, germ-line transcription and DNA recombination. Digestion-circularisation polymerase c hain reaction experiments showed that DNA recombination leading to isotype switching from IgM to IgG1 surface expression is division-dependent and was first detected after B cells had divided three times. Similarly, DNA rearr angement involving the IgE switch region was detectable only after five div ision cycles. These division cycle numbers correlate with the numbers of di visions required before surface expression of the switched isotype [P.D. Ho dgkin, J.-H. Lee, A.B. Lyons, J. Exp. Med. 184 (1996) 277-281]. RT-PCR anal yses also revealed that germ-line transcripts for both IgG1 and IgE increas ed with division number suggesting a threshold expression level may be requ ired for recombination to occur. (C) 1999 Elsevier Science B.V. All rights reserved.