Henry H. Wortis

The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation. Bruton9s tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases, is essential for the full repertoire of IgM signals to be transduced. We examined the ability of BTK to regulate the nuclear factor (NF)-κB/Rel family of transcription factors, as the activation of these factors is required for a B cell response to mIgM. We found greatly diminished IgM- but not CD40-mediated NF-κB/Rel nuclear translocation and DNA binding in B cells from X-linked immunodeficient (xid) mice that harbor an R28C mutation in btk, a mutation that produces a functionally inactive kinase. The defect was due, in part, to a failure to fully degrade the inhibitory protein of NF-κB, IκBα. Using a BTK-deficient variant of DT40 chicken B cells, we found that expression of wild-type or gain-of-function mutant BTK, but not the R28C mutant, reconstituted NF-κB activity. Thus, BTK is essential for activation of NF-κB via the B cell receptor.

To determine directly whether B cell precursors of adult origin are capable of generating CD5⁺ B cells, we reconstituted neonatal C3H.SCID mice with adult C57BL/6 bone marrow and analyzed splenic B cells 10 months later. Surface staining and flow cytometry revealed that the B cells were of donor origin and that 30% were CD5⁺. This confirms that in vivo generated CD5⁺ B cells can be adult derived. After anti‐IgM (but not lipopolysaccharide) stimulation in vitro, virtually all of the B cells from the bone marrow‐reconstituted mice expressed surface CD5. Sequence analysis of expressed V_HDJ_H genes from the CD5⁺ B cells present after anti‐IgM stimulation revealed a high frequency of N nucleotide addition in CDR3 regions. The presence of N nucleotides indicates that these sequences were derived from CD5⁺ B cells of adult origin rather than from long‐lived fetal precursor B cells present in either the adult bone marrow at the time of transfer or adult spleen. These experiments demonstrate conclusively that adult bone marrow contains precursors for CD5⁺ B cells and that unlike fetal liver‐derived precursors these express terminal deoxynucleotidyl transferase.