Immunity to many encapsulated bacterial pathogens involves antibody responses by B cells activated by capsular polysaccharides via surface Ig receptors. B cells activated by Ig cross-linking undergo an ordered chain of events. These events include stimulation of kinase activity, gene and nuclear factor activation, and entry into cell cycle. Mice with the x-linked immunodeficiency gene (xid) possess a mutation in the pleckstrin homology domain of Bruton's tyrosine kinase (Btk). These animals are unresponsive to polysaccharide antigens. Moreover their B cells do not proliferate after Ig cross-linking, although they do enlarge. To determine at which point in the activation pathway xid cells become arrested, both early and late events are being compared in sIg activated xid and wildtype B cells. We have found that early G1 events, such as nuclear factor and proto-oncogene induction occur in xid cells normally. Furthermore, the activity of src related kinases was intact in xid cells. However, induction of cyclin proteins did not occur in xid cells. Their absence correlated with an absence of cyclin mRNA, arguing that nuclear events associated with normal cyclin gene induction are blocked in xid. The cyclin inhibitor p27kip1 was modulated normally in sIg actiavted B cells. Further studies now underway include the construction of Btk-GST fusion proteins to examine Btk functional differences between xid and wild type B cells. These fusion constructs are being used to probe for pleckstrin homology domain binding substrates to determine how the 28R->C xid mutation ablates Btk function. Also, xid-myc transgenic mice have been bred to produce xid B cell lines. A clear understanding of the events associated with sIg mediated activation is required to optimize immune responses to polysaccharide vaccines because these antigens have the ability to trigger B cells directly. Because of their defect, xid mice can be used experimentally to isolate the function of anti-polysaccharide antibodies from other protectiv mechanisms against encapsulated pathogens. This understanding is especially important for vaccination of very young and elderly populations which respond poorly to polysaccharides.
