In rabbit, the development of a preimmune antibody repertoire requires interaction between the gut-associated lymphoid tissues (GALT) and the intestinal microbiota.  By introducing various members of the commensal microflora into surgically-manipulated, germ-free appendix rabbits, we have previously shown that a combination of two commensal species, Bacillus subtilis and Bacteroides fragilis induce B cell proliferation and Ig gene diversification.  However, the mechanism by which these species induce GALT development remains unknown.  To identify the bacterial component(s) of B. subtilis that are required for GALT development, Dr. Sethupathi and I introduce B. subtilis mutants into germ-free appendices and assess their ability to promote GALT development.  In combination with a genetic approach, I am also using a number of biochemical techniques to identify B. subtilis molecules that may directly bind to B cells to stimulate their proliferation and/or Ig gene diversification.

Additionally, I am investigating the mechanism by which the commensal microflora induce a B cell repertoire shift in a particular strain of rabbit, ali/ali, or Alicia.  In Alicia rabbits, at birth nearly all B cells express one V_H allotype, V_Hn.  As the rabbits age the B cell repertoire shifts, such that as an adult the B cells express the V_Ha allotype.  Using Alicia rabbits with germ-free appendices, we have shown that the repertoire shift is due to a selective expansion of the V_Ha allotype B cells by the commensal microbiota.  We hypothesize that a bacterial- or bacterial-induced endogenous superantigen binds to and positively selects the V_Ha B cells.  To test this hypothesis, I have generated V_Hn-scFv and V_Ha-scFv molecules, and am currently using these molecules in various biochemical assays to identify bacterial or endogenous molecules that bind to the V_Ha-scFv but not the V_Hn-scFv.  After we identify such molecules, we will introduce them into germ-free appendices to test whether these molecules induce the B cell repertoire shift.