Role of intestinal microflora in diversification of the rabbit primary antibody repertoire

Rabbits predominantly use only one VH gene in VDJ gene rearrangements, potentially limiting the antibody repertoire. However, the rearranged VDJ genes are then mutated after birth. This diversification process occurs in gut-associated lymphoid tissues (GALT) and depends on the presence of certain types of intestinal bacteria. The goal of my dissertation project is to identify bacteria that can induce diversification of the rabbit primary antibody repertoire and to determine the mechanism by which this is achieved. To identify bacteria that can induce GALT development and a diverse preimmune antibody repertoire in rabbits, we generated a rabbit model in which the appendix had been rendered germfree by microsurgery (GF-Apx rabbits). We introduced bacterial isolates and assessed GALT development and somatic diversification of the Ig genes. Of the bacterial species tried, the combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to an increase in somatic diversification of VDJ-Cmu genes in appendix B cells. GALT development in rabbits does not appear to be the result of an antigen-specific immune response because B. fragilis , which by itself is immunogenic, does not promote GALT development. We therefore introduced into GF-Apx rabbits protein A of Staphylococcus aureus which stimulates B cells in an antigen-nonspecific manner. We found that introduction of protein A alone did not induce GALT development but in combination with B. fragilis it did. This result suggests that two signals are required for GALT development: one signal through the B cell receptor and a second signal provided by B. fragilis.