The rabbit primary antibody repertoire is generated through somatic diversification of a limited initial repertoire in which most VDJ gene rearrangements utilize the 3'-most VH gene segment, VH1. The initial repertoire is diversified by somatic hypermutation and a somatic gene conversion-like mechanism in gut-associated lymphoid tissue (GALT) 1 to 2 months after birth. Studies of germ-free rabbits and of rabbits in which the cecum was ligated at birth to prevent entry of intestinal contents into the appendix have shown that GALT development is dependent on an exogenous factor(s) derived from the gut. Our laboratory has extended these observations by showing, in rabbits in which the appendix was ligated at birth and all other organized GALT was removed (ligApx rabbits), that repertoire diversification is also dependent on an exogenous factor(s) derived from the gut. In order to identify this exogenous factor(s), we have hand-raised sterilely derived rabbits on a sterile diet away from contact with conventional rabbits and their microflora (remote colony rabbits) and found that most of these rabbits do not develop normal GALT or diversify their antibody repertoires. Since these remote colony rabbits are not germ-free, these results suggest that specific members of normal intestinal microflora are required for both normal GALT development and repertoire diversification. Using conventional culture methods to examine differences in the intestinal microflora of conventional and remote colony rabbits, we have not been able to identify bacterial species that drive rabbit GALT development and repertoire diversification. Approaches to the study of microbial diversity based on bacterial 16S rDNA have overcome many of the limitations of culture-based methods and made available sensitive techniques for studying intestinal microflora. We propose in this grant application to use one such approach, in combination with detailed temporal analysis of GALT development and VDJ gene diversification, to identify bacterial species that stimulate rabbit GALT development and repertoire diversification, and determine the mechanism by which they influence these developmental processes.
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