RABBIT ALLOTYPES--STRUCTURE, ORGANIZATION AND REGULATED EXPRESSION OF IG GENES

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Abstract

We study genes of the rabbit immune system by techniques of molecular biology and immunology. The rabbit has a limited number of VH genes that rearrange. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. By six weeks after birth, diversification of rearranged VH genes occurs at least in part, by gene conversion-like events in the appendix suggesting that this organ is a homologue of the avian bursa of Fabricius. We are currently studying the kinetics of diversification of heavy and light chains in developing rabbit appendix. In species such as mouse and human, generation of combinatorial diversity through use of different VH and VL genes in immunoglobulin VHDJH and VLJL rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal as only a few VH genes are rearranged and expressed. To investigate the contribution of combinatorial diversity toward generation of the rabbit V-kappa repertoire, we constructed 5 genomic libraries from rabbit kidney DNA and 1 cDNA library from the bone marrow of a 1 day old rabbit using a series of PCR based strategies. The number of germline V-kappa genes is potentially greater than 141 although our more conservative estimate is at least 39, 28 of which we found expressed as mRNA. The germline V-kappa genes display different lengths of the coding region 3' of Cys 88, resulting in CDR3 length heterogeneity among functional V-kappa-J-kappa sequences ranging from 8 to 15 amino acid. In contrast to limited combinatorial diversity of its heavy chain, the rabbit can draw upon a diverse set of germline V-kappa genes. The kappa light chain has the potential to be a major contributor toward generation of the antibody specificities of the rabbit pre-immune repertoire (Sehgal et al., 1999). In spite of the presence of combinatorial diversity, we found that gene conversion also alters rearranged rabbit V-kappa sequences in splenic germinal centers. We described mechanisms that account for the development of the heterogeneous high affinity anti DNP antibodies that rabbits can produce. We found that in clonal lineages, rearranged V-kappa and VH are further diversified by gene conversion and somatic hypermutation. The positive and negative selection of amino acids in complementarity determining regions observed allows emergence of a variety of different combining site structures. A by-product of the germinal center reaction may be cells with sequences altered by gene conversion that no longer react with the immunizing antigen but are a source of new repertoire. The splenic germinal center would thus play an additional role in adults similar to that of the appendix and other gut associated lymphoid tissues of young rabbits (D. Sehgal et al, 2000 in press).