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. 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. We recently showed that in contrast to limited combinatorial diversity of its heavy chain, the rabbit can draw upon a diverse set of germline V-kappa genes. 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 (2). In order to further investigate the development of the early pre-immune repertoire, we studied the kinetics of diversification of heavy and light chains in developing rabbit appendix between 3 and 9-weeks of age. Single B cells collected using hydraulic micromanipulation and laser capture microdissection were lysed, PCR amplified and products directly sequenced. We found that gene conversion of rearranged heavy and light chain sequences was occurring by 4 weeks of age. Somatic mutations occurred in the D regions that lack known conversion donors; they probably also occurred in the V genes. Small clones were found in 4 to 5 -week appendix, but the majority of cells yielded unique, unrelated sequences. By 5.5 weeks, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found in splenic germinal centers where a specific antigen was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed very different amino acid sequences in each complementarity determining region (CDR) including CDR3 whereas dominant clones from spleen underwent few changes in the CDR3. The variety of different combining sites generated by diversification within a single expanding appendix clone suggests that at least some clonal expansion and selection in appendix may not be driven by specific antigens. Rabbit appendix development requires specific normal gut flora. Rather than acting solely as specific antigens, microbial components may contribute to the development of the broad repertoire found in the B cells through indirect effects on appendix B-cell development and diversification (Sehgal et al. Ms in preparation). We compared techniques of manual hydraulic microdissection with techniques of laser capture microdissection (LCM). For these studies, we used both rabbit and human appendix tissues. Because the rabbit mainly rearranges one VH gene, it is a good control for verifying that we have collected a single cell containing a rearranged VH (1). In order to perfect the methods to collect single cells for PCR amplification and sequencing of rearranged human VH genes, we have been using both the infra red based LCM and another UV laser-based microdissection system,Leica-LMD to collect human appendix B lymphocytes. We also used LCM in a collaborative study on T cells and B cells in mastocytosis patients' tissues (M. Taylor et. al, ms. in preparation) .
Showing the most recent 10 out of 13 publications
