We study genes of the rabbit immune system using techniques of molecular biology and immunology. 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 This resembles chicken antibody formation. 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. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. However, in contrast to chicken, we recently showed that the rabbit can draw upon a diverse set of germline V-kappa genes. In addition to combinatorial diversity, gene conversion further alters rearranged rabbit V-kappa sequences in splenic germinal centers. The young rabbit appendix is a dynamic site for primary B-cell repertoire development and it was of interest to study diversification patterns during clonal expansion. Single appendix B cells from 3 to 9-week old rabbits obtained by hydraulic micromanipulation or laser capture microdissection were lysed, PCR amplified and rearranged heavy and light chain gene products directly sequenced. Gene conversion-like changes were observed to have occurred in the sequences by 3 - 4 weeks of age. Somatic mutations were found in the D regions that lack known conversion donors and probably also occurred in the V genes. The development of clonal variants was also studied. A few small sets of clonally related appendix B cells were found at 3 to 5 weeks; by 5.5 weeks, some larger clones were recovered. The diversification patterns in the clones from appendix were strikingly different from those found previously in splenic germinal centers where an immunizing antigen was driving the expansion and selection process toward high affinity. In addition, clonally related appendix B cells developed different amino acid sequences in each complementarity determining region (CDR) including CDR3 whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign antigens. This diversity of combining sites within B-cell clones supports the proposed role of appendix in generating the preimmune repertoire (3). 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 compared the tedious but successful method of manual hydraulic microdissection with techniques of laser capture microdissection (LCM). For these studies, we used both rabbit and human appendix tissues. Once capability to collect single cells by laser capture microdissection (LCM ) was developed, we modified previous tissue staining and fixation methods so that we could collect cells from a given stained tissue section by HM and LCM and directly compare our success rates using these two methods. Cells were alkaline lysed and after two rounds of nested PCR, products were recovered and directly sequenced. Because each rearrangement of genomic DNA that occurs to form the immunoglobulin heavy-chain-encoding sequence in developing B cells is unique, this system allowed us to verify our success rate in recovering single lymphocytes from tissue sections and amplifying a single allele. The methods developed have now made LCM an efficient alternative to HM for collection of single B cells (1). Both IR and UV lasers have been used for sample collection from tissue sections for genetic analyses. The high peak power densities in nitrogen laser microdissection (337nm) may excite endogenous photosensitizers or some histological dyes and cause DNA or RNA damage, perhaps through two photon mechanisms. We looked for diminished yields as evidence of damage when using LMD to isolate B cells. The Leica-LMD pulsed UV-laser system was used to collect single B cells from human appendix tissue sections, and single or multiple B cells from rabbit tissues, which had been immunohistochemically identified. Circles of different radii were used to assess possible loss of efficiency due to UV damage. The frequency of single allele PCR-amplification was compared between LMD and Hydraulic Micromanipulation (HM). Immunoglobulin VDJ PCR products and sequences obtained were indistinguishable for the two methods. We conclude that UV-based lasers can be used to cut cells individually from tissue sections provided the cutting edge is far enough from the cell membrane (>2.0 microns). In addition, with LMD we successfully isolated mRNA from rabbit splenic germinal centers (GC) containing antigen-specific B cells and determined gene sequences after RT-PCR and cloning. Total DNA and mRNA yields from 100-1000 cells collected in clusters were similar for LMD and HM. LMD appears particularly suited for independent collection of specific clusters of cells from anywhere on a slide and contiguous cell clusters from serial sections (2). The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. A collaboration with Dr. C. Rader and colleagues was established in order to compare different rabbit immune repertoires for the generation and humanization of monoclonal antibodies that bind with strong affinity to antigens involved in tumor angiogenesis. In particular, the diversity of unselected and selected chimeric rabbit/human Fab libraries that were derived from different kappa light chain allotypes was evaluated. Rabbits with particular rare allotypes are excellent sources for therapeutic monoclonal antibodies. Featured among the selected clones is a rabbit/human Fab that binds with a dissociation constant of 1 nM to both human and mouse Tie-2, which will facilitate its evaluation in mouse models of human cancer (C. Rader et. al. submitted).
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