Recruitment of Blood-Borne B-cells to Neonatal Rabbit Appendix Young rabbit appendix is a homologue of chicken bursa of Fabricius; both are crucial sites for preimmune B-cell repertoire development. These are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. Although B cell Ig-gene rearrangements occur in sites such as bone marrow of young rabbits, immature IgM+ B cells undergo further Ig-repertoire diversification in appendix and other gut associated lymphoid tissues. We have now characterized some of the molecules involved in the multi-step recruitment of blood-borne B cells into neonatal rabbit appendix. Expression of peripheral lymph node addressin (PNAd) on appendix high endothelial venules (HEV), and of its counter-receptor, CD62L, on B cells in peripheral blood and in close association with PNAd-positive HEV suggests their role in tethering. Sialyl-Lewis-x, known to be involved in tethering of pre-bursal cells on chicken bursal vasculature, was also found on appendix B cells. The interaction of chemokine receptor CCR7 on peripheral blood B cells and one of its ligands, CCL21, on appendix HEV, could cause activation of integrins such as LFA-1 and alpha 4/beta-1 and lead to firm adhesion of B cells to HEV. We found these integrins expressed on peripheral blood B cells. Simultaneous down-regulation of CCR7 and up-regulation of CXCR5 on appendix B cells compared to peripheral blood suggests a role for CXCR5 in B-cell recruitment into follicles during appendix development. The proportions of appendix B cells expressing CD62L, sialyl-Lewis-x and alpha-4/beta-1 declined between day 3 and 4 weeks after birth while percentages of Lewis-x+ appendix B cells increased. These changes correlate with the stage of repertoire diversification by gene conversion in both rabbits and chickens. We compared the expression of Lewis-x on wild-type and mutant ali/ali appendix B cells. The percentages of Lewis-x+ B cells in ali/ali appendix were significantly lower at one and two weeks compared to wild-type controls. In contrast to the Lewis-x expression profile, the proportions B cells expressing adhesion molecules (CD62L and sialyl-Lewis-x) that are involved in tethering and rolling on HEV were comparable between the wild-type and mutant rabbits. We conclude that migration and recruitment of B cells to appendix was normal in ali/ali rabbits but that their intrinsic defect in rearrangement, due to deletion of the VH1 gene leads to delayed development (1). The Kit activating Mutation in Mast Cells, and Oligoclonal B-Lymphocytes of Mastocytosis Patients Mastocytosis is characterized by focal heterotypic clusters of mast cells and lymphocytes in the bone marrow and by a somatically acquired activating Kit mutation, D816V. The relationship of the occurrence of this mutation to the heterotypic clusters of mast cells and lymphocytes in bone marrow is unknown. We collaborated with the laboratory of Dr. Dean Metcalfe to investigate whether these two unique features of mastocytosis were related. To explore this hypothesis, laser capture microdissected mast cells, B cells and T cells, from both lesional and non lesional areas of bone marrow biopsy tissues from patients with mastocytosis, were examined for the D816V kit mutation. We found mast cells and lymphocytes within focal aggregates in the bone marrow of those with mastocytosis are more frequently positive for the D816V activating mutation. We found that the B cell population in lesional areas of mastocytosis as well as control B cells in normal appendix represented oligoclonal populations. Clonal proliferation is unlikely to be the basis of clustering of B cells in lesional areas of patient bone marrow (M L Taylor et al.,Demonstration that mast cells, T cells and B cells bearing the activating kit mutation D816V occur in clusters within the marrow of patients with mastocytosis. J. Molec. Diagnostics. In press,2004 ). Studies of Rabbit CD5 The variety of combining sites generated within individual clones from appendix follicles of young rabbits suggests that clonal expansion and selection, known to require gut flora, may not be driven by specific antigens but rather through indirect effects of microbial components (superantigens). In addition, there may be survival signals supplied by interactions between B-cell receptor framework regions and endogenous superantigens such as CD5. Although the function of CD5 on B cells is unknown, our studies in the rabbit, suggested that CD5 interaction with VH framework regions of surface immunoglobulins may contribute to survival and expansion of B cells. For further studies of the interaction of CD5 and VH-regions, a fragment of CD5 protein containing the three extracellular domains has been cloned and expressed as recombinant protein. Purified CD5 protein was also used to produce polyclonal goat anti-rabbit CD5. In addition we have generated anti-peptide mAbs specific for each extracellular domain of CD5. These reagents are being used to continue a detailed analysis of the development and selection of rabbit appendix B lymphocytes (R. Pospisil et al., Stable expression of the extracellular domains of rabbit CD5: development and characterization of polyclonal and monoclonal antibodies. Submitted for publication). In collaboration with Drs. David Garboczi and Brandt Burgess, crystallization of CD5 has been accomplished. We hope to also generate co-crystals of CD5 and Fab from the mAbs we characterized. In normal rabbits 3 days after birth, B cells start to form small follicles. As detected by flow cytometry and immunohistochemistry, they are CD79a positive and express IgM but lack the CD5 antigen. By day 8, appendix B cells start to express CD5 and by 2 weeks, the majority of appendix B cells are CD5 positive. These findings contrast to the spleen and PBL where most B cells (IgM+ CD79a+) already express CD5 3 days after birth. There are differences between the kinetics of B cell development in normal compared to mutant ali/ali rabbits. Since mutant ali/ali rabbits lack normal VH framework regions associated with the deleted VH1a2 gene, their B cell development is delayed. B-cell expansion seems to correlate with development of B cells that have undergone gene conversion and consequent expression of VH framework regions more similar to those encoded by the missing VH1a2 gene. B-cell development is not uniform in all appendix follicles of ali/ali rabbits. VHa2+ B cells start to develop in some follicles but not in the others. Once VHa2 positive follicles develop, their B cells also start to up-regulate CD5. B cell growth and expansion in such follicles is also accompanied by apoptotic death suggesting that both positive and negative selection processes are affecting B-cell repertoire formation. Studies of Rabbit Activation Induced Deaminase Studies in mouse, human and chicken suggest that activation-induced deaminase (AID) is involved in the three known processes leading to antibody diversification: somatic hypermutation, gene conversion, and class switch recombination. Developing rabbit appendix provides a particularly good site for studying all three of these B-cell maturation events. We extended our knowledge about AID to another species by cloning and sequencing rabbit AID, and localizing it in cells by immuno-histochemical and -fluorescent staining techniques. We used our antibodies to isolate AID protein associated with possible co-factors, from rabbit appendix B-cell nuclear extracts.
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