Generation of Novel Osteolineage VHL Conditional Knockout Mice to Study B Cell Microenvironments

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PROJECT SUMMARY Our long-term objective is to characterize the microenvironments that influence B lymphocyte development during altered bone homeostasis. B lymphocytes are required for the production of antibodies that are crucial for a robust adaptive immune response. Studies from many groups have determined the stages of maturation, the molecular mechanisms of B cell antigen receptor gene rearrangements and generation of receptor diversity, and mechanisms of self-tolerance in conventional B cells. The microenvironments in the bone marrow (BM) that support B cell progenitors and mature B cells have been described (2-7), and it appears that development of robust antibody responses from B cells is influenced by the relatively low oxygen levels in the germinal centers of the spleen and lymph nodes (11). However, relatively little is known about the mechanisms by which oxygen levels regulate B cell development in the bone marrow. We aim to fill the scientific knowledge gap on the microenvironmental niches that influence the maintenance and function of adult B cell subsets. In the course of our studies to examine the effects of altered bone homeostasis on immune cell development, we discovered that B cell development was severely impaired in mice in which a hypoxia response pathway gene, von-Hippel Lindau (Vhl), is conditionally deleted in osteocytes (12). We hypothesized that Vhl-deficiency in osteocytes results in structural and molecular changes in the vascular architecture in the BM microenvironments, which may alter oxygen tension to levels that support aberrant B development. In support of this, our preliminary analyses suggest that deletion of Vhl in osteocytes results in increased permeability in the vasculature. However, off-target expression of the Dmp1-Cre transgene in osteoblasts clouds this interpretation. Other tissue-specific Cre mice traditionally used to study the skeletal system display off-target or broader expression than expected, diminishing their utility in defining the role of gene deletion in specific cell types. In addition, current mouse models do not permit the study of Vhl-deficiency on immune cell development in postnatal and adult BM microenvironments. MSCs are osteocyte precursors, can support B cell development in the BM (13) but the specific and unique contributions of the MSCs, OBs and OCYs to B cell maturation is unclear. Therefore, we propose to generate a novel inducible Vhl-conditional knockout mouse model, to assess the cell-extrinsic effects of Vhl deletion in MSCs on B cell development. We aim to combine transplantation strategies with high-resolution intravital (live) microscopy of the calvaria and ex vivo long bone imaging to characterize the Vhl-deficient bone marrow microenvironment in order to identify changes that may influence B cell development, such as oxygen tension, blood vessel type and structure, the presence and location of stromal cells or hematopoietic cells, and changes in bone morphology. Finally, we aim to analyze the niches longitudinally over time, in order to determine how the distinct physical properties in the bone change and affects the progression of B cell developmental defects in Vhl-deficient bones as a function of time. This information could be applied to future studies of the effects of irradiation, myeloablative conditioning, or bone-building drugs on bone marrow niches and immune cell development.