In vitro analysis of the microenvironmental regulation of murine B lymphocyte development

grantor: University of TorontoB lymphocyte progenitors develop in close association with stromal cells which produce a variety of growth factors required to sustain the maturation of these precursors to the mature B cell stage. In this thesis, I describe the functional activity of two novel stromal-derived growth factors, Flt3 ligand (FL) and TSLP (Thymic Stromal-Derived Lymphopoietin), on developing B cell progenitors. Using an in vitro assay which supports the growth and differentiation of multipotential and bipotential precursors to the LPS responsive stage of B cell development, I have demonstrated that FL has little activity alone but synergizes with IL-11 and IL-7 to support the differentiation of these lineage uncommitted precursors. At the level of single cells, IL-11 + FL + IL-7 can support the differentiation of both macrophages and B lymphocytes from bipotential precursors. FL further synergizes with IL-7 to stimulate the proliferation of a committed population of early pro-B cells. This synergistic effect is most apparent in day 13 fetal liver cells in which the cohort of differentiating B lineage cells are still at the early pro-B cell stage. I have demonstrated that TSLP functions to support the growth and differentiation of committed B cell progenitors. The proliferative response of pro-B cells to TSLP is not as extensive as that of IL-7. However, progenitors stimulated with TSLP differentiate in secondary cultures containing stromal cells as efficiently as pro-B cells stimulated with IL-7. IL-7 responsive pro-B cells cultured in the presence of stromal cells and IL-7 maintain their pro-B cell characteristics and fail to differentiate to the mitogen responsive stage even though stromal cells are present. The removal of IL-7 from these pro-B cell cultures permits differentiation to the small pre-B cell stage. However, surface $\mu\sp+$ B cell progenitors still require stromal cells to become functionally responsive to LPS. The stromal cell-mediated maturation of surface $\mu\sp+$ B cells is contact-dependent. Furthermore, the differentiation of pre-B cells to the mature B cell stage can occur in the absence of IL-7 or stromal cells if B cell progenitors are cultured in close proximity suggesting that homotypic interactions are important in the final stages of B cell differentiation.Ph.D