The goal of this research is to understand how B lymphocyte production is regulated and to characterize early B cell precursors. Two different long-term bone marrow cultures (LTBMC) are central to these analyses. The Dexter myeloid bone marrow cultures (MBMC) are optimal for myelopoiesis. No B cells are produced in this system but an early B cell precursor is maintained. The Whitlock-Witte lymphoid bone marrow cultures (LBMC) allow long-term B lymphopoiesis. The MBMC B cell precursor can be induced to differentiate into B cells following switching to LBMC conditions.
The first aim of the proposal is to characterize stromal cell lines isolated from the MBMC adherent layer. One, termed S17, supports B cell differentiation while another, S10, does not. The molecular weight of a factor secreted by S17 that induces Ig expression in pre-B cells will be determined by high pressure liquid chromatography. The relative contribution of the factor alone versus direct contact between developing B cells and stromal cells will be examined in diffusion chamber experiments.
This aim will also examine the effects of nonstromal factors as Il-1, Il-2, and gamma-interferon on B cell production by adding these agents to the MBMC-less than LBMC switch system. The second focus of this proposal will examine membrane interactions between B cells and the stromal cells. Initially, the interactions between S17 and developing B cells will be examined by transmission electron microscopy. Components of the stromal cell surface with which B cells interact will then be identified. Stromal cell membrane preparations will be made, separated on I-D gels and the proteins transferred to nitrocellulose filters. B cells and their precursors from LBMC will be incubated on these and the ability of the cells to bind to membrane fractions of a particular molecular weight determined. Once the relevant molecule is identified, monoclonal antibodies will be made to it. An alternative source of immunogen will be whole stromal cell populations.
A third aim of the work will be to identify intermediate stages of B lymphopoiesis. A detailed kinetic analysis of developing cells in the MBMC less than LBMC switch system will be performed using antibodies to surface cytoplasmic antigens. Preliminary observations indicate that cells that may be lymphocyte progenitors from these cultures form colonies in semisolid medium overlayed on a stromal cell feeder layer. The growth conditions of the system will be defined and phenotype of the colony forming cell(s) determined.
The final aim will use cells from the colonies to examine Ig gene utilization during development using CDNA probes to the various Ig heavy chain variable regions. This will allow expression of the Ig repertoire in B cell precursor of defined maturational state to be studied. The data from these studies will provide a knowledge of the basic biology of normal B cell differentiation and contribute to understanding pathological B cell development.
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