Interculture variation and evolution of B lineage lymphocytes in long‐term bone marrow culture

Abstract

A recently described long‐term culture system offers a unique experimental approach for dissecting regulatory mechanisms that control the developmental progression of B‐lineage lymphocytes. Lymphoid cells, including B cells and their precursors, can be maintained for prolonged periods in strict dependence on a layer of adherent cells. However, before this system can yield to interpretable manipulation, much information is needed as to the identity and temporal phenotypic stability of both lymphoid and nonlymphoid cells. The findings reported here provide answers to some of those important questions. Successful establishment of lymphoid cells in culture was extraordinarily dependent on the batch of fetal calf serum used in the medium, and some undesirable serum lots supported cultures that were virtually all myeloid. With standardized culture conditions, various populations of lymphoid cells were identified on the basis of B‐lineage differentiation markers and culture to culture variation was assessed. Lymphocytes that were firmly attached to the adherent cells were carefully compared to nonadherent lymphocytes in terms of cycle status, phenotype, size, and transferrin receptor expression. They were essentially identical in all of these respects and a partitioning of proliferating cells and their progeny in the cultures was therefore not apparent. It is also noteworthy that although a high mitotic rate was maintained, a majority of the cells were small lymphocytes. The outgrowth of identifiable B‐lineage cells (detected with monoclonal 14.8 antibodies) in replicate cultures was initially similar, but the extent of interculture variation increased dramatically during the period 4–6 weeks after initiation of culture. Replicate cultures established from the same marrow cell pool often differed as much as 20‐fold in numbers of 14.8‐positive cells. After this time, the composition of individual cultures evolved much more gradually, and numbers of B cells and pre‐B cells remained relatively constant. This indicates that subsets of lymphocytes become established in each culture dish during a discrete phase.At least two types of supporting adherent cells predominated in these cultures: typical macrophages and very large, nonphagocytic cells resembling adventitial reticular cells. The latter included subpopulations resolved on the basis of alkaline phosphatase content. In contrast to the lymphoid populations, proportions of these adherent cell types were relatively invariant among replicate cultures. The dramatic expansion or contraction of particular B‐lineage populations which occurred prior to 6 weeks of culture did not appear to be reflected in the composition of the adherent layers on which they depended.