Fetal Liver Pro-B and Pre-B Lymphocyte Clones: Expression of Lymphoid-Specific Genes, Surface Markers, Growth Requirements, Colonization of the Bone Marrow, and Generation of B Lymphocytes In Vivo and In Vitro

Abstract

We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220⁺ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220⁺ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, λ5, Vpre-B, cμ, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from cμ, RAG-1, RAG-2, λ5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for cμ, RAG-1, and RAG-2 genes; transcription of the λ5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B-lymphocytes but not T-lymphocytes; moreover, these clones do not express the CD3-γ T-cell-specific gene, nor do they have rearranged γ, δ, or β T-cell antigen receptor genes.