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-i, 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, A5, Vpre-B, c,u, 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 c,, RAG-1, RAG-2, A5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c,u, RAG-1, and RAG-2 genes; transcription of the AS 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--y T-cell-specific gene, nor do they have rearranged y, 8, or j8 T-cell antigen receptor genes.

At day 8-8.5 of mouse development the yolk sac, not the embryo proper, has lymphoid precursor potential in vivo and in vitro.

“…3 and data not shown). We have previously found that FLS4.1 stromal cells, although able to support development and growth of fetal liver pre-B cells, are unable to support differentiation of pre-B cells into surface IgM+ B lymphocytes (12). Therefore, the lack of IgM+ B lymphocytes in the yolk sac cells cultured on FLS4.1 stromal cells was not unexpected.…”

“…Cells from the first culture (Fig. 1 (12) (025% confluency) in six-well Costar plates in the presence of rIL3 (20 units/ml) (12), rIL6 (10-50 units/ml) (12), and rIL7 (500 units/ml) (12), in a final volume of 1.5 ml of culture medium (see above) at 37°C for 8-10 days. One milliliter of fresh culture medium containing rIL7 was added to each well at day 5-6 of culture.…”

“…1, bars A and B). The putative precursor cells were first cultured on monolayers of the subcapsular thymic epithelial clone EH6 in the presence of exogenous rIL3, rIL7, and F [supernatant from the fetal liver stromal line FLS4.1 (12)] at 37°C for 10 days (Fig. 1, bar A).…”

“…Sixteen weeks later, spleen mononuclear cell suspensions free of erythrocytes were prepared by standard procedures. (lacking yolk sac, -3-4 embryos per well) were cultured on monolayers of the EH6 subcapsular thymic epithelial clone (10) on six-well Costar plates in the presence of recombinant interleukin (rIL) 3 (25-50 units/ml) (12), rIL7 (500 units/ml) (12), and F [final concentration 10%; supernatant from FLS4.1 stromal cells (12)] in a final volume of 1.5 ml per well of culture medium [Iscove's modified Dulbecco's medium + 2 mM L-glutamine + 50 ,uM 2-mercaptoethanol + gentamycin at 50 ,g/ml and 7.5% fetal calf serum] at 37°C for 8-10 days.…”

Negative cross-talk between interleukin-3 and interleukin-11 is mediated by suppressor of cytokine signalling-3 (SOCS-3)

“…The mouse pro-B cells Ba\F3 [27] were engineered to express the IL-11 receptor complex. Experimental procedures have been described previously [28].…”