The expression of the mouse VpreB/lambda 5 locus in transformed cell lines and tumors of the B lineage differentiation pathway.

The expression of RNA transcripts from two pre B lymphocyte related genes, VpreB and lambda 5, has been studied in a series of transformed cell lines which appear frozen at different states of B lineage differentiation, from early progenitors to surface Ig positive B cells. In the HAFTL-1 cell line, which arose from fetal liver by transformation with a retrovirus containing the Hras oncogene, Northern analysis of poly A+ mRNA as well as in situ hybridization of RNA in single cells revealed that lambda 5 and VpreB are already expressed at the progenitor stage and increase in expression as the progenitors differentiate to precursor (preB) cells, or are turned off as the progenitors differentiate to myeloid cells. Continued rearrangements of Ig genes in pre B cell lines leading to Ig expression on the surface of NFS-5 pre B cells do not influence the continued expression of VpreB and lambda 5. Surface Ig-positive B lineage cell lines also express the pre B-related genes. Both Ly1+ as well as Ly1- pre B cells are VpreB- and lambda 5-positive. Lipopolysaccharide (LPS) stimulation of 70Z/3 pre B cells does not turn off lambda 5 expression. It therefore appears that, at least in transformed cell lines, the expression of VpreB and lambda 5 is not directly regulated by the expression of microH, kappa L, or lambda L chains, LPS reactivity, or the Ly1 surface antigen. Fusion of plasmacytoma cells with normal pre B cells to generate pre B hybridomas leads to down-regulation of VpreB/lambda 5 expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the interleukin 5-reactive splenic B cell population.

The characteristics of the interleukin (IL) 5-reactive splenic B cell population of C57BL/6 nu/nu mice, with respect to IL 5/IL2 reactivity, cell surface phenotype, VH gene family usage, autoreactivity and the structure of the IL5 receptor (IL5R), were analyzed. It was found that 2%-4% of splenic B cells express relatively high levels of IL 5R as determined by the binding of the anti-IL 5R monoclonal antibody R52.120. Over 90% of the splenic B cells that mature to IgM secretion upon activation with IL5 are comprised in this small subpopulation of B cells. Moreover, the vast majority of splenic B cells that mature to IgM-secreting cells when activated by IL2 also reside in this IL5R+B cell population. The cell surface phenotype of the IL5R+ splenic B cells is IgM+, B220+, Ly-1- and IL2R p55-. Upon activation with IL5 this cell surface phenotype changes, in that a vast majority of the B cells then express the p55 chain of the IL2R, whereas the level of IL5R decreases. VH gene family usage in the IL5-activated splenic B cells was analyzed by in situ hybridization. VH gene family usage was found to be random and not different from the VH genes expressed in LPS-activated B cells. Hybridoma collections from IL5-activated splenic B cells and LPS-activated B cells were screened and compared for the production of autoantibodies and antibodies directed against the haptens (4-hydroxy-3-iodo-5-nitrophenyl)acetyl (NIP) and 2,4,6-trinitrophenyl (TNP). In both collections high, but not significantly different frequencies of autoantibody-(32% IL5, 31.4% LPS) and of anti-hapten antibody (27.8% IL5, 18.6% LPS)-producing hybridomas were found. The structure of the IL5R on IL5-activated B cells was analyzed by 125I-labeled IL5 binding and cross-linking. About 100 high-affinity (10(-11) M) and 1000 low-affinity (10(-9) M) IL5-binding sites are present on IL5-activated splenic B cells, and both high- and low-affinity IL5R are similar to those expressed on the IL5-dependent B13 cell line. Cross-linking of 125I-labeled IL5 to the receptors on IL5-activated B cells revealed one major IL5-binding protein of 45-50 kDa molecular mass and another minor binding protein of 130-140 kDa. The same IL5-binding proteins are present on the IL5-dependent B13 cell line.(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular stages and molecular steps of murine B-cell development.

Development of B cells in fetal liver occurs in one synchronous wave and involves probably no more than four critical divisions. This leads us to suggest that the main pool of proliferating progenitors that replenish the peripheral B-cell pool are progenitors before Ig gene rearrangement, that the four Ig gene rearrangements (DH to JH, VH to DHJH, VK to JK, and V lambda to J lambda) might occur in four critical divisions, and that a stromal-cell-dependent phase of pre-B development in which all rearrangements are made is succeeded by a stromal-cell-independent phase of sIG+ pre-B-cell maturation to mature mitogen-reactive B cells. We speculate on the molecular nature of the tightly controlled steps of Ig rearrangements during pre-B-cell development that might involve the pre-B-cell specific genes Vpre-B and lambda 5 and the B-lineage-specific gene mb-1 in interactions with stromal cells.

Autoreactive B-cell repertoire in mice with chronic graft versus host disease.

A quantitative analysis of the frequencies of autoantibody producing B-cells has been undertaken by producing and analyzing random hybridoma collections generated in fusions with activated B-cells. Activated B-cells were derived from mice injected with LPS and SRBC and normal mice. They were compared to those derived from mice undergoing chronic GVHD. The frequencies of successful fusion events correlate well with the number of activated B-cells used in the fusions, so that it is reasonable to conclude that the hybridoma collections reflect the activated B-cell repertoires in the different animals. The frequencies of hybridomas producing autoantibodies as well as their specificities for self-antigens, were not significantly different between the different collections of hybridomas. Moreover, no difference in VH gene family expression was found in the different collections of autoantibody producing hybridomas. So, the activated autoreactive B-cell repertoires in GVHF1 mice and in normal mice is similar. In contrast to the normal activated autoreactive B-cell repertoires, which make predominantly IgM antibodies, the GVH-activated autoreactive B-cells make predominantly antibodies of the IgG class. Therefore, we conclude that T-cell mediated graft versus host activation does not generally lead to selective expansion of autoreactive B-cells, but appears to play a crucial role in the switch from IgM to IgG production.

B lymphocyte lineage-restricted expression of mb-1, a gene with CD3-like structural properties.

A gene, called m-mb-1, was isolated from a murine pre-B-minus T lymphocyte subtracted library. It was found expressed as mRNA at low to medium abundance in early progenitors of the B lineage, in pre-B and mature B lineage cell lines, in normal resting B lymphocytes and in polyclonally activated B cell blasts. The gene was not expressed in plasmacytomas, in cell lines of the monocyte/macrophage, the T lymphocyte or the fibroblast lineages, nor in thymus, liver, heart, kidney, lung or brain. The nucleotide sequence of the m-mb-1 gene encodes a putative membrane glycoprotein with 220 amino acids, which includes a leader sequence, a putative extracellular domain with two potential N-glycosylation sites, a transmembrane portion and a putative intracellular domain. The partial sequence of a human homologue, h-mb-1, shows nearly 90% homology in nucleotide as well as amino acid sequences to the murine form of a stretch of the putative intracytoplasmic domain. Antibodies raised against a fusion protein of m-mb-1 with protein A, affinity purified for their m-mb-1 specificity, stained pre-B and mature B cell lines on their surface, but did not stain T cell lines and fibroblasts. Antibodies raised against a stretch of 20 amino acids of the putative intracellular domain with 90% homology between the mouse and human protein did not stain the surface of any cell lines tested.(ABSTRACT TRUNCATED AT 250 WORDS)