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Biomedical subjects

F Melchers

Publications and source records attributed to F Melchers.

At least 55 records · Page 3Linked to original sources
PubMed1998

Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity.

The transit of immature to mature sIgM+ B cells, the life span, maturation kinetics and response to polyclonal activators have been analyzed with the help of a new mAb (493), that distinguishes immature, 493+ from mature, 493 B cells in a variety of mouse strains tested. Analysis of the turnover of immature 493+ B cells by bromodeoxyuridine (BrdU) labeling kinetics indicate that only 10-20 % of the cells reach the spleen as immature 493+ cells. The life span of 493+ B cells in bone marrow and spleen is around 4 days. BrdU chase experiments show that most of the immature cells in spleen enter the pool of mature, 493+ B cells where they gain a longer life span of 15-20 weeks. Immature and mature B cells respond equally well to LPS stimulation; anti-CD40, however, stimulates mature B cells better than immature B cells. IgM cross-linking of mature B cells results in proliferation, while it induces apoptosis in immature B cells. This apoptosis of immature cells can be inhibited by costimulation with anti-CD40 or by overexpression of bcl-2. We speculate that Ig receptor ligand-mediated apoptosis (negative selection) plays a major role in the transit of immature B cells from bone marrow to spleen, but only a minor role in the transit from immature B cells to mature B cells in the spleen.

Animals↗
PubMed1998

B-cell development: a comparison between mouse and man.

A common variable immunodeficiency (CVID) patient, who carries mutations on both alleles of the gene encoding the surrogate light chain component lambda 5/14.1, shows a similar phenotype of B-cell deficiency as the lambda 5-deficient mutant mouse. As discussed here by Paolo Ghia and colleagues, this points to a remarkably similar developmental pathway of B cells in humans and mice.

Age Factors↗
PubMed1998

Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression.

Within the pools of muH chain-producing precursor and mature B cells from normal and lambda5-defective mice, the frequency of cells in which both H chain alleles were productively VHDJH rearranged was determined. An equally high percentage (2%-4%) of cells carrying two productively VHDJH-rearranged H chain loci was found in precursor and mature B cell pools of both mouse strains. In all of these cells, one allele encodes a muH chain incapable of forming a surface-expressed pre-B cell receptor. Hence, allelic exclusion is maintained at the level of pre-B cell receptor expression. The surprising conservation of H chain allelic exclusion in lambda5-defective B cells suggests that an alternative form of pre-B cell receptor might function to ensure this allelic exclusion.

Alleles↗
PubMed1998

Fetal liver organ cultures allow the proliferative expansion of pre-B receptor-expressing pre-B-II cells and the differentiation of immature and mature B cells in vitro.

We describe the phenotypic and functional properties of B lineage cells developing in fetal liver organ cultures (FLOC) of mouse embryos at day 14 or 15 of gestation which contain pro/pre-B-I cells. FLOC B cell development proceeds to mature IgM+, IgD+ and CD23+ lipopolysaccharide-reactive B cells within a culture period of 5-6 days. The phenotypes and relative proportions of pro/pre-B-I, pre-B-II, immature and mature B cells from FLOC were similar to that seen in livers freshly isolated from age-matched, i.e. newborn, mice. More importantly, the numbers of cells recovered in the different B lineage subpopulations from FLOC were close to those developed in vivo. Hence, in contrast to single-cell suspension cultures of fetal liver, FLOC allow the proliferative expansion of pre-B cell receptor-expressing pre-B-II cells. FLOC from embryos of mice with targeted mutations in the RAG-2 and lambda5 genes, which cannot expand by proliferative expansion of their pre-B-II compartment in vivo because they cannot express a pre-B cell receptor on their surface, show this same defect in vitro. FLOC are accessible to the action of mAb and cytokines. Thus, addition of anti-IL-7 receptor mAb to FLOC of normal mice inhibits B cell development at the transition of pro/pre-B-I to pre-B-II cells. This inhibition is reversed by addition of excess rIL-7. Addition of IL-7 alone stimulates the proliferation of pro/pre-B-I cells and inhibits their differentiation to pre-B-II and immature B cells, as it does in single-cell suspension cultures. FLOC should be useful to study the effects of other mAb, cytokines, ligands and other molecules on early B cell development.

Animals↗
PubMed1997

A transgenic marker for mouse B lymphoid precursors.

Three lines of transgenic mice have been generated which express human CD25 under the control of the 722-base pair region located immediately 5' of the precursor (pre)-B cell-specific lambda5 gene. All three strains express human CD25 in parallel to endogenous lambda5 on pre-B cells, but not on mature B lymphocytes or other blood cell lineages. High expression of human CD25 on B lineage cells of transgenic mice has allowed the identification of a new B220+CD19-lambda5+ precursor of the B220+CD19+lambda5+ c-kit+ pre-BI cells. Both types of precursors are clonable on stromal cells in the presence of interleukin-7. The CD19- precursors have a sizeable part of their immunoglobulin heavy chain gene loci in germline configuration, while the CD19+ pre-BI cells are predominantly DJH rearranged. The results indicate that random integration of the 722-bp 5' region of the lambda5 gene into the mouse genome confers tissue and differentiation stage-specific expression of a transgene.

Animals↗
PubMed1997

Control of the sizes and contents of precursor B cell repertoires in bone marrow.

Ordered rearrangements of immunoglobulin (Ig) gene loci, first as DH to JH, then as VH to DHJH, and finally as VL to JL segment-specific recombinations occur 'in-frame' and 'out-of-frame'. 'In-frame' rearrangements lead to the expression of truncated DHJH-microC proteins and to microH chains. These H chain proteins have two major effects on precursor B cells. They suppress (as DJC mu proteins) or enhance (as full microH chain) the proliferation of precursor cells at the point where these precursors express these proteins. At the same time, they signal allelic exclusion of the microH chain alleles, so that VH to DHJH rearrangement at the second allele is suppressed. Regulation of precursor B cell proliferation and H chain allelic exclusion is mediated by a pre-B cell receptor that is composed of the microH chains and a surrogate L chain. This surrogate L chain is made up of two proteins encoded by the Vpre-B and lambda 5 genes that are expressed only at the early precursor cell stages just before and when H chain genes are first expressed. They are not found in later B cell development, when L chains are expressed, nor in any other cell of the body tested so far. The physiological roles of surrogate L chain and of the pre-B receptor have been clarified by generating mutant mice in which the lambda 5 gene has been inactivated by targeted disruption. Molecular mechanisms and cellular developments, by which the pre-B receptor controls proliferation and allelic exclusion, are discussed.

Alleles↗
PubMed1997

The chemokine SDF-1, stromal cell-derived factor 1, attracts early stage B cell precursors via the chemokine receptor CXCR4.

In the bone marrow, progenitor (pro-) and precursor (pre-) B cells depend on close contact with stromal cells for growth and maturation. Stromal cell-derived factor 1 (SDF-1), also known as pre-B cell growth-stimulating factor, is produced by bone marrow stromal cells and was reported to act together with interleukin-7 as co-mitogen for pre-B cells. SDF-1 was recently shown to be a chemokine which is chemotactic for different types of leukocytes and acts via the chemokine receptor CXCR4. Using sorted B220+ bone marrow cells and several B cell lines characteristic for different stages of B lymphopoiesis, we now show that SDF-1 is a potent attractant for pro- and pre-B cells, but is inactive on B cells at later stages of development. In early B cell precursors, SDF-1 induced intracellular Ca2+ mobilization and in vitro migration with a potency and efficacy similar to that observed for chemokines acting on blood leukocytes. These responses were mediated via CXCR4 as they could be inhibited by an antireceptor antibody. SDF-1 is the first chemokine shown to act on early-stage B cell precursors. Mice lacking SDF-1 die perinatally and show a severe deficiency in B lymphopoiesis. We propose that SDF-1 released from the stromal cells exerts its critical hematopoietic function by selectively attracting and confining early B cell precursors within the bone marrow microenvironment that provides the necessary factors for growth and differentiation.

Animals↗
PubMed1997

Priming of helper T cell-dependent antibody responses by hemagglutinin-transgenic B cells.

Mice expressing the hemagglutinin (HA) gene of influenza virus PR8 (H1 subtype) under the control of kappa light chain promoter and enhancer have been generated. They express HA in and on B cells, and are tolerant to HA. In vitro, only lipopolysaccharide (LPS) blasts but not resting B cells of transgenic mice can stimulate HA-specific helper T cells of HA-specific alpha/beta T cell receptor (TCR)-transgenic mice. Transfer of HA-transgenic LPS blasts into syngeneic, non-transgenic recipients primes HA-specific antibody responses. Resting, small HA-transgenic B cells, which were purified by fluorescence-activated cell sorting, prime lower antibody responses. Host B cells produce the HA-specific antibody response. The donor HA-transgenic B cells need to express major histocompatibility complex (MHC) class II molecules and need to be alive to induce the antibody response in the host. Most notably, the host antibody response never produces detectable levels of IgM, but only of switched IgG isotypes. Neither resting nor activated HA-transgenic B cells induce tolerance in antibody responses. These results suggest that HA-transgenic B cells, presenting both the intact antigen on the cell surface and peptides of the antigen on MHC class II, are effective inducers of helper T cell responses, and as judged by the Ig-isotype response pattern, which is mainly IgG1, of Th2 type.

Animals↗
PubMed1997

Structure and expression of the c-Myc/Pvt 1 megagene locus.

A chromosomal translocation (Tx) that interrupts the transcription of either c-Myc or Pvt 1 is the principal lesion in many B cell malignancies including Burkitt's Lymphoma (BL), AIDs-NHL, mouse plasmacytoma (Pct) and possibly multiple myeloma (MM). There is a restriction associated with this Tx such that only the immunoglobulin (Ig) heavy chain gene is found juxtaposed to c-Myc and only the Ig light chain gene is found juxtaposed to Pvt 1. Over the past several years, our laboratory has been instrumental in the elucidation of the structure of the mouse Pvt 1 locus as a means of understanding the relationship between these two divergent Txs which, nevertheless, produce indistinguishable disease phenotypes. In the mouse, we have identified a uniform Pvt1/Ig Ck fusion product which is consistently found in all tumors harboring Pvt 1 associated Txs. We have recently constructed transgenic mice harboring a translocated Pvt 1/Ck segment in order to determine whether 1). these mice produce the Pvt 1/Ck fusion product 2). these mice are immunocompromised and 3). these mice develop tumors of a B cell origin.

Amino Acid Sequence↗
PubMed1997

Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor.

The V(H) repertoire on both H chain alleles of normal and lambda5-deficient B lineage cells were analyzed by single-cell PCR. The mu H chains were tested for their capacity to form a pre-B cell receptor. In bone marrow, D-proximal V(H) genes were found preferentially expressed in lambda5-deficient pre-B cells and in a newly identified early c-kit+ cytoplasmic mu H chain+ pre-B cell population of normal mice. Only half of the mu H chains expressed in these cells have the capacity to form a pre-B cell receptor. Representation of the D-proximal V(H) genes was found suppressed on the productive but not on the nonproductive V(H)DJ(H) rearranged alleles of c-kit preB-II cells and splenic lambda5-deficient B cells. More than 95% of the mu H chains expressed in preB-II cells can form a pre-B cell receptor. These results demonstrate that the pre-B cell receptor in normal mice and the B cell receptor in lambda5-deficient mice mediate a shift in the V(H) repertoire.

Alleles↗
PubMed1996

Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci.

CD19+CD10+ human B lineage bone marrow cells were separated into cycling or resting cells, which differ in their expression of CD34, VpreB, recombination activating gene (RAG-1), and terminal deoxynucleotidyl transferase (TdT). Polymerase chain reaction analyses developed for DHJH and VkJk, VkJkK(de) and VkK(de) rearrangements with DNA of single cells and a comparison with B lineage cell development in mouse bone marrow, allow to delineate the human B lymphocyte pathway of development as follows: CD34+VpreB+RAG-1+TdT+, DHJH-rearranged, kL germline cycling pre-B I cells-->CD34-VpreB+microH chain+ (pre-B receptor+) RAG-1-TdT-, VHDHJH-rearranged, kL germline, cycling pre-B II cells-->CD34-VpreB-, intracytoplasmic microH chain+ (pre-B receptor-) RAG-1+/-TdT-, VHDHJH-rearranged, mainly kL germline cycling pre-B II cells-->CD34-VpreB-intracytoplasmic microH chain+, RAG-1+TdT-, VHDHJH-rearranged, VkJk-rearranged, IgM-, resting pre-B II cells CD34+VpreB-, sIgM+, RAG-1+TdT-, VHDHJH- and VkJk-rearranged IgM+ immature B cells-->CD34-, CD10-, sIgM+/sIgD+ mature B cells. This order, for the first time established for human B lineage cells, shows striking similarities with that established for mouse B lineage cells in bone marrow.

Animals↗
PubMed1996

Intrinsic B cell defects in NZB and NZW mice contribute to systemic lupus erythematosus in (NZB x NZW)F1 mice.

We have previously shown that long-term in vitro proliferating fetal liver pre-B cell lines derived from autoimmune-prone (NZB x NZW)F1 (BW) mice, but not normal (B6 x DBA2)F1 mice, can differentiate in severe combined immunodeficient (SCID) mice to produce elevated levels of serum immunoglobulin (Ig) M and IgG, and high titers of antinuclear antibodies The contribution of parental NZB and NZW strains to B cell abnormalities of BW hybrid mice was investigated here by preparing pre-B cells and transferring them into immunodeficient SCID- and RAG-2-targeted mice. We show that transfer of NZB pre-B cells led to a marked IgM hypergammaglobulinemia and to the production of limited amounts of IgG2a. On the other hand, the transfer of NZW pre-B cell lines led to moderately elevated IgM levels and marked hypergammaglobulinemia of IgG2a. High IgM and low IgG anti-DNA titers are found in the recipients of NZB pre-B cells, whereas those receiving NZW pre-B cells contained lower levels of IgM and high titers of IgG anti-DNA. In marked contrast, essentially identical titers of antibodies directed against a non-self-antigen, DNP, are found in all group of pre-B cell recipients. Thus, B-lineage cells of both NZB and NZW parental strains manifest abnormalities associated with the development of this lupus-like disease. Therefore, the present study strongly suggests a complex inheritance of B cell abnormalities in autoimmune-prone (NZB x NZW)F1 mice and emphasizes the critical importance of intrinsic B cell defects in the development of murine systemic lupus erythematosus.

Animals↗
PubMed1996

The half-life of RAG-1 protein in precursor B cells is increased in the absence of RAG-2 expression.

Site-specific recombination of immunoglobulin and T cell receptor gene segments in B and T lymphocytes is dependent on the expression of two recombinant activation genes, Rag-1 and Rag-2. Here, we show that RAG-1 protein turnover in pre-B cells depends on the expression of RAG-2. The apparent half-life of RAG-1 protein is increased when RAG-2 is not expressed in differentiating pre-B cells.

Animals↗
PubMed1996

A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors.

Bone marrow of both normal and rearrangement-deficient mice contains a small population of B220(CD45R)+ cells, which do not express the B lineage marker CD19. Instead, part of this population coexpresses the surface marker CD43 and lacks or expresses very low levels of heat stable antigen (HSA) and BP-1, thus representing a part of Hardy's fraction A (B220(+)-CD43+HSA-, BP-1-) of B lineage development. However, some 20-40% of these B220(+)-CD19- cells also coexpress the NK1.1 surface molecule and do not express genes like VpreB or B29 restricted to the B cell lineage. These cells respond to recombinant interleukin 2 in vitro, and develop into killer cells that can lyse the prototypic NK target tumor cell, YAC-1, as well as syngeneic normal lipopolysaccharide or concanavalin A blasts, providing they lack the surface expression of major histocompatibility complex class I molecules. The implications of these findings for studies on B lymphopoiesis are discussed. It is suggested that the CD19-specific monoclonal antibody is more reliable, as in humans, than B220(CD45R) to detect B lineage cells in mice.

Animals↗
PubMed1996

The murine VpreB1 and VpreB2 genes both encode a protein of the surrogate light chain and are co-expressed during B cell development.

The surrogate light chain is composed of two polypeptides, VpreB and lambda 5. In the mouse there are two VpreB genes which are 99% identical within the coding regions. Extensive restriction enzyme mapping and sequencing of these two genes showed that only the coding region and immediate 5' and 3' flanking sequences exhibited such high homology. More distal sequences have diverged considerably. The region 5' of the respective gene directed transcription of a reporter gene in a pre-B cell line, indicating that it contained promoter, and perhaps enhancer function. The VpreB2 gene is functional, as it directed the production in COS cells of a 16-kDa protein that assembled with lambda 5 and was recognized by a VpreB-specific monoclonal antibody. Using transfected COS cells expressing either VpreB1 or VpreB2, a PCR assay was developed to examine the steady state level of transcripts from each gene. When this assay was applied to a number of cell lines representing early stages of B cell differentiation, co-expression of the two genes was observed in every case. VpreB1 and VpreB2 were co-expressed in the fetal liver of CB17 mice, where peak expression of each gene occurred at days 16-17 of gestation. Similarly, adult bone marrow from several strains of mice expressed both genes. In sorted bone marrow cells expression of both VpreB genes was detected in pro-B/pre-BI and large pre-BII cells, while the RNA steady state levels were at least 100-fold lower in small pre-BII and immature/mature B cells. Finally, single-cell reverse transcriptase-polymerase chain reaction on such sorted bone marrow cells detected VpreB1 and VpreB2 expression in at least 30% of all pro-B/pre-BI cells and large Ig heavy chain, surrogate light chain (pre-B receptor) expressing pre-BII cells. These results demonstrate that the control of expression of the two VpreB genes overlaps during development. They suggest that both VpreB1 and VpreB2 polypeptides can assemble with lambda 5 and mu to form pre-B cell receptor complexes.

Animals↗