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Biomedical subjects

F Melchers

Publications and source records attributed to F Melchers.

At least 73 records · Page 4Linked to original sources
PubMed1996

The SCID but not the RAG-2 gene product is required for S mu-S epsilon heavy chain class switching.

We have investigated the capacity of precursor B cells from normal (BDF1) and V(D)J recombinase-deficient (RAG-27) or defective (SCID) mice to be induced by a CD40-specific monoclonal antibody and IL-4 to epsilon H chain gene transcription and to S mu-S epsilon switch recombination. In differentiating precursor B cells from all three strains of mice, the development of similar numbers of CD19+, CD23+, CD40+, and MHC class II+ expressing B lineage cells and similar levels of epsilon H chain gene transcription were induced. Efficient S mu-S epsilon switching occurred in normal and RAG-2-deficient, but not in SCID, precursor B cells. Thus, the transcription of the epsilon H chain is independent of the RAG-2 and the SCID gene product, while the S mu-S epsilon switch recombination requires the SCID gene-encoded DNA-dependent protein kinase, but not the RAG-2 protein.

Animals↗
PubMed1996

Profound reduction of mature B cell numbers, reactivities and serum Ig levels in mice which simultaneously carry the XID and CD40 deficiency genes.

It has been known for some time that single mutant nude or CD40T mice have apparently normal numbers of cells in the precursor compartments of bone marrow and the mature B cell compartments of the periphery. X-linked immunodeficiency (XID) mice are deficient only in some of the sIgM+sIgD+ B cells. We have investigated further the contributions of the xid mutation, of the T cell deficiency of nude of the inability of CD40T cells to cooperate with T cells in the generation of the precursor and the mature B cell compartments in mice. Double mutant XID/nu and XID/CD40T mice have precursor B cell compartments that are no more deficient than the single mutant XID mice. However, the peripheral B cell compartments of both XID/mu and XID/CD40T are even more deficient than those of single mutant XID mice. While 10% of the peripheral B cells of wild-type or CD40T, one-third of XID and half of XID/nu mice turn over rapidly, as many as three-quarters of those in XID/CD40T are short-lived. Total numbers of sIgM+sIgD+ B cells in the spleen are at best 10-15% of normal mice at 6-8 weeks of age in XID, XID/nu and XID/CD40T mice. They remain that low at 3 months of age in XID/CD40T mice, while in XID mice these peripheral B cells slowly build up in numbers with age. As expected, double mutant XID/CD40T mice do not respond to the T-dependent antigen keyhole limpet hemocyanin. Only the responses to the T-independent type I antigen, TNP-lipopolysaccharide (LPS), appear to be normal. In vitro, their splenic B cells respond poorly to LPS or to IgM-specific antibody in either the absence or presence of cytokines. Most notably, serum IgM, IgG2b or IgG3 levels are severely depressed, while IgG1, IgG2a and IgA levels are < 10 micrograms/ml. These results suggest a model of mature B cell development in which the peripheral, mature B cell compartments are generated in two parallel, not tandemly organized pathways. They could be selected and/or stimulated at the transition from immature to mature B cells: in btk controlled or in CD40 controlled ways.

Animals↗
PubMed1995

Interleukin-3 and interleukin-7 are alternative growth factors for the same B-cell precursors in the mouse.

Clones and lines of precursor (pre) B cells can be established by limiting dilutions of unseparated cell suspensions of fetal liver or bone marrow on stromal cells in the presence of interleukin (IL)-7. When IL-3 is used instead of IL-7, cultures are regularly overgrown by different precursor cells of the myeloid lineage, as well as by adherent cells that inhibit pre-B-cell expansion. However, in the presence of either IL-7 or IL-3, clones of pre-B cells can be established on stroma cells at frequencies near one in one when the cultures are initiated with cell sorter purified CD45RO (B220)+/c-kit+ fetal liver or bone marrow derived pre-B cells. Clones grown on stromal cells in the presence of IL-7 can be regrown in IL-3, and vice versa. Pre-B cells that proliferate on stromal cells in the presence of IL-7 or IL-3 have the same phenotype, ie, are B220+ c-kit+, CD43+, and surrogate light chain+. Removal of the growth factors (IL-7, respectively IL-3) from the cultures results in differentiation to surface immunoglobulin (slg) positive, c-kit-, CD43-, surrogate light chain- B cells, a fraction of which is lipopolysaccharide (LPS) responsive as shown by IgM secretion. These results show that IL-7 and IL-3 stimulate largely overlapping populations of precursor B cells from bone marrow to proliferate for long periods of time in the presence of stromal cells. Thus, IL-7 and IL-3 are alternative growth factors for the same pre-BI cell.

Animals↗
PubMed1995

Precursor B cells of mouse bone marrow express two different complexes with the surrogate light chain on the surface.

Two monoclonal antibodies raised against the complex of mu heavy (H) chain and Vpre-B/lambda 5 surrogate light (L) chains recognize surrogate L chain in different conformations on normal pre-B cells. One, LM34 recognizes free lambda 5 protein and free lambda 5/Vpre-B surrogate L chains and binds to surrogate L chains on the surface of early, pro-B and pre-B-I cells where the surrogate L chain is associated with a gp130/gp35-65 complex of proteins. It also recognizes the surrogate L chain associated with the mu H chain on pre-B-II cells. The other monoclonal antibody, SL156, does not recognize free surrogate L chain or its components, nor its complex with gp130/gp35-65 on pro-B and pre-B-I cells. However, it does bind to a conformational epitope on the surrogate light chain/mu H chain complex on a subpopulation of pre-B-II cells and on mu H chain-positive pre-B cell lines. On mouse precursor B cells prepared ex vivo on ice, expression of the surrogate L chain is very low and almost undetectable. Incubation of the precursor cells for 1 h at 37 degrees C up-regulates the surface expression of surrogate L chain associated with gp130/gp35-65 (early complex) as well as the mu H chain/surrogate L chain complex. These results reconcile some of the apparently discrepant results on surface expression of the surrogate L chain obtained with human and mouse bone marrow pre-B cells, and show that a surrogate L chain/mu H chain-containing pre-B cell receptor can be expressed also on the surface of mouse pre-B-II cells.

Animals↗
PubMed1995

An interleukin-6 transgene expressed in B lymphocyte lineage cells overcomes the T cell-dependent establishment of normal levels of switched immunoglobulin isotypes.

Long-term proliferating, stromal cell/interleukin (IL)-7-reactive precursor B cell lines established from fetal liver and bone marrow of human IL-6-transgenic B6Ld46 mice produce and secrete human IL-6. When transplanted into severe-combined immunodeficient (SCID) or Rag2 knockout (Rag2-T) mice, these pre-B-cell lines establish a part of the B cell compartment but yield no T cells, as do pre-B cell lines from genetically matched non-transgenic mice. Within 2 to 3 months after transplantation, the serum of mice transplanted with pre-B cells from normal mice contains normal levels of IgM (200-600 micrograms/ml) but 10-100-fold lower levels of the IgG subclasses and of IgA. In contrast, the sera of mice transplanted with IL-6 transgenic pre-B cells contain not only IgM, but also IgG and IgA at nearly normal levels. The results indicate that at least a part of the plasmacytosis and elevated IgG production observed previously in the IL-6-transgenic mice appears to be due to a T cell-independent activation of IgG and IgA production by the IL-6-secreting pre-B cells and their differentiated progeny in the immunodeficient hosts.

Animals↗
PubMed1995

Immature B cells from human and mouse bone marrow can change their surface light chain expression.

The capacity of bone marrow-derived surface immunoglobulin-positive (sIg+) human and mouse immature B cells, generated either in vitro or in vivo, to change their light (L) chain expression, has been assayed by the number of cells which change in vitro from one type of L chain to the other type, or to no sIg at all. Immature sIg+ B cells were generated in vitro from sIg- precursor cells from human or mouse bone marrow. The immature sIg+ cells expressed RAG-1. Human sIg+ cells expressed kappa and lambda L chains in ratios between 1:1 and 3:1, whereas in mouse cells, this ratio ranged from 10:1 to 20:1. Upon reculture of the human and mouse kappa+ sIg+ cells, about half of them remained kappa+, a quarter became lambda+, and another quarter became sIg-. Between 1 and 3% expressed both kappa and lambda chains. Of the human lambda+ cells, about two-thirds remained lambda+, only 1 to 2% became kappa+, while the other third became sIg-. Again, between 1 and 3% expressed both kappa and lambda L chains. These results indicate that expression of sIgM in the B cell membrane does not terminate L chain gene rearrangement, and that some order exists in kappa versus lambda gene rearrangements. Hence, human and mouse kappa+ immature B cells can become lambda+, but very few of the lambda+ cells can become kappa+, and both can become sIg-. Further, human CD10+/sIg+ kappa+ and lambda+ cells and mouse B220low/sIglow kappa+ cells enriched from bone marrow, i.e. immature B cells differentiated in vivo, changed their Ig phenotype upon in vitro culture, but in lower frequencies. By contrast, human and mouse mature B cells did not change their L chain or Ig phenotype. Hence, at least a part of the sIg+ immature B cells in bone marrow retain the capacity to change their L chain and Ig phenotype, and this capacity is lost when they become mature, peripheral B cells.

Adolescent↗
PubMed1995

Positive and negative selection events during B lymphopoiesis.

Early in B-cell development, large numbers of cells have to be generated, each of which expresses only one type of B-cell receptor (i.e. Ig) on its surface. This is achieved by the surface expression of a pre-B cell receptor containing a mu heavy chain/surrogate light chain which differentially provides signals for two responses of precursor B cells at this stage of development. On the one hand, it signals inhibition of further rearrangements of variable heavy chain to diverse-joining heavy chain loci to achieve allelic exclusion at the heavy-chain locus. On the other hand, it signals proliferative expansion by factors between 20 and 100. Later in B-cell development, tolerance to autoantigens must be established and maintained. Tolerance is achieved by developmental arrest and induction of secondary light-chain gene rearrangements in those IgM+ immature B cells that are reactive to autoantigens presented in the primary B-cell generating organs. Even later in development, when mature surface (s)IgM+/sIgD+ B cells encounter autoantigens presented to them in the periphery, either deletion or anergy of the autoantigen-reactive cells occurs. Anergic cells have a sIg-dependent, sIg-proximal defect in signaling and are short-lived. Anergy can be broken in vitro by polyclonal activation via ligation of CD40 in the presence of IL-4. A small part of the remaining immature B cells not reactive to autoantigens are selected to become mature, antigen-reactive sIgM+/sIgD+ B cells. Molecules which might guide such positive selection of B cells still remain to be identified.

Animals↗
PubMed1995

In-vitro analyses of mechanisms of B-cell development.

B-cell lymphopoiesis in vivo is very complex due to the influences of cooperating cells, cytokines and other receptor-ligand interactions which appear to occur developmentally at different cellular stages. Therefore in-vitro models will help to unravel this complex situation. Here, we review our and others' work on in-vitro models of B-cell development. The role of stromal cells, cytokines, surrogate light chain and products of rearranged Ig-loci in the developmentally different cellular stages will be discussed.

Animals↗
PubMed1995

Down-regulation of RAG1 and RAG2 gene expression in preB cells after functional immunoglobulin heavy chain rearrangement.

Two waves of immunoglobulin gene rearrangements, first of the heavy, then of the light chain chain gene loci form functional immunoglobulin genes during B cell development. In mouse bone marrow the differential surface expression of B220 (CD45R), c-kit, CD25, and surrogate light chain as well as the cell cycle status allows FACS separation of the cells in which these two waves of rearrangements occur. The gene products of two recombination activating genes, RAG1 and RAG2 are crucial for this rearrangement process. Here, we show that the expression of the RAG genes is twice up- and down-regulated, at the transcriptional level for RAG1 and RAG2, and at the postranscriptional level for RAG2 protein. Expression levels are high in D-->JH and VH-->DJH rearranging proB and preB-I cells, low in preB cells expressing the preB cell receptor on the cell surface, and high again in VL-->JL rearranging small preB-II cells. In immature B cells expressing on the cell surface RAG1 and RAG2 mRNA is down-regulated, whereas RAG2 protein levels are maintained. Down-regulation of RAG1 and RAG2 gene expression after productive rearrangement at one heavy chain allele might be part of the mechanisms that prevent further rearrangements at the other allele.

Animals↗
PubMed1995

Induction of sterile transcription from the kappa L chain gene locus in V(D)J recombinase-deficient progenitor B cells.

B cell development in RAG-2-deficient (RAG-2T) mice is impeded at an early stage, due to the inability of these animals to rearrange their endogenous ig gene loci. Expression of an E mu-bcl-2 transgene in these mice did not change this phenotype. However, stromal cell/IL-7-reactive B cell progenitors (pro-B cells) were found in fetal live and bone marrow of RAG-2T and RAG-2T/E mu-bcl-2 transgenic mice in numbers comparable to normal mice. Like cells from normal mice they are c-kit+, surrogate L chain+ and CD25-, and can proliferate in vitro for long periods of time. Upon IL-7 deprivation, they can be induced to differentiate into c-kit-, surrogate L chain- and CD25+ cells that are no longer clonable on stromal cells and IL-7. Furthermore, sterile transcription from the kappa L chain gene loci is induced. The latter was also observed with pro-B cells directly isolated ex vivo from the bone marrow of RAG-2-deficient animals. The results suggest that progenitor B cell differentiation can occur in cells from V(D)J recombinase-deficient mice to the stage where kL chain gene rearrangements would normally be initiated. It further indicates that some molecular programs of early B cell differentiation can take place in the absence of Ig gene rearrangements.

Animals↗
PubMed1995

The status of Ig loci rearrangements in single cells from different stages of B cell development.

Differential expression of c-kit, CD25 (TAC), surrogate L chain and cytoplasmic muH chain, and surface expression of IgM and IgD allows the separation of B220 (CD45+) B cell subpopulations. PCR analyses with DNA of single cells developed by others and by us have been used to monitor the conformation of the Ig H and L chain gene loci in these different B lineage subpopulations. The results of these analyses indicate that B220+/c-kit+/CD25- cells are the precursors of large B220+/CD25+/sIgM- which, in turn, are the precursors of small B220+/CD25+/sIgM- cells. The majority of B220+/c-kit+/CD25- cells are DHJH-rearranged, with L chain loci in germline configuration and are thus pre-B I cells. More than 90% of all large B220+/CD25+/sIgM- cells have at least one H chain locus VHDHJH rearranged; half of them have also the second locus VHDHJH rearranged and are thus large pre-B II cells. Rearrangements of at least one allele of the kappa L chain loci become detectable in 65% of the small B220+/CD25+/sIgM- cells, 67% of the immature B and > 75% of the mature B cells. The ratio of kappa L to lambda L gene rearrangements in all three subpopulations is approximately 10:1, indicating that the kappa L/lambda L ratio is established as soon as rearrangements are made.

Animals↗
PubMed1995

Stimulation by T cell independent antigens can relieve the arrest of differentiation of immature auto-reactive B cells in the bone marrow.

The pair of microH-chain and kappa L-chain transgenes encoding the Sp6 TNP/DNA-specific IgM was bred onto the rearrangement-deficient genetic background of RAG-2T mice, and onto the kappa L-chain expression-deficient background of iE kappa T mice. Bone marrow of Sp6 transgenic RAG-2T mice contained normal numbers of B220(CD45R)+c-kit+ pro/preB-I-like cells and normal numbers of B220(CD45R)+TAC+ preB-II-like cells. Most strikingly, the numbers of immature sIgM+ B cells in the bone marrow were at least five-fold lower than normal, while mature B cells were almost undetectable in bone marrow as well as spleen. Hence, B cell development in these mice appears to be arrested at the transition from preB-II to immature B cells. The contents of bone marrow and spleen of the different precursors, immature and mature B cell compartments in Sp6iE kappa T mice were found to be similar to those of normal mice except that all sIg+ cells expressed lambda L-chains, of which 40% coexpressed the transgenic kappa L-chain. It indicates that the repertoire of lambda L-chain rearrangements and the lambda L-chains expressed from it suffices to relieve the arrest of differentiation seen in Sp6RAG-2T mice. The T cell-independent antigen TNP-Ficoll elicited within 5 days a response of the Sp6RAG-2T mice to develop to IgM-secreting cells and to fill the serum pool with the Sp6 transgenic IgM to 100 micrograms/ml, i.e. to normal serum levels of IgM in normal mice. TNP-Ficoll appears to interfere with the arrest of differentiation. Two scenarios for this arrest of differentiation and its relief by the T-independent antigen TNP-Ficoll are discussed.

Animals↗