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Biomedical subjects

F Melchers

Publications and source records attributed to F Melchers.

At least 37 records · Page 2Linked to original sources
PubMed1999

Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors.

The mechanisms controlling the commitment of haematopoietic progenitors to the B-lymphoid lineage are poorly understood. The observations that mice deficient in E2A and EBF lack B-lineage cells have implicated these two transcription factors in the commitment process. Moreover, the expression of genes encoding components of the rearrangement machinery (RAG1, RAG2, TdT) or pre-B-cell receptor (lambda5, VpreB, Igalpha, Igbeta) has been considered to indicate B-lineage commitment. All these genes including E2A and EBF are expressed in pro-B cells lacking the transcription factor Pax5. Here we show that cloned Pax5-deficient pro-B cells transferred into RAG2-deficient mice provide long-term reconstitution of the thymus and give rise to mature T cells expressing alpha/beta-T-cell receptors. The bone marrow of these mice contains a population of cells of Pax5-/- origin with the same phenotype as the donor pro-B cells. When transferred into secondary recipients, these pro-B cells again home to the bone marrow and reconstitute the thymus. Hence, B-lineage commitment is determined neither by immunoglobulin DJ rearrangement nor by the expression of E2A, EBF, lambda5, VpreB, Igalpha and Igbeta. Instead, our data implicate Pax5 in the control of B-lineage commitment.

Animals↗
PubMed1999

Continued RAG expression in late stages of B cell development and no apparent re-induction after immunization.

Models of B-cell development in the immune system suggest that only those immature B cells in the bone marrow that undergo receptor editing express V(D)J-recombination-activating genes (RAGs). Here we investigate the regulation of RAG expression in transgenic mice carrying a bacterial artificial chromosome that encodes a green fluorescent protein reporter instead of RAG2. We find that the reporter is expressed in all immature B cells in the bone marrow and spleen. Endogenous RAG messenger RNA is expressed in immature B cells in bone marrow and spleen and decreases by two orders of magnitude as they acquire higher levels of surface immunoglobulin M (IgM). Once RAG expression is stopped it is not re-induced during immune responses. Our findings may help to reconcile a series of apparently contradictory observations, and suggest a new model for the mechanisms that regulate allelic exclusion, receptor editing and tolerance.

Alleles↗
PubMed1999

Effect of deregulated IL-7 transgene expression on B lymphocyte development in mice expressing mutated pre-B cell receptors.

Deregulated overexpression of IL-7 under the control of the promoter of the Ealpha gene of MHC class II in IL-7-transgenic mice changes B cell development in wild-type mice and in mutants which limit B cell development at various cellular stages. While the introduction of deregulated IL-7 production does not change the size of the pro-B and pre-B I compartments in the bone marrow of wild-type and lambda5-/- mice, it increases these compartments 2.5- to fivefold in mice which cannot make immature and mature B cells, i. e. in RAG-2-/-, tmmuH-/-, and RAG-2-/- mice expressing a transgenic muH chain. Excessive IL-7 production also increases four- to fivefold the pre-B II compartment in all those mouse strains where it can be formed (i. e. in wild-type, lambda5-/- and muH chain-transgenic RAG-2-/- mice), while no pre-B- II-like cells appear in excessively IL-7-stimulated bone marrow of mice devoid of pre-B II cells (i. e. in tmmuH-/- and RAG-2-/- mice). In the spleen of all IL-7-transgenic mice significant numbers of both pro-B and pre-B I cells are detectable and increased numbers of pre-B II and immature B cells appear in the spleen of mouse strains which are capable of making them. The capacity of the spleen to accommodate expanded numbers of these B-lineage cells as well as mature B cells is much larger than that of the bone marrow of the IL-7-transgenic mice probably because the bone limits cellular expansion and provokes spillover into the peripheral lymphoid organs.

Animals↗
PubMed1999

Three chemokines with potential functions in T lymphocyte-independent and -dependent B lymphocyte stimulation.

Three clustered mouse chemokine genes, ABCD-1, -2 and -3, are all expressed highly in dendritic cells and, at various levels, in activated B cells. T cell-independently activated B cells express ABCD-1 and -2, but not -3. T cell-dependently activated B cells express all three. ABCD-1 attracts activated CD8+ cytotoxic T cells and CD4+ helper T cells of type 1 and 2. ABCD-2 preferentially attracts type 2 helper T cells, while ABCD-3 does not attract T cells at all. Both ABCD-1 and ABCD-2 bind to the same receptor (CCR4). In addition, ABCD-1 binds to a second, unknown, receptor on a separate T cell population. The three chemokines might guide T cell-independent as well as -dependent responses with two types of CD4+ T cells.

Amino Acid Sequence↗
PubMed1999

The roles of preB and B cell receptors in the stepwise allelic exclusion of mouse IgH and L chain gene loci.

Membrane-bound preBCR of wild-type mice, and probably also preBCR-like V(preB) muH chain complexes in lambda5-deficient mice, signal allelic exclusion so that < 0.1% of all preB-II cells and all subsequent B lineage cells express two muH chains on their surface. On the other hand a large number of muH chains which are originally generated at the transition of preB-I to preB-II cells cannot pair with surrogate L chains, cannot form a preBCR on the surface and, hence, allow two H chain alleles to be productively rearranged in one B-lineage cell. By contrast membrane-bound BCR on immature B cells does not signal allelic or isotypic exclusion Of Ig kappaL and lambdaL chain gene loci. This allows the rearrangement machinery to remain active, and secondary L chain rearrangements on one kappaL chain allele are frequently observed. Rapid selection of fitting H/L chain pairs, forming BCR on the surface, allows B-lineage cells to enter the mature B cell pool where the rearrangement machinery is shut off, securing allelic exclusion of L chain loci in most B cells.

Alleles↗
PubMed1999

Mutations affecting either generation or survival of cells influence the pool size of mature B cells.

The mature B cell compartment of MHC class II-deficient B6 I-Aalpha(-/-) and the btk-defective CBA/N mouse strain is 4- to 5-fold smaller than in wild-type B6 mice. The defect in B6 I-Aalpha(-/-) mice is intrinsic to B cells and due to a 4- to 5-fold reduced lifespan, which however can be normalized by an I-Ealpha(d) transgene, but only when expressed early during B cell development. The reduced number of mature B cells in the btk-defective CBA/N mouse is due to a 4- to 5-fold lower number of immature splenic B cells entering the mature compartment. The combined defects of reduced lifespan and impaired generation in double mutant mice result in a severe deficiency in the mature B cell pool.

Animals↗
PubMed1999

Four of five RAG-expressing JCkappa-/- small pre-BII cells have no L chain gene rearrangements: detection by high-efficiency single cell PCR.

Single cell PCR assays have been further developed that detect over 80% of all VkappaJkappa, VkappaRS, and VlambdaJlambda rearrangements at efficiencies between 70% and 90%. These IgL chain gene rearrangement assays were used with small pre-BII cells that develop in comparably high numbers in the bone marrow of wild-type, Ckappa-deficient, and JCkappa-deficient homozygous and heterozygous mice. In all of these mice, only 15%-25% of all small pre-BII cells carry VlambdaJlambda rearrangements. These results confirm that lambdaL chain gene rearrangements occur independently of kappaL chain gene rearrangement and expression. They also show that a large part of the small pre-BII cells that express the rearrangement machinery can develop without IgL chain gene rearrangements.

Animals↗
PubMed1999

Frequencies of multiple IgL chain gene rearrangements in single normal or kappaL chain-deficient B lineage cells.

PCR analyses of the kappaL chain locus in single B-lineage cells of wild-type, Ckappa-, or JCkappa-deficient homozygous or heterozygous mice often detect multiple in- and out-of-frame rearrangements at the kappaL and lambdaL loci. They are most frequent in small pre-BII cells and equally so in wild-type and kappaL chain-deficient cells. Hence, kappaL chain production appears not to inhibit secondary rearrangements. Around 20% of all small preBII cells express IgL chains in their cytoplasm. Cells with a first productive rearrangement on one allele are favored to enter the immature B cell compartment. Thus, allelic exclusion might be secured by control of accessibility of IgL chain loci for rearrangement and by rapid selection of cells with a fitting over those with a nonfitting IgL chain.

Alleles↗
PubMed1999

Partial block in B lymphocyte development at the transition into the pre-B cell receptor stage in Vpre-B1-deficient mice.

The surrogate light chain (SL) is composed of two polypeptides, Vpre-B and lambda5. In large pre-BII cells the SL chain associates with Ig mu heavy chain (muH) to form the pre-B cell receptor (pre-BCR). In mice there are two Vpre-B genes which are 98% identical within the coding regions. The two genes are co-expressed at the RNA level and encode functional proteins that can assemble with lambda5. However, it is not known whether both gene products serve the same function in vivo. Here we have established mice that lack the Vpre-B1 gene (VpreB1(-/-)), but still express the Vpre-B2 gene, both as RNA and protein. In Vpre-B1(-/-) mice, the bone marrow cellularity and the percentage of B220+ cells is normal. However, among the B220+ cells, the percentage of pre-BI cells is increased, and the percentage of pre-BII and immature B cells is slightly decreased, suggesting that the lack of Vpre-B1 causes a partial block at the transition from pre-BI to pre-BII cells, i.e. into the pre-BCR stage. The number of cells that produce a functional pre-BCR is thus lower, but the cells that reach this stage are normal as they can be expanded by proliferation and then differentiate into more mature cells. The spleens of Vpre-B1 homozygous mutant mice show normal numbers of B and T lymphocytes. Moreover, the Ig loci are allelicly excluded and the homozygous mutant mice respond with normal levels of antigen-specific antibodies to T-dependent antigens. These results demonstrate that VpreB2 alone is capable of supporting B lymphocyte development in the bone marrow and can give rise to immuno-competent cells in the periphery.

Animals↗
PubMed1998

Activated murine B lymphocytes and dendritic cells produce a novel CC chemokine which acts selectively on activated T cells.

Genes were isolated using the suppression subtractive hybridization method by stimulation of pro/pre B cells with anti-CD40 and interleukin (IL)-4 to mature S mu-Sepsilon-switched cells. One of the strongly upregulated genes encodes a novel murine CC chemokine we have named ABCD-1. The ABCD-1 gene has three exons separated by 1. 2- and 2.7-kb introns. It gives rise to a 2.2-kb transcript containing an open reading frame of 276 nucleotides. Two polyadenylation sites are used, giving rise to cDNAs with either 1550 or 1850 bp of 3' untranslated regions. The open reading frame encodes a 24 amino acid-long leader peptide and a 68 amino acid-long mature protein with a predicted molecular mass of 7.8 kD. ABCD-1 mRNA is found in highest quantities in activated splenic B lymphocytes and dendritic cells. Little chemokine mRNA is present in lung, in unstimulated splenic cells, in thymocytes, and in lymph node cells. No ABCD-1 mRNA is detected in bone marrow, liver, kidney, or brain, in peritoneal exudate cells as well as in the majority of all unstimulated B lineage cells tested. It is also undetectable in Concanavalin A-activated/IL-2-restimulated splenic T cells, and in bone marrow-derived IL-2-induced natural killer cells and IL-3-activated macrophages. Recombinant ABCD-1 revealed a concentration-dependent and specific migration of activated splenic T lymphoblasts in chemotaxis assays. FACS(R) analyses of migrated cells showed no preferential difference in migration of CD4(+) versus CD8(+) T cell blasts. Murine as well as human T cells responded to ABCD-1. Freshly isolated cells from bone marrow, thymus, spleen, and lymph node, IL-2-activated NK cells, and LPS-stimulated splenic cells, all did not show any chemotactic response. Thus, ABCD-1 is the first chemokine produced in large amounts by activated B cells and acting selectively on activated T lymphocytes. Therefore, ABCD-1 is expected to play an important role in the collaboration of dendritic cells and B lymphocytes with T cells in immune responses.

Amino Acid Sequence↗