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S Lebecque

Publications and source records attributed to S Lebecque.

At least 55 records · Page 3Linked to original sources
PubMed1997

Germinal center founder cells display propensity for apoptosis before onset of somatic mutation.

B lymphocytes undergo affinity maturation of their antigen receptors within germinal centers. These anatomical structures develop in secondary lymphoid organs from the clonal expansion of a few antigen-specific founder B cells, whose isolation and characterization are reported here. Human germinal center founder cells express the naive B cell markers surface IgM and IgD as well as the germinal center B cell markers CD10 and CD38. They express low levels of Bcl-2, high levels of Fas, and undergo rapid apoptosis in culture. The smaller nonproliferating sIgM+IgD+CD38+ B cells displayed a lower level of somatic mutation in their immunoglobulin variable region genes compared with the large proliferating ones. Unmutated sIgM+IgD-CD38+ tonsillar B cells may thus represent germinal center founder cells in which the program for apoptotic cell death is triggered before the onset of somatic mutation, allowing the selection of the germline antibody repertoire at an early stage.

ADP-ribosyl Cyclase↗
PubMed1997

Follicular dendritic cells specifically express the long CR2/CD21 isoform.

This paper describes an antibody (mAb 7D6) that specifically recognizes human follicular dendritic cells (FDCs). By expression cloning, a cDNA clone encoding for the long human CR2/ CD21 isoform (CD21L) that contains an additional exon (10a) was isolated. We demonstrated that FDCs selectively express CD21L, while B cells selectively express the short CR2/CD21 lacking exon 10a (CD21S). By screening mouse Ltk- cells transfected with the CD21L cDNA, we further showed that the other two anti-human FDC mAbs DRC-1 and KiM4 also recognize CD21L. Thus, CD21L represents the first characterized human FDC-specific molecule, which may confer unique functions of FDCs in germinal center development.

Animals↗
PubMed1997

Molecular cloning of human RP105.

RP105 is a 105-kDa type I membrane protein of the leucine-rich repeat (LRR) family. Anti-RP105 sensitizes B cells to antigen-receptor-mediated apoptosis, but protects B cells from radiation-induced apoptosis and stimulates B cell proliferation. The sequence of the mouse RP105 has been reported. Here, we report the characterization of the human RP105. The 2.6-kb cDNA encodes a protein of 661 amino acids which displays 78% homology with mouse RP105. The 22 LRR and the 9 potential N-linked glycosylation sites within the extracellular region are conserved. While previous studies have shown that RP105 is expressed on surface IgM+IgD+2 B cells in mice, human RP105 was shown to be expressed on all subsets of mature B cells and dendritic cells. Human RP105 gene was mapped to the long arm of chromosome 5, where numerous cytokines and receptors have been localized.

Animals↗
PubMed1997

Identification and analysis of a novel member of the ubiquitin family expressed in dendritic cells and mature B cells.

Using a cDNA subtraction technique, a novel member of the ubiquitin family was isolated from human dendritic cells. This gene encodes a diubiquitin protein containing tandem head to tail ubiquitin-like domains, with the conservation of key functional residues. Expression of this 777-bp mRNA was restricted to dendritic cells and B cells, with strong expression in mature B cells. Southern blot analysis indicated that a single copy of this gene is present. In situ hybridization on tonsillar tissue showed expression in epithelial cells and isolated cells within the germinal center. With respect to an expressed-sequence tag (EST) this cDNA could be localized to the major histocompatibility complex class I region of chromosome 6. Comparative analysis and the expression pattern of this gene suggests a function in antigen processing and presentation.

Amino Acid Sequence↗
PubMed1997

Polymerase chain reaction-based identification of a novel serpin from human dendritic cells.

A subtraction library of CD40-stimulated human tonsil dendritic cells has been constructed using a polymerase chain reaction approach adapted for low numbers of cells. From this library we identified a cDNA for a serine protease inhibitor, a serpin, which is absent from monocytes, B cells and T cells but expressed in CD40-activated monocyte- and progenitor cell-generated dendritic cells. In addition, the serpin is expressed in a lung fibroblast cell line and keratinocytes. Its mRNA is detected only in tonsil and thymus. The serpin described here reportedly functions as a megakaryocyte maturation factor in the presence of interleukin (IL)-3 and IL-11. This suggests that dendritic cells may promote the immune response by protecting IL-3 and IL-11 or other essential proteins from degradation.

Amino Acid Sequence↗
PubMed1997

Immunologic characterization of monoclonal antibodies that modulate human IgE binding to the major birch pollen allergen Bet v 1.

BACKGROUND: Bet v 1 and homologous proteins represent major allergens for almost 95% of patients allergic to tree pollen and approximately 70% of those allergic to fruits and vegetables. As yet, no continuous (sequential) IgE epitopes have been determined for Bet v 1, and evidence has accumulated that Bet v 1 IgE epitopes belong to the conformational (discontinuous) type. OBJECTIVE: A panel of 85 mouse monoclonal anti-Bet v 1 antibodies was raised as a tool with which to study the interaction of human IgE antibodies with Bet v 1. METHODS: The epitopes of selected monoclonal antibodies (mAbs) were characterized by mapping with synthetic overlapping peptides and by cross-competition experiments. Cross-reactivity of Bet v 1-specific mAbs with tree and plant food allergens was investigated by Western blotting. The influence of Bet v 1-specific mAbs on the IgE-Bet v 1 interaction was studied by competition assays with immobilized purified recombinant Bet v 1 and by basophil histamine release experiments. RESULTS: Antibodies that increased the IgE binding to Bet v 1 up to fivefold could be defined, whereas others inhibited IgE binding to Bet v 1 up to 99% and competed with the Bet v 1-induced histamine release from patients' basophils. CONCLUSION: The activity of the enhancing antibodies is interpreted as a stabilization of Bet v 1 states/IgE epitopes, which are either more accessible for certain IgE antibodies or are recognized with higher affinity. Those mAbs that competed with the Bet v 1-IgE interaction, if humanized or produced as recombinant antibody fragments, might be considered as potential tools for local allergy therapy.

Allergens↗
PubMed1997

Induction of somatic mutation in a human B cell line in vitro.

Both the B cell-surface trigger(s) and the intracellular molecular mechanism(s) of somatic hypermutation in immunoglobulin (Ig) variable region genes remain unknown, partly because of the lack of a simple and reproducible in vitro model. Here, we show that upon surface immunoglobulin cross-linking followed by co-culture with activated cloned T cells, the Burkitt's lymphoma cell line BL2 is induced to mutate its IgV(H) gene. Repeated activation of BL2 cells increased the frequency of mutation. The in vitro-induced mutations, which do not affect the IgM constant region, are point mutations distributed over the entire V(H)DJ(H) gene segment and do not show evidence of antigen-driven selection.

Amino Acid Sequence↗
PubMed1996

Somatic mutations in human Ig variable genes correlate with a partially functional CD40-ligand in the X-linked hyper-IgM syndrome.

X-linked hyper-IgM (HIGM-1) syndrome is a rare disorder resulting from mutations in the CD40-ligand (CD40L) gene. This defect is associated with normal or elevated serum levels of IgM, and with low to undetectable levels of serum IgG, IgA, and IgE. We analyzed the somatic mutation status in Ig V genes from three unrelated HIGM-1 patients by reverse-transcription PCR and sequence analysis. Two patients (B.S. and P.S.) expressed unmutated VH6 genes. In contrast, one patient (A.T.) was found to express mutated VH6 genes. Whether the presence of somatic mutations in this patient was related to a functional CD40L was assessed by deriving T cell clones from his peripheral blood cells. Upon activation, these T cell clones expressed weakly and transiently surface CD40L, and were able to induce limited isotype switch of normal native B cells, indicating residual CD40L function. Altogether, our results 1) confirm the central role played by CD40L in the generation of somatic mutation (patients B.S. and P.S.), 2) provide an unusual illustration of the relative dissociation between somatic mutation and isotype switching (patient A.T.), and 3) demonstrate a further complexity of the X-linked HIGM syndrome that may occur despite a partially functional CD40L.

B-Lymphocytes↗
PubMed1996

T cell interleukin-17 induces stromal cells to produce proinflammatory and hematopoietic cytokines.

Analysis of the cDNA encoding murine interleukin (IL) 17 (cytotoxic T lymphocyte associated antigen 8) predicted a secreted protein sharing 57% amino acid identity with the protein predicted from ORF13, an open reading frame of Herpesvirus saimiri. Here we report on the cloning of human IL-17 (hIL-17), the human counterpart of murine IL-17. hIL-17 is a glycoprotein of 155 amino acids secreted as an homodimer by activated memory CD4+ T cells. Although devoid of direct effects on cells of hematopoietic origin, hIL-17 and the product of its viral counterpart, ORF13, stimulate epithelial, endothelial, and fibroblastic cells to secrete cytokines such as IL-6, IL-8, and granulocyte-colony-stimulating factor, as well as prostaglandin E2. Furthermore, when cultured in the presence of hIL-17, fibroblasts could sustain the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These observations suggest that hIL-17 may constitute (a) an early initiator of the T cell-dependent inflammmatory reaction; and (b) an element of the cytokine network that bridges the immune system to hematopoiesis.

Amino Acid Sequence↗
PubMed1996

Four IgG anti-islet human monoclonal antibodies isolated from a type 1 diabetes patient recognize distinct epitopes of glutamic acid decarboxylase 65 and are somatically mutated.

The selective destruction by an autoimmune process of the beta cells in the pancreas is the hallmark of the type 1 insulin-dependent diabetes mellitus. What triggers islet cell-specific autoreactive T and B cells, however, remains unclear. Identification of the targets of the anti-islet cell autoantibodies frequently found in insulin-dependent diabetes mellitus patients and analysis of their sequences should provide some insights into the nature of this disease. We have combined EBV transformation with CD40 activation of peripheral B cells from one patient with insulin-dependent diabetes mellitus to isolate four B cell clones that secrete islet cell-specific autoantibodies, These four human monoclonal autoantibodies are of the IgG1 isotype, and they each recognize a different epitope of the glutamic acid decarboxylase enzyme. Analysis of their variable gene sequences shows that, while clonally unrelated, three of the four human monoclonal autoantibodies use a member of the VH4 family, and two have rearranged the same delta light chain variable gene. The IgG1 isotype of the four autoantibodies as well as the presence of somatic mutations in both heavy and light chain genes provide concrete evidence for their derivation by a T cell-dependent immune response.

Adult↗
PubMed1996

Sequential triggering of apoptosis, somatic mutation and isotype switch during germinal center development.

Using an approach similar to that used to study primary B-lymphocyte development within bone marrow and primary T-lymphocyte development within thymus, the peripheral B-cell maturation pathway within secondary lymphoid tissue (human tonsils) was analysed on the expression of discrete surface antigens. sIgD and CD38 permit the identification of four subpopulations of tonsillar B lymphocytes, including sIgD+ CD38-, sIgD+, CD38+, sIgD-CD38+ and sIgD-CD38- B cells. Further phenotypic, functional and Ig gene analysis (IgV gene sequences, expression of sterile transcripts and DNA switch circles) allowed us to conclude the following: (1) sIgM+ IgD+ CD38- B cells are naive B cells (Bm1 + 2), which carry unmutated antigen-receptors; (2) sIgM+ IgD+ CD38+ B cells are germinal center founder cells (Bm2'), which become prone to undergo apoptosis before the onset of somatic mutation; (3) sIgM-IgD+ CD38+ are germinal center B cells (Bm 3 delta), that have accumulated the highest number of somatic mutations ever reported in normal B cells; these cells may have undergone C mu-deletion by homologous recombination through sigma mu-sigma delta sequences: (4) sIgD-CD38+ CD77+ B cells are centroblasts (Bm3), in which somatic mutation machinery is activated; (5) sIgD-CD38+ CD77- B cells are centrocytes (Bm4), in which the isotype switching machinery is activated; (6) sIgD-CD38- cells (Bm5) represent somatically mutated resting memory B cells. In conclusion, human peripheral B-cell subpopulations corresponding to the differentiation stages before, during and after the triggering of apoptosis program, somatic mutation and isotype switch have been identified and isolated using a combination of surface markers.

Apoptosis↗
PubMed1996

Within germinal centers, isotype switching of immunoglobulin genes occurs after the onset of somatic mutation.

Human tonsillar B cells were separated into naive IgD+CD38-CD23- (Bm1) and IgD+CD38-CD23 (Bm2), germinal center IgD-CD38+CD23- centroblasts (Bm3) and IgD-CD38+CD77- centrocytes (Bm4) and memory IgD-CD38- (Bm5) subsets. Previous IgVH sequence analysis concluded that the triggering of somatic mutations occurs during the transition from Bm2 subset into the Bm3 subset. To determine the initiation of isotype switching, sterile transcript expression was analyzed by amplification, cloning, and sequencing. A selective sterile I gamma, I alpha, and I epsilon expression was observed at centrocyte (Bm4) stage, suggesting that isotype switch is triggered within germinal centers, after somatic mutation is initiated with centroblasts (Bm3). Finally, the high level of 5'S gamma-S mu 3' DNA switching circles observed in germinal center B cells indicates that within human tonsils, germinal center is a major location for isotype switching.

B-Lymphocytes↗
PubMed1996

Normal human IgD+IgM- germinal center B cells can express up to 80 mutations in the variable region of their IgD transcripts.

Somatic hypermutation in immunoglobulin variable region genes occurs within germinal centers. Here, we describe a subset of germinal center dark zone centroblasts that express only sIgD and have accumulated up to 80 mutations per heavy chain variable region (IgVH delta gene). Over half of the hypermutated IgVH delta sequences were found to be clonally related. This level of mutation is not observed in either IgVH gamma transcripts from the same sample or IgVH delta transcripts from peripheral blood, suggesting that these cells neither undergo isotype switch nor mature into circulating memory B cells. Optimal growth of these cells in vitro depends on CD40 ligand, T cell cytokines, and a fibroblast stroma, a combination possibly mimicking the dark zone microenvironment. Our hypothesis is that these cells may be sequestered within germinal centers, where their somatic mutation machinery is triggered. The isolation of these hypermutated B cells may represent a critical step for studying both the biology and biochemistry of somatic hypermutation.

ADP-ribosyl Cyclase↗
PubMed1995

Anti-CD40 plus interleukin-4-activated human naive B cell lines express unmutated immunoglobulin genes with intraclonal heavy chain isotype variability.

Combination of anti-CD40 antibody and interleukin-4 (IL-4) induces B cell clonal expansion reminiscent of the T-dependent proliferation following antigenic challenge in vivo. We have analyzed the usage of CH genes and the presence or absence of somatic mutations within the progeny of a single human naive B cell activated with anti-CD40 + IL-4. To address this issue, single-cell cultures of naive (sIgD+) tonsillar B lymphocytes expressing the VH1-restricted G8 idiotype were set up. After culture and RNA extraction, VH1+ Ig mRNA were reverse-transcribed, amplified by polymerase chain reaction and sequenced. A single sIgD+ B cell could generate clones expressing mu, gamma 1, gamma 3, or epsilon, illustrating that the progeny of a single cell can express different isotypes in response to the same stimulus in vitro. The rate of somatic mutations affecting the immunoglobulin variable heavy chain gene was indistinguishable from the background of errors introduced by Taq polymerase.

Antibodies, Anti-Idiotypic↗
PubMed1993

Role of cytokines in human B lymphocyte growth and differentiation.

B lymphocytes express at their surface the CD40 antigen which belongs to the NGF receptor superfamily. The crosslinking of the CD40 antigen using a mouse fibroblastic cell line expressing the human Fc receptor (Fc gamma RII/CDw32) and anti-CD40 monoclonal antibody induces resting B lymphocytes to enter a state of sustained proliferation. Addition of IL-4 to such cultures results in the generation of factor dependent long-term normal human B cell lines and in the secretion of IgE following isotype switching. Addition of IL-10 results in limited cell proliferation but most importantly in very high immunoglobulin production which results from the differentiation of B cells into plasma cells. The combination of IL-10 and TGF beta induces naive sIgD+ sIgM+ B cells to secrete IgA1 and IgA2 as a consequence of isotype switching. The extracellular domain of CD40 binds with high affinity and high specificity to a 32 kDa glycoprotein transiently expressed on activated T cells. This interaction of the CD40 antigen on B cells with its ligand on T cells represents a key step in T cell dependent B cell activation.

Animals↗
PubMedDate not supplied

Generation and characterization of a human monoclonal autoantibody that acts as a high affinity interleukin-1 alpha specific inhibitor.

Interleukin-1 (IL-1) defines two polypeptides, IL-1 alpha and IL-1 beta, that possess a wide spectrum of biological effects. Two natural antagonists of IL-1 action have been characterized: the IL-1 receptor antagonist (IL-1Ra) and a soluble form of the type II IL-1 receptor. Neutralizing autoantibodies to IL-1 alpha have also been detected in sera of healthy individuals and patients with autoimmune or inflammatory diseases. To characterize such antibodies molecularly, we attempted to generate B cell clones producing anti-IL-1 alpha human monoclonal antibody (HuMAb) by combining Epstein-Barr virus-immortalization and CD40-activation of B lymphocytes from individuals with circulating anti-IL-1 alpha. We describe herein the generation and properties of a natural IgG4/kappa anti-IL-1 alpha monoclonal autoantibody, HuMAb X3, that bound specifically to human IL-1 alpha, but not to IL-1 beta and IL-1Ra, with a high affinity (Kd = 1.2 x 10(-10)M). HuMAb X3 inhibited IL-1 alpha binding to IL-1 receptors and neutralized biological activities of both recombinant and natural forms of IL-1 alpha. A recombinant form of HuMAb X3 was found to display identical specific IL-1 alpha antagonism. The presence of somatic mutations within X3 variable regions suggests an antigen-driven affinity maturation. This study extends the demonstration of the presence of high affinity neutralizing anti-IL-1 alpha autoantibodies that can function as a third type of IL-1 antagonist.

Amino Acid Sequence↗