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M Fougereau

Publications and source records attributed to M Fougereau.

At least 37 records · Page 2Linked to original sources
PubMed1994

The human pre-B cell receptor: structural constraints for a tentative model of the pseudo-light (psi L) chain.

In human pre-B cells, the mu chain is associated with a surrogate light chain composed of the lambda-like and Vpre-B gene products. This pre-B cell receptor presumably triggers early steps of B cell differentiation, We have determined the NH2-terminal amino acid sequence of the lambda-like chain, showing that the mature chain results from the cleavage of a leader segment of 44 residues, leaving a polypeptide of 169 amino acids having partial features of the Ig light chain domains, with the exception of the first 50 amino acid NH2-terminal region. We have completed the nucleotide sequence of the Vpre-B gene, which appears to contain 126 residues in its mature form of which the 24 COOH-terminal portion was not Ig-related. Analysis of transfectants has provided direct evidence that lambda-like and Vpre-B chains assemble together even in the absence of heavy chain, prompting the search for a structural basis of this interaction. Comparison with the domain organization of the regular Ig lambda chain suggests that most of the psi L chain can be accommodated within a CL-VL-like structure, with an extra "subdomain" contributed by the non-Ig-like portions of both the lambda-like and Vpre-B polypeptides.

Amino Acid Sequence↗
PubMed1993

Reconstitution of human immunoglobulin VH repertoire after bone marrow transplantation mimics B-cell ontogeny.

Reconstitution of the human Ig repertoire after allogeneic and autologous bone marrow transplantation (BMT) was followed by analysis of the expression of the six VH families using in situ hybridization on BM cells at 30, 60, and 90 days postgraft. Our results indicate that during the early post-transplantation period, the expressed repertoire of VH is dramatically affected: VH3, the major expressed family in adult B cells, is decreased two- to threefold and is compensated by transient overexpression of the other families, especially VH4, VH5, and VH6. Similar results were observed in allogeneic and autologous grafted patients. Kinetics of reconstitution mimics normal repertoire development described in ontogeny, although normalization, as compared with the adult pattern, may take as long as 1 year.

Adult↗
PubMed1993

Mechanisms that generate human immunoglobulin diversity operate from the 8th week of gestation in fetal liver.

The repertoire of immunoglobulin expressed very early in human development was approached by cloning and sequencing 55 rearranged and 11 germ-line VH transcripts, after amplification by polymerase chain reaction of cDNA libraries derived from two fetal livers at 8 and 13 weeks of gestation. All families with the exception of VH2, were expressed as soon as 8 weeks, with preferential usage of certain germ-line genes. Very few somatic mutations, randomly localized, were identified. By contrast, in a series of clones derived from the same VDJ rearrangement using the VH6 family, extensive mutations had taken place, mostly accumulated in the third complementarity-determining region (CDR3) suggesting that the specialized enzymatic machinery was at hand very early during human development. Some other characteristics of the fetal repertoire also emerged, namely increased usage of JH3 and JH2, as compared to the adult pattern, where JH4 is dominant and reduced length of the D/CDR3 regions. All D gene families were identified, and their usage frequently involved D-D fusions. N diversity was present very early, and increased with age. Identification of germ-line transcripts pertaining to all six VH families including pseudogenes, in the E55 library, revealed a population very different as compared to rearranged gene transcripts. This suggests that a large portion of VH locus is accessible for transcription, bringing no evidence of correlation between preferential rearrangement of a given VH gene and its localization in the locus.

Antibody Diversity↗
PubMed1993

Structure, biosynthesis, and transduction properties of the human mu-psi L complex: similar behavior of preB and intermediate preB-B cells in transducing ability.

In human preB cells, the mu chain is associated with a surrogate light chain composed of the VpreB and lambda-like gene products. Using anti-peptide antibodies directed against VpreB and lambda-like epitopes, we identified the discrete components of the mu-psi L (pseudo-light) chain complex in various preB cell lines, and in intermediate preB-B cells that co-expressed the psi L and the kappa chain. The lambda-like gene product was identified as a single band at 20 kDa, disulfide linked to the mu chain. VpreB was detected at 16 kDa and, depending upon the cell lines, an isoform of this polypeptide was also present at 15 kDa. In addition, lambda-like--VpreB chain complexes not associated with mu were identified both in cell lysates and culture supernatants. Pulse-chase experiments indicated that VpreB was transiently associated with two new polypeptides of molecular weights 17.5 and 36 kDa. Expression of mu-psi L and co-expression of mu-psi L and microL at the surface of preB and intermediate preB-B cells respectively was detected by cytofluorimetry. The signal transduction ability of the complex in both types of cells was shown by measuring the calcium mobilization and the phosphorylation of tyrosyl residues upon stimulation by anti-mu. Signal events were similar in both cases, but differed from those induced in a mature B cell line. This points to a definite function of the preB cell receptor and suggests that the intermediate preB-B cell line still lacks some molecular components that condition initiation of a mature B cell transduction signal.

Amino Acid Sequence↗
PubMed1993

Bone marrow cells in X-linked agammaglobulinemia express pre-B-specific genes (lambda-like and V pre-B) and present immunoglobulin V-D-J gene usage strongly biased to a fetal-like repertoire.

Expression of Ig and Ig-related genes has been studied in bone marrow cells from two patients with severe form of X-linked agammaglobulinemia (XLA). Phenotypic analysis revealed the presence of pre-B cells, in the absence of mature B cell markers. The pre-B-specific genes, lambda-like and V pre-B, were normally transcribed. Sequence analysis of 48 distinct V-D-J cDNA clones directly derived from XLA bone marrow cells indicated that they had characteristics of an early fetal pre-B repertoire. All VH families were identified, with a strong bias in the gene usage: a few VH genes were largely overexpressed, either germline or slightly mutated; most genes had been located 3' of the VH locus and were also used in fetal liver (8-13 wk of gestation). Short D regions, (resulting from D-D fusion, making usage of all D genes in both orientations with utilization of the three reading frames), restricted N diversity, and a fetal JH usage pattern were also observed. Taken together, our data suggest that the XLA defect does not alter V-D-J rearrangements nor the expression of mu, lambda-like, and V pre-B transcripts and most likely results in a poor efficiency of some critical steps of the B cell maturation.

Adult↗
PubMed1992

IGM kappa/lambda EBV human B cell clone: an early step of differentiation of fetal B cells or a distinct B lineage?

In agreement with the clonal theory, one B lymphocyte synthesizes one antibody due to allelic and isotypic exclusion. We analyzed an EBV B-cell clone, E29.1, derived from an 11 week-old embryo, and secreting both IgM kappa and IgM lambda. Structural analysis of produced IgM, indicated that lambda-containing pentamers could be considered hybrid molecules, expressing both the kappa and lambda. chains, with a kappa/lambda ratio between 5 and 10. It was also found that 60% of the lambda chains were secreted in free form, presumably as a result of a better affinity of mu chains for kappa chains. The sequence of the three transcripts had an entirely ORF (Open Reading Frame), and were very close to germline sequences, with, however, an additional codon between V kappa and J kappa gene which has never been described in adult myeloma protein or cDNA human sequence. This observation is suggestive of N diversity taking place in kappa chains. The possible role of Kde (kappa deleting element) recombination onto kappa/lambda locus activation was analyzed on a collection of 23 lambda clones. The status of rearrangement of kappa genes indicated that 35% of these clones had retained, at least, one kappa allele without the Kde recombination, four lambda clones had one kappa allele in germline configuration. Different hypotheses of maturation from pre-B cell to B cell with activation of light chain genes are discussed.

Aging↗
PubMed1992

Organization and expression of the pseudo-light chain genes in human B-cell ontogeny.

In pre-B cells, the mu chain is expressed at the cell surface in association with a "light chain surrogate" encoded by the V pre-B and the lambda-like genes. This mu-psi-L complex presumably triggers early steps of the B cell differentiation, possibly by controlling the Ig gene rearrangements. In the humans, the lambda-like complex contains 3 genes, located in the 22q11.2-q12.3 band, telomeric to the IGCL locus, with which they share a similar organization, pointing to a common genetic origin. Only one lambda-like gene, 14.1, is functional and specifically expressed with V pre-B in pre-B cells. This expression starts in cells which still have the IGH locus in germline configuration (pro-B stage) and ceases as soon as the IGL loci rearrange. These pre-B specific transcripts provide useful markers of cells of the B lineage in both physiological and pathological situations.

B-Lymphocytes↗
PubMed1991

Lambda-like and V pre-B genes expression: an early B-lineage marker of human leukemias.

V pre-B and lambda-like genes are selectively expressed in human pre-B cells and encode polypeptide chains that associate in a mu-pseudolight chain complex that may regulate some crucial steps of early B-cell differentiation. We have followed by polymerase chain reaction and Northern blot analysis the expression of these "pre-B-specific" genes in correlation with the status (rearranged v germline) of Ig gene loci (H, kappa, lambda) in a panel of 32 leukemias pertaining mostly to the B lineage and including a number of ambiguously characterized samples. All cells that had rearranged the H locus only expressed V pre-B and lambda-like transcripts, in agreement with a pre-B status. In this group, some biphenotypic leukemias (mostly My/B) might, in fact, be already engaged in the B lineage. Rearrangement of V kappa or V lambda loci correlated with the disappearance of the pre-B gene products. In a pre-B acute lymphoblastic leukemia cell line that was induced to mature to the B-cell stage in culture upon kappa gene rearrangement, the mu-pseudolight chain complex was actually replaced by the classical mu-kappa molecule. Finally, V pre-B and lambda-like genes were found expressed in two leukemic cells that had retained all Ig loci in germline configuration. This finding raises the possibility of having an early pro-B progenitor in which V pre-B and lambda-like products associate with a H chain surrogate in a complex that would trigger an early event of B-cell differentiation such as the H locus rearrangements.

Antigens, CD↗
PubMed1991

Ig repertoire of human polyspecific antibodies and B cell ontogeny.

A total of 463 EBV Ig-secreting clones were derived from embryonic tissues, cord blood, and adult peripheral blood. Subcloning and analysis of the H and K loci (germline vs rearranged DNA status) of 44 primary clones insured clonality in at least 92% of cases. Whatever the cell origin, a somewhat constant proportion of clones (i.e., 11 to 16%) expressed polyspecific antibodies when tested on a panel of nine Ag, including self-Ag. The VH and VK repertoires have been studied using VH1-VH6 and VK1-VK4 family-specific probes. For all EBV clones the VH and VK utilization was similar to that of the normal untransformed population. A correlation was observed between the level of expression and the gene number for VH, whereas a clear distortion appeared for VK. Moreover, the usage pattern of VH and VK families of the polyspecific clones did not significantly differ from that of clones of unknown specificity, suggesting that polyspecificity was not linked to a restricted repertoire.

Adult↗
PubMed1991

Early occurrence of immunoglobulin isotype switching in human fetal liver.

A cDNA library prepared from a human fetal liver of the first trimester of gestation was screened with Ig C mu, C gamma, C kappa and C lambda probes. Ten heavy chain clones were isolated and characterized by restriction mapping and partial sequencing. The absence of Ig light chain clone and the presence of pre-B-specific lambda-like transcripts suggest that the immune compartment of this cDNA library was mostly derived from pre-B cells. Three transcripts of mu, gamma 2 and gamma 4 isotypes contained a V-D-J-C region with an open reading frame and used members of the VHIV, VHIII and VHI families, respectively. Seven clones were derived from sterile transcripts, one C mu and six C gamma. In addition to C mu exons, the sterile mu transcript contained the 5' flanking germline region. By contrast, the gamma sterile transcripts used a 5' sequence that was spliced from the I gamma 1 region onto the first C gamma 1 exon. In addition several of these transcripts were derived from alternative splicing. The simultaneous expression of both sterile and functional gamma transcripts suggests that the switch mechanism operates in normal fetal liver very early in ontogeny.

Antigenic Variation↗
PubMed1991

The human pre-B-specific lambda-like cluster is located in the 22q11.2-22q12.3 region, distal to the IgC lambda locus.

The chromosomal location of the lambda-like gene cluster, a gene family selectively expressed in human pre-B cells, was analyzed by in situ hybridization with a probe specific for the lambda-like genes. This cluster mapped in the q11.2-q12.3 region of chromosome 22. The use of Burkitt lymphoma and myelogenous leukemia cell lines with translocations in the 22q11 region led to a refinement in the location according to the following order: cen----BCRL2, VpreB, IgV lambda 1----BCRL4, IgV lambda----IgC lambda----BCR----BCRL3, lambda-like----tel. Unlike those of the mouse system, the pre-B-specific genes VpreB and lambda-like do not belong to the same transcriptional unit.

Animals↗
PubMed1991

Organization and expression of the lambda-like genes that contribute to the mu-psi light chain complex in human pre-B cells.

Lambda-like genes encode a polypeptide chain that associates with VpreB and mu chain in the so-called mu-psi light chain complex specifically expressed in pre-B cells. In humans, the lambda-like gene cluster contains three genes, termed 14.1, 16.1, and F lambda 1. The 14.1 gene contains three exons and was previously sequenced. The 16.1 and F lambda 1 have been isolated from cosmid libraries. They both contain only the exons 2 and 3 that appear highly homologous to their 14.1 counterparts. The lambda-like gene cluster has been mapped on chromosome 22, close and distal to the BCR gene, in the order (14.1; F lambda 1) and 16.1. Hybridization with the 14.1 exon 1 probe did not detect an equivalent on 16.1 and F lambda 1 genes, but instead, only revealed the X6 gene, that was previously identified 5 kb upstream of the IGLC locus. It appears, therefore, that the gene organization of the lambda and lambda-like loci is strikingly similar, with a three-exon-containing gene 5' of a series of J lambda-C lambda or J lambda-C lambda-like tandemly organized genes. Analysis of the transcripts in a 8 week old human fetal liver clearly indicated that 14.1 was the only functional gene of the lambda-like cluster. Various forms of transcripts resulting from alternate splicing have been characterized. The major component consisted of a full-length (exons 1-2-3) mRNA, whereas a minor (1-3) transcript was identified. Only the full-length transcript could encode a functional polypeptide chain corresponding to the 22 kDa light chain surrogate.

Amino Acid Sequence↗
PubMed1991

Individual germinal centres of myasthenia gravis human thymuses contain polyclonal activated B cells that express all the Vh and Vk families.

Using in situ hybridization, we analysed the immunoglobulin repertoire expressed by the B cells present in myasthenia gravis thymuses from four patients. B cells, mostly in activated state, were clustered in germinal centres, in which multiple isotypes were identified. A majority of cells expressed IgG as compared with IgM, with a roughly similar contribution of kappa and lambda chains. Hybridization with the six VH and the 4 VK human family probes was observed in serial sections, providing additional evidence that individual germinal centres were polyclonal. The thymic B cell repertoire closely reflected the VH and the VK family usage of normal peripheral blood lymphocytes with the preferential utilization of VH3, VK1 and VK3.

Adult↗
PubMed1990

Poly(Glu60Ala30Tyr10) (GAT)-induced IgG monoclonal antibodies cross-react with various self and non-self antigens through the complementarity determining regions. Comparison with IgM monoclonal polyreactive natural antibodies.

Previous studies have shown that the antibodies of the preimmune repertoire are able to bind to various auto- and xenoantigens including chemical haptens. Sequence analysis of two such murine monoclonal IgM natural autoantibodies showed that they are encoded by unmutated germ-line variable regions of the light and heavy chain (V alpha and VH) genes which were also found in various murine immune responses, like phenyl-oxazolone, dinitrophenyl, arsonate, phosphorylcholine and influenza virus hemagglutinin. These data raised the question as to whether induced antibodies possessing germ-line sequence are also able to react with autoantigens. To study this problem, anti-poly(Glu60Ala30Tyr10) (GAT) and anti-alprenolol (Alp) monoclonal antibodies, carrying similar VH and V alpha genes and the same IgG1 isotype, were examined for their capacity to react with several self and non-self antigens. The results showed that: (a) the anti-GAT antibodies tested reacted with different autoantigens, such as murine tubulin, actin and myosin as well as trinitrophenyl (TNP) and bovine serum albumin. Similarly, one of the anti-Alp showed weak reactivities for myosin, DNA, actin and TNP; (b) in contrast two other anti-Alp antibodies did not react with any of the tested antigens. Since the major differences between the oligoreactive anti-GAT and the monoreactive anti-Alp antibodies are in the complementarity determining regions (CDR) our results suggest that the observed cross-reactions are mediated by hypervariable loops. Sequence comparison of these antibodies indicate a possible correlation between cross-reactivity and the presence of aromatic and charged amino acids in the CDR.

Amino Acid Sequence↗
PubMed1990

Human immunoglobulin VH and VK repertoire revealed by in situ hybridization.

We report in this paper the first analysis of the expression pattern of Ig VH and VK families in human adult normal peripheral B lymphocytes, by in situ hybridization using specific VH1 to VH6 and VK1 to VK4 probes, which cover the known human V gene families reported to date. The major families were VH3 and VK1, with the respective gradient VH3 greater than VH4 greater than VH1 greater than VH5 greater than VH6 greater than VH2, and VK1 greater than VK3 greater than VK4 greater than VK2. Using a large sampling of EBV clones, we found that the pattern of VH and VK family usage was similar. The expression level correlated fairly with the estimated gene number for the VH, but diverged noticeably for the K chains. Taken together with the fact that the level of light chain expression (K + lambda) was about two-fold that of heavy chains, these results suggest that the VH and the VK repertoires are not regulated by a similar selective process.

Autoradiography↗
PubMed1990

The immunoglobulin lambda-like gene cluster (14.1, 16.1 and F lambda 1) contains gene(s) selectively expressed in pre-B cells and is the human counterpart of the mouse lambda 5 gene.

A human immunoglobulin (Ig)-related gene, covering approximately 8 kb, was isolated from a cosmid genomic library, by hybridization with a C lambda probe and with a lambda-like probe. This gene was identified as 14.1 It belongs to the human lambda-like cluster which is composed of three genes (14.1, 16.1 and F lambda 1) that do not rearrange. Sequence data indicate that 14.1 is organized similarly to the mouse lambda 5 gene. It contains three exons with lengths of 69, 38, and 106 codons as compared with 65, 38, and 106 for exons 1, 2, and 3 of mouse lambda 5, respectively. The corresponding homology values were 61, 66 and 75.5%. Using a 14.1 specific probe containing exon 1, we showed that this gene was selectively expressed in human pre-B cell lines. It is likely to encode a 213-amino acid lambda-like light chain that would associate with mu chains and play an important role in the early steps of B cell differentiation.

Amino Acid Sequence↗
PubMed1990

Rapid expansion of human immunoglobulin repertoire (VH, V kappa, V lambda) expressed in early fetal bone marrow.

We have isolated pre-B- and B-cell clones after transformation by Epstein-Barr virus (EBV) of human fetal bone marrow cells between weeks 8 and 13 of gestation. These clones were characterized for immunoglobulin (Ig) chain synthesis, the status (rearranged versus germ line) of the heavy (H) chain, kappa, and lambda loci, and of the Ig mRNA transcripts by using specific variable (V), VH, V kappa, and V lambda probes covering the almost complete IgV repertoire. Mature B cells could already be identified in the 8-week-old bone marrow, with both kappa and lambda isotypes being present. Nevertheless, at this stage, most of the clones had Xhe characteristics of pre-B cells, as indicated by the presence of mu transcripts (either functional or sterile) in the absence of L chains. The kinetics of gene rearrangements were compatible with the classical scheme H----kappa----lambda. A rapid expansion of the expressed repertoire occurred between the weeks 8 and 11, with 95% of the EBV clones having the characteristics of mature B cells. V gene family usage was analyzed for the three loci and compared with the pattern of expression observed at 30 weeks of gestation and in adult clones. The "adult" pattern was rapidly acquired for the H and kappa loci, with the major subgroups being VH3 and V kappa 1. When the expression of the repertoire was "normalized," the pattern of V usage correlated fairly well with the estimated number of VH genes, but differed noticeably for the kappa chains, suggesting that the VH and V kappa repertoires are not regulated by similar processes.

Amino Acid Sequence↗