Analysis of somatic hypermutations of immunoglobulin gene rearrangements in childhood acute lymphoblastic leukemia.

The current study investigated the presence, frequency, and status of somatic hypermutations as well as their role in children with B lineage ALL. The obtained sequences were analyzed using IMGT/V-QUEST. Totally, 150 IGH sequences were evaluated; 139 from the 111 patients at the time of diagnosis and 11 from 8/111 patients at the time of relapse. The findings of the current report revealed the presence of somatically mutated V genes in childhood B lineage ALL. A higher frequency of somatic hypermutations was noted in unproductive rearrangements and was generally attributed to nucleotide mutation type, region, and IGHV gene subgroup biases.

Characterization of human FCGR3B*02 (HNA-1b, NA2) cDNAs and IMGT standardized description of FCGR3B alleles.

The low-affinity Fc gamma receptor IIIb (Fc gamma RIIIb and CD16b) is constituted by a unique FCGR3B polypeptide chain that comprises two extracellular immunoglobulin-like domains, and is expressed as a glycosylphosphatidyl inositol-anchored receptor on the neutrophils. The FCGR3B chain bears allotypes that define the human neutrophil antigen-1 (HNA-1 and NA) system involved in major post-transfusional reactions. FCGR3B is highly homologous to FCGR3A, which is expressed as a transmembrane receptor on natural killer cells and monocytes/macrophages. Its transcription products were not yet fully characterized. In the present work, we sequenced FCGR3B cDNAs with complete 3' untranslated region from purified granulocytes of HNA-1b/HNA-1b (NA2/NA2) genotyped donors. We characterized two FCGR3B cDNAs of different lengths corresponding to two polyadenylation sites. This result was corroborated by data raised by serial analysis of gene expression (SAGE). FCGR3B allele polymorphisms, from this article [FCGR3B*02 (HNA-1b, NA2)] and from the literature, are described for the first time according to the IMGT standardized nomenclature and to the IMGT unique numbering for C-LIKE-DOMAIN (http://imgt.cines.fr). These rules, described in the IMGT Scientific chart, are based on the IMGT-ONTOLOGY concepts. IMGT allele alignments and IMGT Collier de Perles graphical two-dimensional representations are provided for the two Ig-like domains (or C-LIKE-DOMAINs) [D1] and [D2] of FCGR3B*02. The standardized description of FCGR3B allele polymorphisms was approved by the IMGT Nomenclature Committee (IMGT-NC) and is freely available in IMGT repertoire at IMGT, http://imgt.cines.fr.

IMGT databases, web resources and tools for immunoglobulin and T cell receptor sequence analysis, http://imgt.cines.fr.

IMGT, the international ImMunoGeneTics database((R)) (http://imgt.cines.fr), is a high-quality integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates, created in 1989, by LIGM, at the Université Montpellier II, CNRS, Montpellier, France. IMGT provides a common access to standardized data which include nucleotide and protein sequences, oligonucleotide primers, gene maps, genetic polymorphisms, specificities, 2D and 3D structures. IMGT includes several databases (IMGT/LIGM-DB, IMGT/3Dstructure-DB, IMGT/HLA-DB), Web resources ('IMGT Marie-Paule page') and interactive tools (IMGT/V-QUEST, IMGT/JunctionAnalysis). IMGT expertly annotated data and tools described in this paper are particularly useful for the analysis of the IG and TR rearrangements in leukemia, lymphoma and myeloma, and in translocations involving the antigen receptor loci. IMGT is freely available at http://imgt.cines.fr.

IMGT, the international ImMunoGeneTics database.

IMGT, the international ImMunoGeneTics database (http://imgt.cines. fr:8104 ), is a high-quality integrated database specialising in Immunoglobulins (Ig), T cell Receptors (TcR) and Major Histocompatibility Complex (MHC) molecules of all vertebrate species, created in 1989 by Marie-Paule Lefranc, Université Montpellier II, CNRS, Montpellier, France (lefranc@ligm.igh.cnrs.fr ). At present, IMGT includes two databases: IMGT/LIGM-DB, a comprehensive database of Ig and TcR from human and other vertebrates, with translation for fully annotated sequences, and IMGT/HLA-DB, a database of the human MHC referred to as HLA (Human Leucocyte Antigens). The IMGT server provides a common access to expertized genomic, proteomic, structural and polymorphic data of Ig and TcR molecules of all vertebrates. By its high quality and its easy data distribution, IMGT has important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutic approaches (antibody engineering), genome diversity and genome evolution studies. IMGT is freely available at http://imgt.cines.fr:8104. The IMGT Index is provided at the IMGT Marie-Paule page (http://imgt.cines.fr:8104/textes/IMGTindex.html).

The human immunoglobulin heavy diversity (IGHD) and joining (IGHJ) segments.

The 'Human Immunoglobulin Heavy Diversity (IGHD) and Joining (IGHJ) segments', fifth report of the 'IMGT Locus on Focus' section, comprises six tables entitled: (1) 'Human germline IGHD segments at 14q32.33'; (2) 'Human IGHD alleles'; (3) 'Human germline IGHJ segments at 14q32.33'; (4) 'Human IGHJ alleles'; (5) 'Human germline IGHD orphons on chromosome 15 (15q11.2)'; (6) 'Correspondence between the different human IGHD nomenclatures', and two figures: (1) 'Protein display of human IGH D-REGIONs'; (2) 'Protein display of human IGH J-REGIONs'. These tables and figures are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cnusc.fr:8104) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.

IMGT, the international ImMunoGeneTics database.

IMGT, the international ImMunoGeneTics database (http://imgt.cnusc. fr:8104), is a high-quality integrated database specialising in Immunoglobulins (Ig), T cell Receptors (TcR) and Major Histocompatibility Complex (MHC) molecules of all vertebrate species, created in 1989 by Marie-Paule Lefranc, Université Montpellier II, CNRS, Montpellier, France (lefranc@ligm.igh.cnrs.fr). IMGT comprises three databases: LIGM-DB, a comprehensive database of Ig and TcR, MHC/HLA-DB, and PRIMER-DB (the last two in development); a tool, IMGT/DNAPLOT, developed for sequence analysis and alignments; and expertised data based on the IMGT scientific chart, the IMGT repertoire. By its high quality and its easy data distribution, IMGT has important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutic approaches (antibody engineering), genome diversity and genome evolution studies. IMGT is freely available at http://imgt.cnusc. fr:8104

The human immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments.

'Human Immunoglobulin Kappa Variable (IGKV) Genes and Joining (IGKJ) Segments', second report of the 'IMGT Locus on Focus' section, comprises five tables entitled: (1) 'Number of human germline IGKV genes at 2p12 and potential repertoire'; (2) 'Human germline IGKV gene table'; (3) 'Human IGKV allele table'; (4) 'Human germline IGKJ table' and (5) 'Human IGKJ allele table'. These tables are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cnusc.fr:8104) created by Marie-Paule Lefranc, CNRS, Université Montpellier II, Montpellier, France.

The mouse (Mus musculus) immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments.

The 'Mouse (Mus musculus) Immunoglobulin Kappa Variable (IGKV) Genes and Joining (IGKJ) Segments', third report of the 'IMGT Locus on Focus' section, comprises four tables entitled: (1) 'Mouse (Mus musculus) germline IGKV gene table'; (2) 'Correspondence between Mouse (Mus musculus) IMGT IGKV subgroups and previous designations'; (3) 'Mouse (Mus musculus) germline IGKJ table', and (4) 'Mouse (Mus musculus) IGKJ allele table'. These tables are available at the IMGT Marie-Paule page from IMGT, the international ImMunoGeneTics database (http://imgt.cnusc.fr:8104) created by Marie-Paule Lefranc, CNRS, Université Montpellier II, Montpellier, France.

PreB1 (CD10-) acute lymphoblastic leukemia: immunophenotypic and genomic characteristics, clinical features and outcome in 38 adults and 26 children. The Groupe dEtude Immunologique des Leucémies.

The less differentiated stage (CD10-) of B-lineage acute lymphoblastic leukaemia (ALL) described as preB1-ALL in the GEIL nomenclature, accounts for less than 10% of ALL. It is classically considered to be associated with translocation (4;11)(q21;q23), and to have a poor prognosis. We report an extensive immunophenotypic, genomic and clinical study of a series of 64 preB-1 ALL patients, representing 6.3% of a cohort of consecutive ALLs. The engagement of preB1-ALL cells in the B-lineage was confirmed by their B-lineage score, equal to or higher than 2. In addition, more than 90% of the cases tested showed rearranged IGH genes. Translocation (4;11) was the most frequent karyotypic anomaly seen, but only accounted for 24% of the preB1-ALL cases tested. Expression of the myeloid associated antigen CD15 was also found with high incidence in this subset. Clinical and biological features at presentation showed more significant differences between preB1- and T-ALL than between preB1- and preB2-ALL (CD10+). However, outcome characteristics of the 22 children with preB1-ALL confirmed the worse prognosis of this entity.

IMGT, the International ImMunoGeneTics database.

IMGT, the international ImMunoGeneTics database, is an integrated database specialising in Immunoglobulins (Ig), T cell Receptors (TcR) and Major Histocompatibility Complex (MHC) of all vertebrate species, created by Marie-Paule Lefranc, CNRS, Montpellier II University, Montpellier, France (lefranc@ligm.crbm.cnrs-mop.fr). IMGT includes three databases: LIGM-DB (for Ig and TcR), MHC/HLA-DB and PRIMER-DB (the last two in development). IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 23 000 Immunoglobulin and T cell Receptor sequences from 78 species. MHC/HLA-DB contains Class I and Class II Human Leucocyte Antigen alignment tables. An IMGT tool, DNAPLOT, developed for Ig, TcR and MHC sequence alignments, is also available. IMGT works in close collaboration with the EMBL database. IMGT goals are to establish a common data access to all immunogenetics data, including nucleotide and protein sequences, oligonucleotide primers, gene maps and other genetic data of Ig, TcR and MHC molecules, and to provide a graphical user friendly data access. IMGT has important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT is freely available at http://imgt.cnusc.fr:8104

Detection of antigen receptor gene rearrangements in lymphoproliferative malignancies by fluorescent polymerase chain reaction.

Monoclonal rearrangements of antigen receptor genes in lymphoproliferative diseases are characterized by the specific sequence and the length of their junctional region, which can be used as markers of the proliferating clone. PCR techniques have greatly simplified routine detection of monoclonal rearrangements. But on the one hand, identification of the sequences requires sequencing methods and on the other hand, sizing of rearrangements by conventional analysis of PCR products on agarose or nondenaturing polyacrylamide gels may be uncertain. We have developed an approach based on amplification of rearranged IGH, TCRG and TCRD locus by fluorescent PCR associated to a computerized analysis of generated PCR products allowing their objective sizing. We tested this method on DNA samples from patients with acute lymphoblastic leukemia and chronic lymphocytic leukemia, whose pattern of IGH and TCRG rearrangements had been previously identified by Southern blot techniques. TCRG-PCR assay allowed detection of 100% of rearranged samples. No false-negative results were found but a high rate (60%) of Southern-negative and PCR-positive samples were identified. TCRD PCR-assay detected VD1-JD1 or VD2-D2/3 rearrangements in both acute lymphoblastic leukemia and chronic lymphocytic leukemia samples. IGH PCR assay permitted detection of all known rearranged samples. The sensitivity of these three different PCR assays (1% leukemic cells) was equivalent to that of other published PCR protocols. These results show the validity and reliability of the fluorescent PCR method for routine detection of IGH, TCRG and TCRD rearrangements. Sizing of PCR products by computerized analysis was also validated. It provides additional information on rearrangement patterns in lymphoproliferative diseases, as clonal rearrangements can be recognized by their size. This can be of great interest in various circumstances, particularly for detection and follow-up of oligoclonality.

IMGT, the international ImMunoGeneTics database: a new design for immunogenetics data access.

IMGT, the international ImMunoGeneTics database is an integrated database specializing in Immunoglobulins (Ig), T-cell receptors (TcR) and MHC molecules of all vertebrate species, created by Marie-Paule Lefranc, University of Montpellier, CNRS, Montpellier, France (Nucleic Acids Research, Database issue, Vol 26, January 1998). IMGT includes three databases: LIGM-DB (for Ig and TcR), MHC/HLA-DB and IMGT/PRIMER-DB (an Ig, TcR and MHC-related primer database), the last two in development. IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 24.000 Immunoglobulin and T cell Receptor sequences from 81 different species. MHC/HLA-DB contains class I and class II Human Leucocyte Antigen alignment tables. An IMGT tool, DNAPLOT, developed for Ig, TcR and MHC sequence analysis, is also available. IMGT goals are to establish a common data access to all immunogenetics data, including nucleotide and protein sequences, oligonucleotide primers, gene maps and other genetic data of Ig, TcR and MHC molecules, from all species, and to provide a graphical user friendly data access. IMGT has important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. In this paper, we describe our approach for the data modelisation, the automation of the annotation procedure and control of data quality in LIGM-DB database. IMGT is freely available on the CNUSC WWW server at Montpellier: http://imgt.cnusc.fr: 8104 (contact: Denys.Chaume@cnusc.fr) and on the EBI servers: http://www.ebi.ac.uk/imgt (contact: malik@ebi.ac.uk) and ftp.ebi.ac.uk/pub/databases/imgt. LIGM-DB users are encouraged to report errors or suggestions to giudi@ligm.crbm.cnrs-mop.fr. IMGT initiator and coordinator: Marie-Paule Lefranc, lefranc@ligm.crbm.cnrs-mop.fr. (fax: +33(0)467040231).

B lymphocytes of xeroderma pigmentosum or Cockayne syndrome patients with inherited defects in nucleotide excision repair are fully capable of somatic hypermutation of immunoglobulin genes.

Recent experiments have strongly suggested that the process of somatic mutation is linked to transcription initiation. It was postulated that a mutator factor loads onto the RNA polymerase and, during elongation, causes transcriptional arrest that activates DNA repair, thus occasionally causing errors in the DNA sequence. We report the analysis of the role of one of the known DNA repair systems, nucleotide excision repair (NER), in somatic mutation. Epstein-Barrvirus-transformed B cells from patients with defects in NER (XP-B, XP-D, XP-V, and CS-A) were studied. Their heavy and light chain genes show a high frequency of point mutations in the variable (V), but not in the constant (C) regions. This suggests that these B cells can undergo somatic hypermutation despite significant defects in NER. Thus, it is doubtful that NER is an essential part of the mechanism of somatic hypermutation of Ig genes. As an aside, NER seems also not involved in Ig gene switch recombination.

A simple luciferase assay for signal transduction activity detection of epidermal growth factor displayed on phage.

Studies on receptor-ligand interactions are important for the design of agonists or antagonists of natural ligands. We developed a luciferase reporter assay to screen epidermal growth factor receptor (EGFR) binding molecules rapidly for their ability to stimulate or inhibit signal transduction. Human EGF displayed on fd filamentous phage presented an activity similar to soluble EGF when tested for binding to the EGFR, for induction of cell cycle progression or in the luciferase assay. Two libraries of human EGF variants displayed on phage were constructed in which the aspartic acid residue at position 46 or the arginine residue at position 41 were randomised. EGF mutants displayed on phage were screened in parallel for binding to the EGFR using an ELISA assay and for transducing activity using the luciferase assay. Regarding the 46 position, most of the mutants retained the ability to bind the EGFR and their transducing activity corresponded perfectly with their binding. For the more crucial 41 position, only the wild-type EGF was able to bind the EGFR. Our approach allowed a simple determination of crucial positions and paved the way for identification of agonists with altered transduction activity.

IMGT, the international ImMunoGeneTics database.

IMGT, the international ImMunoGeneTics database, is an integrated database specializing in immunoglobulins, T-cell receptors (TcR) and major histocompatibility complex (MHC) of all vertebrate species, initiated and co-ordinated by Marie-Paule Lefranc, CNRS, Montpellier II University, Montpellier, France (lefranc@ligm.crbm.cnrs-mop.fr). IMGT includes two databases: LIGM-DB (for immunoglobulins and TcR) and MHC/HLA-DB. IMGT comprises expertly annotated sequences and alignment tables. LIGM-DB contains more than 19 000 immunoglobulin and TcR sequences from 78 species. MHC/HLA-DB contains class I and class II human leukocyte antigen alignment tables. An IMGT tool, DNAPLOT, developed for immunoglobulins, TcR and MHC sequence alignments, is also available. IMGT works in close collaboration with the EMBL database. IMGT goals are to establish a common data access to all immunogenetics data, including sequences, oligonucleotide primers, gene maps and other genetic data of immunoglobulins, TcR and MHC molecules, and to provide a graphical user-friendly data access. IMGT will have important implications in medical research (repertoire in autoimmune diseases, AIDS, leukemias, lymphomas), therapeutical approaches (antibody engineering), genome diversity and genome evolution studies. IMGT can be accessed at http://imgt.cnusc.fr:8104 and http://www.ebi.ac.uk/IMGT

Cassette baculovirus vectors for the production of chimeric, humanized, or human antibodies in insect cells.

Plasmid cassette-transfer vectors pBHuC chi and pBHuC gamma l have been designed which enable the construction of recombinant baculoviruses directing the co-expression of complete immunoglobulin in insect cells. We describe the application of these vectors for the expression of a human/mouse chimeric monoclonal antibody of potential immunosuppressive clinical value derived from a mouse anti-human CD29 monoclonal antibody (Mu-K20). The chimeric K20 light and heavy chains produced in sf9 insect cells were correctly processed and assembled into a normal immunoglobulin which is secreted into the culture medium of infected cells. The chimeric mAb Ch-K20-sf9 reproduces in vitro the functional properties of the parental mouse K20, including affinity and inhibition of lymphocyte proliferation. These results demonstrate that the baculovirus/insect cell expression system is suitable for the expression of fully active monoclonal antibodies of therapeutic value. Our generic cassette approach makes this system a very flexible and convenient one for the rapid production of either chimeric, humanized or human mAb with heavy and light chains of any isotype.

First study of immunoglobulin and T cell receptor gene rearrangements in chronic and acute lymphoblastic leukemias from Tunisia.

We report the first characterization at the immunological and molecular level of 12 cases of chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) from Tunisia. Our results show biallelic IgH gene rearrangement in B-CLL (6/6). A high ratio of T-ALL (4/6) was observed in Tunisian ALL leukemias. One T-ALL expressed CD10 (common ALL) which has already been found in some other cases of T-ALL. We report the occurrence of T cell receptor (TCR) beta and/or gamma gene rearrangements in two precursor B-ALL patients who had normally rearranged Ig genes. In one precursor B-ALL case, multiple rearranged IgH and TCR gamma bands allowed the identification of three clones. Such an oligoclonal ALL is interesting since only rare biclonal TCR beta or gamma gene rearrangements have been described.