Publications by authors named "Marie-Paule Lefranc"

147 Publications

Profiling the T Cell Receptor Alpha/Delta Locus in Salmonids.

Front Immunol 2021 18;12:753960. Epub 2021 Oct 18.

Immunology Laboratory, Biomedical Research Center (CINBIO), University of Vigo, Vigo, Spain.

In jawed vertebrates, two major T cell populations have been characterized. They are defined as α/β or γ/δ T cells, based on the expressed T cell receptor. Salmonids (family ) include two key teleost species for aquaculture, rainbow trout () and Atlantic salmon ( which constitute important models for fish immunology and important targets for vaccine development. The growing interest to decipher the dynamics of adaptive immune responses against pathogens or vaccines has resulted in recent efforts to sequence the immunoglobulin (IG) or antibodies and T cell receptor (TR) repertoire in these species. In this context, establishing a comprehensive and coherent locus annotation is the fundamental basis for the analysis of high-throughput repertoire sequencing data. We therefore decided to revisit the description and annotation of TRA/TRD locus in Atlantic salmon and two strains of rainbow trout (Swanson and Arlee) using the now available high-quality genome assemblies. Phylogenetic analysis of functional TRA/TRD V genes from these three genomes led to the definition of 25 subgroups shared by both species, some with particular feature. A total of 128 TRAJ genes were identified in , the majority with a close counterpart in . Analysis of expressed TRA repertoire indicates that most TRAV gene subgroups are expressed at mucosal and systemic level. The present work on TRA/TRD locus annotation along with the analysis of TRA repertoire sequencing data show the feasibility and advantages of a common salmonid TRA/TRD nomenclature that allows an accurate annotation and analysis of high-throughput sequencing results, across salmonid T cell subsets.
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http://dx.doi.org/10.3389/fimmu.2021.753960DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8559430PMC
October 2021

Biological controls for standardization and interpretation of adaptive immune receptor repertoire profiling.

Elife 2021 05 26;10. Epub 2021 May 26.

Sorbonne Université U959, Immunology-Immunopathology-Immunotherapy (i3), Paris, France.

Use of adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread, providing new insights into the immune system with potential broad clinical and diagnostic applications. However, like many high-throughput technologies, it comes with several problems, and the AIRR Community was established to understand and help solve them. We, the AIRR Community's Biological Resources Working Group, have surveyed scientists about the need for standards and controls in generating and annotating AIRR-seq data. Here, we review the current status of AIRR-seq, provide the results of our survey, and based on them, offer recommendations for developing AIRR-seq standards and controls, including future work.
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http://dx.doi.org/10.7554/eLife.66274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154019PMC
May 2021

The T Cell Receptor (TRB) Locus in : From Sequence to Structure of the Alpha/Beta Heterodimer in the Human/Dolphin Comparison.

Genes (Basel) 2021 04 14;12(4). Epub 2021 Apr 14.

Department of Biology, University of Bari, "Aldo Moro", 70124 Bari, Italy.

The bottlenose dolphin () belongs to the Cetartiodactyla and, similarly to other cetaceans, represents the most successful mammalian colonization of the aquatic environment. Here we report a genomic, evolutionary, and expression study of T cell receptor beta (TRB) genes. Although the organization of the dolphin TRB locus is similar to that of the other artiodactyl species, with three in tandem D-J-C clusters located at its 3' end, its uniqueness is given by the reduction of the total length due essentially to the absence of duplications and to the deletions that have drastically reduced the number of the germline TRBV genes. We have analyzed the relevant mature transcripts from two subjects. The simultaneous availability of rearranged T cell receptor α (TRA) and TRB cDNA from the peripheral blood of one of the two specimens, and the human/dolphin amino acids multi-sequence alignments, allowed us to calculate the most likely interactions at the protein interface between the alpha/beta heterodimer in complex with major histocompatibility class I (MH1) protein. Interacting amino acids located in the complementarity-determining region according to IMGT numbering (CDR-IMGT) of the dolphin variable V-alpha and beta domains were identified. According to comparative modelization, the atom pair contact sites analysis between the human MH1 grove (G) domains and the T cell receptor (TR) V domains confirms conservation of the structure of the dolphin TR/pMH.
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http://dx.doi.org/10.3390/genes12040571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8070946PMC
April 2021

Genomic analysis of a second rainbow trout line (Arlee) leads to an extended description of the IGH VDJ gene repertoire.

Dev Comp Immunol 2021 05 12;118:103998. Epub 2021 Jan 12.

Université Paris-Saclay, INRAE, UVSQ, VIM, 78350, Jouy-en-Josas, France. Electronic address:

High-throughput sequencing technologies brought a renewed interest for immune repertoires. Fish Ab and B cell repertoires are no exception, and their comprehensive analysis can both provide new insights into poorly understood immune mechanisms, and identify markers of protection after vaccination. However, the lack of genomic description and standardized nomenclature of IG genes hampers accurate annotation of Ig mRNA deep sequencing data. Complete genome sequences of Atlantic salmon and rainbow trout (Swanson line) recently allowed us to establish a comprehensive and coherent annotation of Salmonid IGH genes following IMGT standards. Here we analyzed the IGHV, D, and J genes from the newly released genome of a second rainbow trout line (Arlee). We confirmed the validity of salmonid IGHV subgroups, and extended the description of the rainbow trout IGH gene repertoire with novel sequences, while keeping nomenclature continuity. This work provides an important resource for annotation of high-throughput Ab repertoire sequencing data.
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http://dx.doi.org/10.1016/j.dci.2021.103998DOI Listing
May 2021

IMGT Biocuration and Comparative Analysis of and TRA/TRD Loci.

Genes (Basel) 2020 12 28;12(1). Epub 2020 Dec 28.

IMGT®, The International ImMunoGeneTics Information System®, Institut de Génétique Humaine (IGH), Centre National de la Recherche Scientifique (CNRS), Université de Montpellier (UM), 34000 Montpellier, France.

The adaptive immune response provides the vertebrate immune system with the ability to recognize and remember specific pathogens to generate immunity, and mount stronger attacks each time the pathogen is encountered. T cell receptors are the antigen receptors of the adaptive immune response expressed by T cells, which specifically recognize processed antigens, presented as peptides by the highly polymorphic major histocompatibility (MH) proteins. T cell receptors (TR) are divided into two groups, αβ and γδ, which express distinct TR containing either α and β, or γ and δ chains, respectively. The TRα locus (TRA) and TRδ locus (TRD) of bovine () and the sheep () have recently been described and annotated by IMGT biocurators. The aim of the present study is to present the results of the biocuration and to compare the genes of the TRA/TRD loci among these ruminant species based on the repertoire. The comparative analysis shows similarities but also differences, including the fact that these two species have a TRA/TRD locus about three times larger than that of humans and therefore have many more genes which may demonstrate duplications and/or deletions during evolution.
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http://dx.doi.org/10.3390/genes12010030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7824213PMC
December 2020

Antimalarial antibody repertoire defined by plasma IG proteomics and single B cell IG sequencing.

JCI Insight 2020 11 19;5(22). Epub 2020 Nov 19.

Laboratory of Malaria Immunology and Vaccinology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland, USA.

Plasma antimalarial Ab can mediate antiparasite immunity but has not previously been characterized at the molecular level. Here, we develop an innovative strategy to characterize humoral responses by integrating profiles of plasma immunoglobulins (IGs) or Abs with those expressed on B cells as part of the B cell receptor. We applied this strategy to define plasma IG and to determine variable (V) gene usage after vaccination with the Plasmodium falciparum zygote antigen Pfs25. Using proteomic tools coupled with bulk immunosequencing data, we determined human antigen-binding fragment [F(ab')2] peptide sequences from plasma IG of adults who received 4 doses of Pfs25-EPA/Alhydrogel. Specifically, Pfs25 antigen-specific F(ab')2 peptides (Pfs25-IG) were aligned to cDNA sequences of IG heavy (IGH) chain complementarity determining region 3 from a data set generated by total peripheral B cell immunosequencing of the entire vaccinated population. IGHV4 was the most commonly identified IGHV subgroup of Pfs25-IG, a pattern that was corroborated by V heavy/V light chain sequencing of Pfs25-specific single B cells from 5 vaccinees and by matching plasma Pfs25-IG peptides and V-(D)-J sequences of Pfs25-specific single B cells from the same donor. Among 13 recombinant human mAbs generated from IG sequences of Pfs25-specific single B cells, a single IGHV4 mAb displayed strong neutralizing activity, reducing the number of P. falciparum oocysts in infected mosquitoes by more than 80% at 100 μg/mL. Our approach characterizes the human plasma Ab repertoire in response to the Pfs25-EPA/Alhydrogel vaccine and will be useful for studying circulating Abs in response to other vaccines as well as those induced during infections or autoimmune disorders.
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http://dx.doi.org/10.1172/jci.insight.143471DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710313PMC
November 2020

Higher-order connections between stereotyped subsets: implications for improved patient classification in CLL.

Blood 2021 03;137(10):1365-1376

Department of Internal Medicine, Hematology and Oncology, Faculty of Medicine, Masaryk University-University Hospital Brno, Brno, Czech Republic.

Chronic lymphocytic leukemia (CLL) is characterized by the existence of subsets of patients with (quasi)identical, stereotyped B-cell receptor (BcR) immunoglobulins. Patients in certain major stereotyped subsets often display remarkably consistent clinicobiological profiles, suggesting that the study of BcR immunoglobulin stereotypy in CLL has important implications for understanding disease pathophysiology and refining clinical decision-making. Nevertheless, several issues remain open, especially pertaining to the actual frequency of BcR immunoglobulin stereotypy and major subsets, as well as the existence of higher-order connections between individual subsets. To address these issues, we investigated clonotypic IGHV-IGHD-IGHJ gene rearrangements in a series of 29 856 patients with CLL, by far the largest series worldwide. We report that the stereotyped fraction of CLL peaks at 41% of the entire cohort and that all 19 previously identified major subsets retained their relative size and ranking, while 10 new ones emerged; overall, major stereotyped subsets had a cumulative frequency of 13.5%. Higher-level relationships were evident between subsets, particularly for major stereotyped subsets with unmutated IGHV genes (U-CLL), for which close relations with other subsets, termed "satellites," were identified. Satellite subsets accounted for 3% of the entire cohort. These results confirm our previous notion that major subsets can be robustly identified and are consistent in relative size, hence representing distinct disease variants amenable to compartmentalized research with the potential of overcoming the pronounced heterogeneity of CLL. Furthermore, the existence of satellite subsets reveals a novel aspect of repertoire restriction with implications for refined molecular classification of CLL.
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http://dx.doi.org/10.1182/blood.2020007039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7976441PMC
March 2021

Immunoglobulins or Antibodies: IMGT Bridging Genes, Structures and Functions.

Biomedicines 2020 Aug 31;8(9). Epub 2020 Aug 31.

IMGT®, The International ImMunoGeneTics Information System®, Laboratoire d'ImmunoGénétique Moléculaire LIGM, Institut de Génétique Humaine IGH, Université de Montpellier UM, Centre National de la Recherche Scientifique CNRS, UMR 9002 CNRS-UM, 141 Rue de la Cardonille, CEDEX 5, 34396 Montpellier, France.

IMGT, the international ImMunoGeneTics information system founded in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS), marked the advent of immunoinformatics, a new science at the interface between immunogenetics and bioinformatics. For the first time, the immunoglobulin (IG) or antibody and T cell receptor (TR) genes were officially recognized as 'genes' as well as were conventional genes. This major breakthrough has allowed the entry, in genomic databases, of the IG and TR variable (V), diversity (D) and joining (J) genes and alleles of and of other jawed vertebrate species, based on the CLASSIFICATION axiom. The second major breakthrough has been the IMGT unique numbering and the IMGT Collier de Perles for the V and constant (C) domains of the IG and TR and other proteins of the IG superfamily (IgSF), based on the NUMEROTATION axiom. IMGT-ONTOLOGY axioms and concepts bridge genes, sequences, structures and functions, between biological and computational spheres in the IMGT system (Web resources, databases and tools). They provide the IMGT Scientific chart rules to identify, to describe and to analyse the IG complex molecular data, the huge diversity of repertoires, the genetic (alleles, allotypes, CNV) polymorphisms, the IG dual function (paratope/epitope, effector properties), the antibody humanization and engineering.
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http://dx.doi.org/10.3390/biomedicines8090319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555362PMC
August 2020

Evolution of the T-Cell Receptor (TR) Loci in the Adaptive Immune Response: The Tale of the TRG Locus in Mammals.

Genes (Basel) 2020 06 5;11(6). Epub 2020 Jun 5.

Department of Biology, University of Bari "Aldo Moro", 70124 Bari, Italy.

T lymphocytes are the principal actors of vertebrates' cell-mediated immunity. Like B cells, they can recognize an unlimited number of foreign molecules through their antigen-specific heterodimer receptors (TRs), which consist of αβ or γδ chains. The diversity of the TRs is mainly due to the unique organization of the genes encoding the α, β, γ, and δ chains. For each chain, multi-gene families are arranged in a TR locus, and their expression is guaranteed by the somatic recombination process. A great plasticity of the gene organization within the TR loci exists among species. Marked structural differences affect the TR γ (TRG) locus. The recent sequencing of multiple whole genome provides an opportunity to examine the TR gene repertoire in a systematic and consistent fashion. In this review, we report the most recent findings on the genomic organization of TRG loci in mammalian species in order to show differences and similarities. The comparison revealed remarkable diversification of both the genomic organization and gene repertoire across species, but also unexpected evolutionary conservation, which highlights the important role of the T cells in the immune response.
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http://dx.doi.org/10.3390/genes11060624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7349638PMC
June 2020

IMGT® Biocuration and Comparative Study of the T Cell Receptor Beta Locus of Veterinary Species Based on TRB.

Front Immunol 2020 5;11:821. Epub 2020 May 5.

IMGT®, The International ImMunoGeneTics Information System®, Centre National de la Recherche Scientifique (CNRS), Institut de Génétique Humaine (IGH), Université de Montpellier (UM), Montpellier, France.

IMGT®, the international ImMunoGeneTics information system® is the global reference in immunogenetics and immunoinformatics. By its creation in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS), IMGT® marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. IMGT® is specialized in the immunoglobulins (IG) or antibodies, T cell receptors (TR), major histocompatibility (MH), and proteins of the IgSF and MhSF superfamilies. T cell receptors are divided into two groups, αβ and γδ TR, which express distinct TR containing either α and β, or γ and δ chains, respectively. The TRβ locus (TRB) was recently described and annotated by IMGT® biocurators for several veterinary species, i.e., cat (), dog (), ferret (), pig (), rabbit (), rhesus monkey (), and sheep (). The aim of the present study is to compare the genes of the TRB locus among these different veterinary species based on . The results reveal that there are similarities but also differences including the number of genes by subgroup which may demonstrate duplications and/or deletions during evolution.
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http://dx.doi.org/10.3389/fimmu.2020.00821DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216736PMC
March 2021

Topology and expressed repertoire of the Felis catus T cell receptor loci.

BMC Genomics 2020 Jan 6;21(1):20. Epub 2020 Jan 6.

Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA, USA.

Background: The domestic cat (Felis catus) is an important companion animal and is used as a large animal model for human disease. However, the comprehensive study of adaptive immunity in this species is hampered by the lack of data on lymphocyte antigen receptor genes and usage. The objectives of this study were to annotate the feline T cell receptor (TR) loci and to characterize the expressed repertoire in lymphoid organs of normal cats using high-throughput sequencing.

Results: The Felis catus TRG locus contains 30 genes: 12 TRGV, 12 TRGJ and 6 TRGC, the TRB locus contains 48 genes: 33 TRBV, 2 TRBD, 11 TRBJ, 2 TRBC, the TRD locus contains 19 genes: 11 TRDV, 2 TRDD, 5 TRDJ, 1 TRDC, and the TRA locus contains 127 genes: 62 TRAV, 64 TRAJ, 1 TRAC. Functional feline V genes form monophyletic clades with their orthologs, and clustering of multimember subgroups frequently occurs in V genes located at the 5' end of TR loci. Recombination signal (RS) sequences of the heptamer and nonamer of functional V and J genes are highly conserved. Analysis of the TRG expressed repertoire showed preferential intra-cassette over inter-cassette rearrangements and dominant usage of the TRGV2-1 and TRGJ1-2 genes. The usage of TRBV genes showed minor bias but TRBJ genes of the second J-C-cluster were more commonly rearranged than TRBJ genes of the first cluster. The TRA/TRD V genes almost exclusively rearranged to J genes within their locus. The TRAV/TRAJ gene usage was relatively balanced while the TRD repertoire was dominated by TRDJ3.

Conclusions: This is the first description of all TR loci in the cat. The genomic organization of feline TR loci was similar to that of previously described jawed vertebrates (gnathostomata) and is compatible with the birth-and-death model of evolution. The large-scale characterization of feline TR genes provides comprehensive baseline data on immune repertoires in healthy cats and will facilitate the development of improved reagents for the diagnosis of lymphoproliferative diseases in cats. In addition, these data might benefit studies using cats as a large animal model for human disease.
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http://dx.doi.org/10.1186/s12864-019-6431-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945721PMC
January 2020

Standardized IMGT® Nomenclature of Salmonidae IGH Genes, the Paradigm of Atlantic Salmon and Rainbow Trout: From Genomics to Repertoires.

Front Immunol 2019 12;10:2541. Epub 2019 Nov 12.

Virologie et Immunologie Moléculaires (VIM), Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Jouy-en-Josas, France.

In teleost fish as in mammals, humoral adaptive immunity is based on B lymphocytes expressing highly diverse immunoglobulins (IG). During B cell differentiation, IG loci are subjected to genomic rearrangements of V, D, and J genes, producing a unique antigen receptor expressed on the surface of each lymphocyte. During the course of an immune response to infections or immunizations, B cell clones specific of epitopes from the immunogen are expanded and activated, leading to production of specific antibodies. Among teleost fish, salmonids comprise key species for aquaculture. Rainbow trout () and Atlantic salmon () are especially important from a commercial point of view and have emerged as critical models for fish immunology. The growing interest to capture accurate and comprehensive antibody responses against common pathogens and vaccines has resulted in recent efforts to sequence the IG repertoire in these species. In this context, a unified and standardized nomenclature of salmonid IG heavy chain (IGH) genes is urgently required, to improve accuracy of annotation of adaptive immune receptor repertoire dataset generated by high-throughput sequencing (AIRRseq) and facilitate comparisons between studies and species. Interestingly, the assembly of salmonids IGH genomic sequences is challenging due to the presence of two large size duplicated IGH loci and high numbers of IG genes and pseudogenes. We used data available for Atlantic salmon to establish an IMGT standardized nomenclature of IGH genes in this species and then applied the IMGT rules to the rainbow trout IGH loci to set up a nomenclature, which takes into account the specificities of Salmonid loci. This unique, consistent nomenclature for Salmonid IGH genes was then used to construct IMGT sequence reference directories allowing accurate annotation of AIRRseq data. The complex issues raised by the genetic diversity of salmon and trout strains are discussed in the context of IG repertoire annotation.
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http://dx.doi.org/10.3389/fimmu.2019.02541DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6866254PMC
November 2020

IMGT and 30 Years of Immunoinformatics Insight in Antibody V and C Domain Structure and Function.

Antibodies (Basel) 2019 Apr 11;8(2). Epub 2019 Apr 11.

IMGT®, the international ImMunoGeneTics information system®, University of Montpellier, CNRS, Laboratoire d'ImmunoGénétique Moléculaire LIGM, Institut de Génétique Humaine IGH, UMR 9002 CNRS-UM, 141 rue de la Cardonille, 34396 Montpellier CEDEX 5, France.

At the 10th Human Genome Mapping (HGM10) Workshop, in New Haven, for the first time, immunoglobulin (IG) or antibody and T cell receptor (TR) variable (V), diversity (D), joining (J), and constant (C) genes were officially recognized as 'genes', as were the conventional genes. Under these HGM auspices, IMGT, the international ImMunoGeneTics information system, was created in June 1989 at Montpellier (University of Montpellier and CNRS). The creation of IMGT marked the birth of immunoinformatics, a new science, at the interface between immunogenetics and bioinformatics. The accuracy and the consistency between genes and alleles, sequences, and three-dimensional (3D) structures are based on the IMGT Scientific chart rules generated from the IMGT-ONTOLOGY axioms and concepts: IMGT standardized keywords (IDENTIFICATION), IMGT gene and allele nomenclature (CLASSIFICATION), IMGT standardized labels (DESCRIPTION), IMGT unique numbering and IMGT Collier de Perles (NUMEROTATION). These concepts provide IMGT immunoinformatics insights for antibody V and C domain structure and function, used for the standardized description in IMGT web resources, databases and tools, immune repertoires analysis, single cell and/or high-throughput sequencing (HTS, NGS), antibody humanization, and antibody engineering in relation with effector properties.
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http://dx.doi.org/10.3390/antib8020029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6640715PMC
April 2019

The T cell receptor (TRA) locus in the rabbit (Oryctolagus cuniculus): Genomic features and consequences for invariant T cells.

Eur J Immunol 2019 12 13;49(12):2146-2158. Epub 2019 Aug 13.

Virologie et Immunologie Moléculaires (VIM), Institut National de la Recherche Agronomique (INRA), Université Paris-Saclay, Jouy-en-Josas, France.

The rabbit has been widely used in immunology and infectiology. Rabbit immunoglobulins have been extensively studied, leading to the discovery of their idiotypes, allotypic diversity, and of the diversification of the primary repertoire by hyperconversion. Much less is known about rabbit T cell receptors (TR), especially TRA. This isotype is particularly important for innate-like T cells, which typically express invariant TRA (iTRA). The presence of such cells in the rabbit remains an enigma. Rabbit NKT cells seem to be very rare, and lagomorphs lack MAIT cells. TRAV1, the variable gene expressed in the iTRA of these cells across most mammals, and MR1, the MH1-like receptor that present riboflavin derivatives to MAIT cells, are missing in rabbit. An alternative iTRA has been identified, that may be expressed by new innate-like T cells. To facilitate TRA repertoire analyses in rabbit, we report here a full description of TRA and TRD loci and a subgroup definition based on IMGT® classification. Rabbit TRA rearrangements follow the same temporal pattern that is observed in mouse and human. Rare transcripts expressing TRDV/TRDD/TRDJ rearrangements spliced to TRAC were detected. TRA and TRD genes have been made available in IMGT and IMGT/HighV-QUEST, allowing easy analysis of TRA/TRD RepSeq.
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http://dx.doi.org/10.1002/eji.201948228DOI Listing
December 2019

Inferred Allelic Variants of Immunoglobulin Receptor Genes: A System for Their Evaluation, Documentation, and Naming.

Front Immunol 2019 18;10:435. Epub 2019 Mar 18.

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW, Australia.

Immunoglobulins or antibodies are the main effector molecules of the B-cell lineage and are encoded by hundreds of variable (V), diversity (D), and joining (J) germline genes, which recombine to generate enormous IG diversity. Recently, high-throughput adaptive immune receptor repertoire sequencing (AIRR-seq) of recombined V-(D)-J genes has offered unprecedented insights into the dynamics of IG repertoires in health and disease. Faithful biological interpretation of AIRR-seq studies depends upon the annotation of raw AIRR-seq data, using reference germline gene databases to identify the germline genes within each rearrangement. Existing reference databases are incomplete, as shown by recent AIRR-seq studies that have inferred the existence of many previously unreported polymorphisms. Completing the documentation of genetic variation in germline gene databases is therefore of crucial importance. Lymphocyte receptor genes and alleles are currently assigned by the Immunoglobulins, T cell Receptors and Major Histocompatibility Nomenclature Subcommittee of the International Union of Immunological Societies (IUIS) and managed in IMGT, the international ImMunoGeneTics information system (IMGT). In 2017, the IMGT Group reached agreement with a group of AIRR-seq researchers on the principles of a streamlined process for identifying and naming inferred allelic sequences, for their incorporation into IMGT. These researchers represented the AIRR Community, a network of over 300 researchers whose objective is to promote all aspects of immunoglobulin and T-cell receptor repertoire studies, including the standardization of experimental and computational aspects of AIRR-seq data generation and analysis. The Inferred Allele Review Committee (IARC) was established by the AIRR Community to devise policies, criteria, and procedures to perform this function. Formalized evaluations of novel inferred sequences have now begun and submissions are invited via a new dedicated portal (https://ogrdb.airr-community.org). Here, we summarize recommendations developed by the IARC-focusing, to begin with, on human IGHV genes-with the goal of facilitating the acceptance of inferred allelic variants of germline IGHV genes. We believe that this initiative will improve the quality of AIRR-seq studies by facilitating the description of human IG germline gene variation, and that in time, it will expand to the documentation of TR and IG genes in many vertebrate species.
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http://dx.doi.org/10.3389/fimmu.2019.00435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431624PMC
September 2020

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

J Pathol 2019 04 30;247(4):416-421. Epub 2019 Jan 30.

IMGT®, the international ImMunoGeneTics Information System®, Université de Montpellier, LIGM, Institut de Génétique Humaine IGH, UMR CNRS UM, Montpellier, France.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5209DOI Listing
April 2019

Use of IMGT Databases and Tools for Antibody Engineering and Humanization.

Methods Mol Biol 2018 ;1827:35-69

IMGT®, The International ImMunoGeneTics Information System®, Laboratoire d'ImmunoGénétique Moléculaire (LIGM), Institut de Génétique Humaine (IGH), UMR 9002 CNRS-UM, Université de Montpellier, Montpellier Cedex 5, France.

IMGT, the international ImMunoGeneTics information system ( http://www.imgt.org ), was created in 1989 by Marie-Paule Lefranc (Université de Montpellier and CNRS) to manage the huge diversity of the antigen receptors, immunoglobulins (IG) or antibodies, and T cell receptors (TR). The founding of IMGT marked the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. Standardized sequence and structure analysis of antibody using IMGT databases and tools allow one to bridge, for the first time, the gap between antibody sequences and three-dimensional (3D) structures. This is achieved through the IMGT Scientific chart rules, based on the IMGT-ONTOLOGY concepts of classification (IMGT gene and allele nomenclature), description (IMGT standardized labels), and numerotation (IMGT unique numbering and IMGT Collier de Perles). IMGT is acknowledged as the global reference for immunogenetics and immunoinformatics, and its standards are particularly useful for antibody engineering and humanization. IMGT databases for antibody nucleotide sequences and genes include IMGT/LIGM-DB and IMGT/GENE-DB, respectively, and nucleotide sequence analysis is performed by the IMGT/V-QUEST and IMGT/JunctionAnalysis tools and for NGS by IMGT/HighV-QUEST. In this chapter, we focus on IMGT databases and tools for amino acid sequences, two-dimensional (2D) and three-dimensional (3D) structures: the IMGT/DomainGapAlign and IMGT Collier de Perles tools and the IMGT/2Dstructure-DB and IMGT/3Dstructure-DB database. IMGT/mAb-DB provides the query interface for monoclonal antibodies (mAb), fusion proteins for immune applications (FPIA), and composite proteins for clinical applications (CPCA) and related proteins of interest (RPI) and links to the proposed and recommended lists of the World Health Organization International Nonproprietary Name (WHO INN) programme, to IMGT/2Dstructure-DB for amino acid sequences, and to IMGT/3Dstructure-DB and its associated tools (IMGT/StructuralQuery, IMGT/DomainSuperimpose) for crystallized antibodies.
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http://dx.doi.org/10.1007/978-1-4939-8648-4_3DOI Listing
April 2019

Coupling of Single Molecule, Long Read Sequencing with IMGT/HighV-QUEST Analysis Expedites Identification of SIV gp140-Specific Antibodies from scFv Phage Display Libraries.

Front Immunol 2018 1;9:329. Epub 2018 Mar 1.

Biology Department, Boston College, Chestnut Hill, MA, United States.

The simian immunodeficiency virus (SIV)/macaque model of human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome pathogenesis is critical for furthering our understanding of the role of antibody responses in the prevention of HIV infection, and will only increase in importance as macaque immunoglobulin (IG) gene databases are expanded. We have previously reported the construction of a phage display library from a SIV-infected rhesus macaque () using oligonucleotide primers based on human IG gene sequences. Our previous screening relied on Sanger sequencing, which was inefficient and generated only a few dozen sequences. Here, we re-analyzed this library using single molecule, real-time (SMRT) sequencing on the Pacific Biosciences (PacBio) platform to generate thousands of highly accurate circular consensus sequencing (CCS) reads corresponding to full length single chain fragment variable. CCS data were then analyzed through the international ImMunoGeneTics information system (IMGT)/HighV-QUEST (www.imgt.org) to identify variable genes and perform statistical analyses. Overall the library was very diverse, with 2,569 different IMGT clonotypes called for the 5,238 IGHV sequences assigned to an IMGT clonotype. Within the library, SIV-specific antibodies represented a relatively limited number of clones, with only 135 different IMGT clonotypes called from 4,594 IGHV-assigned sequences. Our data did confirm that the IGHV4 and IGHV3 gene usage was the most abundant within the rhesus antibodies screened, and that these genes were even more enriched among SIV gp140-specific antibodies. Although a broad range of VH CDR3 amino acid (AA) lengths was observed in the unpanned library, the vast majority of SIV gp140-specific antibodies demonstrated a more uniform VH CDR3 length (20 AA). This uniformity was far less apparent when VH CDR3 were classified according to their clonotype (range: 9-25 AA), which we believe is more relevant for specific antibody identification. Only 174 IGKV and 588 IGLV clonotypes were identified within the VL sequences associated with SIV gp140-specific VH. Together, these data strongly suggest that the combination of SMRT sequencing with the IMGT/HighV-QUEST querying tool will facilitate and expedite our understanding of polyclonal antibody responses during SIV infection and may serve to rapidly expand the known scope of macaque V genes utilized during these responses.
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http://dx.doi.org/10.3389/fimmu.2018.00329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5837965PMC
March 2019

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

MAbs 2018 May/Jun;10(4):539-546. Epub 2018 Mar 29.

p Technische Universität Braunschweig, Institute of Biochemistry, Biotechnology and Bioinformatics , Spielmannstr. 7, Braunschweig , Germany.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.
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http://dx.doi.org/10.1080/19420862.2018.1445456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5973764PMC
August 2019

Pacific Biosciences Sequencing and IMGT/HighV-QUEST Analysis of Full-Length Single Chain Fragment Variable from an Selected Phage-Display Combinatorial Library.

Front Immunol 2017 20;8:1796. Epub 2017 Dec 20.

CRMSB, UMR 5536, CNRS, Bordeaux, France.

Phage-display selection of immunoglobulin (IG) or antibody single chain Fragment variable (scFv) from combinatorial libraries is widely used for identifying new antibodies for novel targets. Next-generation sequencing (NGS) has recently emerged as a new method for the high throughput characterization of IG and T cell receptor (TR) immune repertoires both and . However, challenges remain for the NGS sequencing of scFv from combinatorial libraries owing to the scFv length (>800 bp) and the presence of two variable domains [variable heavy (VH) and variable light (VL) for IG] associated by a peptide linker in a single chain. Here, we show that single-molecule real-time (SMRT) sequencing with the Pacific Biosciences RS II platform allows for the generation of full-length scFv reads obtained from an selection of scFv-phages in an animal model of atherosclerosis. We first amplified the DNA of the phagemid inserts from scFv-phages eluted from an aortic section at the third round of the selection. From this amplified DNA, 450,558 reads were obtained from 15 SMRT cells. Highly accurate circular consensus sequences from these reads were generated, filtered by quality and then analyzed by IMGT/HighV-QUEST with the functionality for scFv. Full-length scFv were identified and characterized in 348,659 reads. Full-length scFv sequencing is an absolute requirement for analyzing the associated VH and VL domains enriched during the panning rounds. In order to further validate the ability of SMRT sequencing to provide high quality, full-length scFv sequences, we tracked the reads of an scFv-phage clone P3 previously identified by biological assays and Sanger sequencing. Sixty P3 reads showed 100% identity with the full-length scFv of 767 bp, 53 of them covering the whole insert of 977 bp, which encompassed the primer sequences. The remaining seven reads were identical over a shortened length of 939 bp that excludes the vicinity of primers at both ends. Interestingly these reads were obtained from each of the 15 SMRT cells. Thus, the SMRT sequencing method and the IMGT/HighV-QUEST functionality for scFv provides a straightforward protocol for characterization of full-length scFv from combinatorial phage libraries.
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http://dx.doi.org/10.3389/fimmu.2017.01796DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5742356PMC
December 2017

Immunogenetic factors driving formation of ultralong VH CDR3 in Bos taurus antibodies.

Cell Mol Immunol 2019 01 4;16(1):53-64. Epub 2017 Dec 4.

Department of Molecular Medicine, The Scripps Research Institute, 92037, La Jolla, CA, USA.

The antibody repertoire of Bos taurus is characterized by a subset of variable heavy (VH) chain regions with ultralong third complementarity determining regions (CDR3) which, compared to other species, can provide a potent response to challenging antigens like HIV env. These unusual CDR3 can range to over seventy highly diverse amino acids in length and form unique β-ribbon 'stalk' and disulfide bonded 'knob' structures, far from the typical antigen binding site. The genetic components and processes for forming these unusual cattle antibody VH CDR3 are not well understood. Here we analyze sequences of Bos taurus antibody VH domains and find that the subset with ultralong CDR3 exclusively uses a single variable gene, IGHV1-7 (VHBUL) rearranged to the longest diversity gene, IGHD8-2. An eight nucleotide duplication at the 3' end of IGHV1-7 encodes a longer V-region producing an extended F β-strand that contributes to the stalk in a rearranged CDR3. A low amino acid variability was observed in CDR1 and CDR2, suggesting that antigen binding for this subset most likely only depends on the CDR3. Importantly a novel, potentially AID mediated, deletional diversification mechanism of the B. taurus VH ultralong CDR3 knob was discovered, in which interior codons of the IGHD8-2 region are removed while maintaining integral structural components of the knob and descending strand of the stalk in place. These deletions serve to further diversify cysteine positions, and thus disulfide bonded loops. Hence, both germline and somatic genetic factors and processes appear to be involved in diversification of this structurally unusual cattle VH ultralong CDR3 repertoire.
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http://dx.doi.org/10.1038/cmi.2017.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318308PMC
January 2019

Reproducibility and Reuse of Adaptive Immune Receptor Repertoire Data.

Front Immunol 2017 1;8:1418. Epub 2017 Nov 1.

Department of Microbiology, Boston University School of Medicine, Boston, MA, United States.

High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1-3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of this perspective is to provide an overview of the AIRR Community's founding principles and present the progress that the AIRR Community has made in developing standards of practice and data sharing protocols. Finally, and most important, we invite all interested parties to join this effort to facilitate sharing and use of these powerful data sets ([email protected]).
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http://dx.doi.org/10.3389/fimmu.2017.01418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671925PMC
November 2017

Comprehensive annotation and evolutionary insights into the canine (Canis lupus familiaris) antigen receptor loci.

Immunogenetics 2018 04 19;70(4):223-236. Epub 2017 Sep 19.

Wellcome Trust Sanger Institute, Hinxton, UK.

Dogs are an excellent model for human disease. For example, the treatment of canine lymphoma has been predictive of the human response to that treatment. However, an incomplete picture of canine (Canis lupus familiaris) immunoglobulin (IG) and T cell receptor (TR)-or antigen receptor (AR)-gene loci has restricted their utility. This work advances the annotation of the canine AR loci and looks into breed-specific features of the loci. Bioinformatic analysis of unbiased RNA sequence data was used to complete the annotation of the canine AR genes. This annotation was used to query 107 whole genome sequences from 19 breeds and identified over 5500 alleles across the 550 genes of the seven AR loci: the IG heavy, kappa, and lambda loci; and the TR alpha, beta, gamma, and delta loci. Of note was the discovery that half of the IGK variable (V) genes were located downstream of, and inverted with respect to, the rest of the locus. Analysis of the germline sequences of all the AR V genes identified greater conservation between dog and human than mouse with either. This work brings our understanding of the genetic diversity and expression of AR in dogs to the same completeness as that of mice and men, making it the third species to have all AR loci comprehensively and accurately annotated. The large number of germline sequences serves as a reference for future studies, and has allowed statistically powerful conclusions to be drawn on the pressures that have shaped these loci.
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http://dx.doi.org/10.1007/s00251-017-1028-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871656PMC
April 2018

IG and TR single chain fragment variable (scFv) sequence analysis: a new advanced functionality of IMGT/V-QUEST and IMGT/HighV-QUEST.

BMC Immunol 2017 06 26;18(1):35. Epub 2017 Jun 26.

IMGT®, the international ImMunoGeneTics information system®, Laboratoire d'ImmunoGénétique Moléculaire LIGM, Institut de Génétique Humaine IGH, UMR 9002, CNRS, Montpellier University, Montpellier, France.

Background: IMGT®, the international ImMunoGeneTics information system® ( http://www.imgt.org ), was created in 1989 in Montpellier, France (CNRS and Montpellier University) to manage the huge and complex diversity of the antigen receptors, and is at the origin of immunoinformatics, a science at the interface between immunogenetics and bioinformatics. Immunoglobulins (IG) or antibodies and T cell receptors (TR) are managed and described in the IMGT® databases and tools at the level of receptor, chain and domain. The analysis of the IG and TR variable (V) domain rearranged nucleotide sequences is performed by IMGT/V-QUEST (online since 1997, 50 sequences per batch) and, for next generation sequencing (NGS), by IMGT/HighV-QUEST, the high throughput version of IMGT/V-QUEST (portal begun in 2010, 500,000 sequences per batch). In vitro combinatorial libraries of engineered antibody single chain Fragment variable (scFv) which mimic the in vivo natural diversity of the immune adaptive responses are extensively screened for the discovery of novel antigen binding specificities. However the analysis of NGS full length scFv (~850 bp) represents a challenge as they contain two V domains connected by a linker and there is no tool for the analysis of two V domains in a single chain.

Methods: The functionality "Analyis of single chain Fragment variable (scFv)" has been implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST for the analysis of the two V domains of IG and TR scFv. It proceeds in five steps: search for a first closest V-REGION, full characterization of the first V-(D)-J-REGION, then search for a second V-REGION and full characterization of the second V-(D)-J-REGION, and finally linker delimitation.

Results: For each sequence or NGS read, positions of the 5'V-DOMAIN, linker and 3'V-DOMAIN in the scFv are provided in the 'V-orientated' sense. Each V-DOMAIN is fully characterized (gene identification, sequence description, junction analysis, characterization of mutations and amino changes). The functionality is generic and can analyse any IG or TR single chain nucleotide sequence containing two V domains, provided that the corresponding species IMGT reference directory is available.

Conclusion: The "Analysis of single chain Fragment variable (scFv)" implemented in IMGT/V-QUEST and, for NGS, in IMGT/HighV-QUEST provides the identification and full characterization of the two V domains of full-length scFv (~850 bp) nucleotide sequences from combinatorial libraries. The analysis can also be performed on concatenated paired chains of expressed antigen receptor IG or TR repertoires.
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http://dx.doi.org/10.1186/s12865-017-0218-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5485737PMC
June 2017

Targeting the Wnt Pathway and Cancer Stem Cells with Anti-progastrin Humanized Antibodies as a Potential Treatment for K-RAS-Mutated Colorectal Cancer.

Clin Cancer Res 2017 Sep 9;23(17):5267-5280. Epub 2017 Jun 9.

Accompagnement Pharma, Luxembourg, Luxembourg.

Patients with metastatic colorectal cancer suffer from disease relapse mainly due to cancer stem cells (CSC). Interestingly, they have an increased level of blood progastrin, a tumor-promoting peptide essential for the self-renewal of colon CSCs, which is also a direct β-catenin/TCF4 target gene. In this study, we aimed to develop a novel targeted therapy to neutralize secreted progastrin to inhibit Wnt signaling, CSCs, and reduce relapses. Antibodies (monoclonal and humanized) directed against progastrin were produced and selected for target specificity and affinity. After validation of their effectiveness on survival of colorectal cancer cell lines harboring B-RAF or K-RAS mutations, their efficacy was assessed and , alone or concomitantly with chemotherapy, on CSC self-renewal capacity, tumor recurrence, and Wnt signaling. We show that anti-progastrin antibodies decrease self-renewal of CSCs both and , either alone or in combination with chemotherapy. Furthermore, migration and invasion of colorectal cancer cells are diminished; chemosensitivity is prolonged in SW620 and HT29 cells and posttreatment relapse is significantly delayed in T84 cells, xenografted nude mice. Finally, we show that the Wnt signaling activity is decreased, and, in transgenic mice developing Wnt-driven intestinal neoplasia, the tumor burden is alleviated, with an amplification of cell differentiation in the remaining tumors. Altogether, these data show that humanized anti-progastrin antibodies might represent a potential new treatment for K-RAS-mutated colorectal patients, for which there is a crucial unmet medical need. .
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http://dx.doi.org/10.1158/1078-0432.CCR-17-0533DOI Listing
September 2017

Chronic Lymphocytic Leukemia with Mutated IGHV4-34 Receptors: Shared and Distinct Immunogenetic Features and Clinical Outcomes.

Clin Cancer Res 2017 Sep 23;23(17):5292-5301. Epub 2017 May 23.

Department of Hematology, Rigshospitalet, Copenhagen, Denmark.

We sought to investigate whether B cell receptor immunoglobulin (BcR IG) stereotypy is associated with particular clinicobiological features among chronic lymphocytic leukemia (CLL) patients expressing mutated BcR IG (M-CLL) encoded by the IGHV4-34 gene, and also ascertain whether these associations could refine prognostication. In a series of 19,907 CLL cases with available immunogenetic information, we identified 339 IGHV4-34-expressing cases assigned to one of the four largest stereotyped M-CLL subsets, namely subsets #4, #16, #29 and #201, and investigated in detail their clinicobiological characteristics and disease outcomes. We identified shared and subset-specific patterns of somatic hypermutation (SHM) among patients assigned to these subsets. The greatest similarity was observed between subsets #4 and #16, both including IgG-switched cases (IgG-CLL). In contrast, the least similarity was detected between subsets #16 and #201, the latter concerning IgM/D-expressing CLL. Significant differences between subsets also involved disease stage at diagnosis and the presence of specific genomic aberrations. IgG subsets #4 and #16 emerged as particularly indolent with a significantly ( < 0.05) longer time-to-first-treatment (TTFT; median TTFT: not yet reached) compared with the IgM/D subsets #29 and #201 (median TTFT: 11 and 12 years, respectively). Our findings support the notion that BcR IG stereotypy further refines prognostication in CLL, superseding the immunogenetic distinction based solely on SHM load. In addition, the observed distinct genetic aberration landscapes and clinical heterogeneity suggest that not all M-CLL cases are equal, prompting further research into the underlying biological background with the ultimate aim of tailored patient management. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-3100DOI Listing
September 2017

High-Throughput Immunogenetics for Clinical and Research Applications in Immunohematology: Potential and Challenges.

J Immunol 2017 05 17;198(10):3765-3774. Epub 2017 Apr 17.

Institute of Applied Biosciences, Center for Research and Technology Hellas, GR-57001 Thessaloniki, Greece.

Analysis and interpretation of Ig and TCR gene rearrangements in the conventional, low-throughput way have their limitations in terms of resolution, coverage, and biases. With the advent of high-throughput, next-generation sequencing (NGS) technologies, a deeper analysis of Ig and/or TCR (IG/TR) gene rearrangements is now within reach, which impacts on all main applications of IG/TR immunogenetic analysis. To bridge the generation gap from low- to high-throughput analysis, the EuroClonality-NGS Consortium has been formed, with the main objectives to develop, standardize, and validate the entire workflow of IG/TR NGS assays for 1) clonality assessment, 2) minimal residual disease detection, and 3) repertoire analysis. This concerns the preanalytical (sample preparation, target choice), analytical (amplification, NGS), and postanalytical (immunoinformatics) phases. Here we critically discuss pitfalls and challenges of IG/TR NGS methodology and its applications in hemato-oncology and immunology.
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http://dx.doi.org/10.4049/jimmunol.1602050DOI Listing
May 2017

Human Immunoglobulin Heavy Gamma Chain Polymorphisms: Molecular Confirmation Of Proteomic Assessment.

Mol Cell Proteomics 2017 05 6;16(5):824-839. Epub 2017 Mar 6.

From the ‡Institut de Recherche pour le Développement, UMR 216 Mère et enfant face aux infections tropicales, Paris, France;

Immunoglobulin G (IgG) proteins are known for the huge diversity of the variable domains of their heavy and light chains, aimed at protecting each individual against foreign antigens. The IgG also harbor specific polymorphism concentrated in the CH2 and CH3-CHS constant regions located on the Fc fragment of their heavy chains. But this individual particularity relies only on a few amino acids among which some could make accurate sequence determination a challenge for mass spectrometry-based techniques.The purpose of the study was to bring a molecular validation of proteomic results by the sequencing of encoding DNA fragments. It was performed using ten individual samples (DNA and sera) selected on the basis of their Gm (gamma marker) allotype polymorphism in order to cover the main immunoglobulin heavy gamma (IGHG) gene diversity. Gm allotypes, reflecting part of this diversity, were determined by a serological method. On its side, the IGH locus comprises four functional IGHG genes totalizing 34 alleles and encoding the four IgG subclasses. The genomic study focused on the nucleotide polymorphism of the CH2 and CH3-CHS exons and of the intron. Despite strong sequence identity, four pairs of specific gene amplification primers could be designed. Additional primers were identified to perform the subsequent sequencing. The nucleotide sequences obtained were first assigned to a specific IGHG gene, and then IGHG alleles were deduced using a home-made decision tree reading of the nucleotide sequences. IGHG amino acid (AA) alleles were determined by mass spectrometry. Identical results were found at 95% between alleles identified by proteomics and those deduced from genomics. These results validate the proteomic approach which could be used for diagnostic purposes, namely for a mother-and-child differential IGHG detection in a context of suspicion of congenital infection.
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http://dx.doi.org/10.1074/mcp.M116.064733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5417824PMC
May 2017
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