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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 (Felis catus), dog (Canis lupus familiaris), ferret (Mustela putorius furo), pig (Sus scrofa), rabbit (Oryctolagus cuniculus), rhesus monkey (Macaca mulatta), and sheep (Ovis aries). The aim of the present study is to compare the genes of the TRB locus among these different veterinary species based on Homo sapiens. 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.

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 (c) 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Immunoglobulin gene sequence analysis in chronic lymphocytic leukemia : updated ERIC recommendations

Purpose: 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. Experimental Design: 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. Results: 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 (P < 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). Conclusions: 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. Clin Cancer Res; 23(17); 5292–301. ©2017 AACR.

An unresolved issue in chronic lymphocytic leukemia (CLL) is whether IGHV3-21 gene usage, in general, or the expression of stereotyped B-cell receptor immunoglobulin defining subset # 2 (IGHV3-21/IGLV3-21), in particular, determines outcome for IGHV3-21-utilizing cases. We reappraised this issue in 8593 CLL patients of whom 437 (5%) used the IGHV3-21 gene with 254/437 (58%) classified as subset # 2. Within subset # 2, immunoglobulin heavy variable (IGHV)-mutated cases predominated, whereas non-subset # 2/IGHV3-21 was enriched for IGHV-unmutated cases (P =.002). Subset # 2 exhibited significantly shorter time-to-first-treatment (TTFT) compared with non-subset # 2/IGHV3-21 (22 vs 60 months, P =.001). No such difference was observed between non-subset # 2/IGHV3-21 vs the remaining CLL with similar IGHV mutational status. In conclusion, IGHV3-21 CLL should not be axiomatically considered a homogeneous entity with adverse prognosis, given that only subset # 2 emerges as uniformly aggressive, contrasting non-subset # 2/IGVH3-21 patients whose prognosis depends on IGHV mutational status as the remaining CLL.

The HIV vaccine-induced neutralizing antibodies (Abs) display low rates of mutation in their variable regions. To determine the range of neutralization mediated by similar human monoclonal Abs (mAbs) but derived from unselected chronically HIV-1 infected subjects, we tested a panel of 66 mAbs specific to V3, CD4 binding site (CD4bs) and V2 regions. The mAbs were tested against 41 pseudoviruses, including 15 tier 1 and 26 tier 2, 3 viruses, showing that the neutralization potency and breadth of anti-V3 mAbs were significantly higher than those of the anti-CD4bs and anti-V2 mAbs, and only anti-V3 mAbs were able to neutralize some tier 2, 3 viruses. The percentage of mutations in the variable regions of the heavy (VH) and light (VL) chains varied broadly in a range from 2% to 18% and correlated moderately with the neutralization breadth of tier 2, 3 viruses. There was no correlation with neutralization of tier 1 viruses as some mAbs with low and high percentages of mutations neutralized the same number of viruses. The electrostatic interactions between anti-V3 mAbs and the charged V3 region may contribute to their neutralization because the isoelectric points of the VH CDR3 of 48 anti-V3 mAbs were inversely correlated with the neutralization breadth of tier 2, 3 viruses. The results demonstrate that infection-induced antibodies to CD4bs, V3 and V2 regions can mediate cross-clade neutralization despite low levels of mutations which can be achieved by HIV-1 vaccine-induced antibodies.

IMGT/V-QUEST, for 'V-QUEry and STandardization', is an integrated software program which analyses the immunoglobulin (IG) and T cell receptor (TR) rearranged nucleotide sequences. The extraordinary diversity of the IG and TR repertoires (10(12) antibodies and 10(12) TR per individual) results from several mechanisms at the DNA level: the combinatorial diversity of the variable (V), diversity (D) and joining (J) genes, the N-diversity and, for IG, the somatic mutations. IMGT/V-QUEST identifies the V, D and J genes and alleles by alignment with the germline IG and TR gene and allele sequences of the IMGT reference directory. IMGT/V-QUEST delimits the structurally important features, frameworks and complementarity-determining regions (the last of these forming the antigen binding site), on the basis of the IMGT unique numbering. The tool localizes the somatic mutations of the IG rearranged sequences. IMGT/V-QUEST also dynamically displays a graphical two-dimensional representation, or IMGT Collier de Perles, of the IG and TR variable regions. Moreover, IMGT/V-QUEST can interact with IMGT/JunctionAnalysis for the detailed description of the V-J and V-D-J junctions, and with IMGT/PhyloGene for the construction of phylogenetic trees. IMGT/V-QUEST is currently available for human and mouse, and partly for non-human primates, sheep, chondrichthyes and teleostei. IMGT/V-QUEST is freely available at http://imgt.cines.fr.

IMGT(®), the international ImMunoGeneTics information system(®) (http://www.imgt.org), created in 1989 (Centre National de la Recherche Scientifique, Montpellier University), is acknowledged as the global reference in immunogenetics and immunoinformatics. The accuracy and the consistency of the IMGT(®) data are based on IMGT-ONTOLOGY which bridges the gap between genes, sequences, and three-dimensional (3D) structures. Thus, receptors, chains, and domains are characterized with the same IMGT(®) rules and standards (IMGT standardized labels, IMGT gene and allele nomenclature, IMGT unique numbering, IMGT Collier de Perles), independently from the molecule type (genomic DNA, complementary DNA, transcript, or protein) or from the species. More particularly, IMGT(®) tools and databases provide a highly standardized analysis of the immunoglobulin (IG) or antibody and T cell receptor (TR) V and C domains. IMGT/V-QUEST analyzes the V domains of IG or TR rearranged nucleotide sequences, integrates the IMGT/JunctionAnalysis and IMGT/Automat tools, and provides IMGT Collier de Perles. IMGT/HighV-QUEST analyzes sequences from high-throughput sequencing (HTS) (up to 150,000 sequences per batch) and performs statistical analysis on up to 450,000 results, with the same resolution and high quality as IMGT/V-QUEST online. IMGT/DomainGapAlign analyzes amino acid sequences of V and C domains and IMGT/3Dstructure-DB and associated tools provide information on 3D structures, contact analysis, and paratope/epitope interactions. These IMGT(®) tools and databases, and the IMGT/mAb-DB interface with access to therapeutical antibody data, provide an invaluable help for antibody engineering and antibody humanization.

Upon infection, B-lymphocytes expressing antibodies specific for the intruding pathogen develop clonal responses triggered by pathogen recognition via the B-cell receptor. The constant region of antibodies produced by such responding clones dictates their functional properties. In teleost fish, the clonal structure of B-cell responses and the respective contribution of the three isotypes IgM, IgD and IgT remain unknown. The expression of IgM and IgT are mutually exclusive, leading to the existence of two B-cell subsets expressing either both IgM and IgD or only IgT. Here, we undertook a comprehensive analysis of the variable heavy chain (VH) domain repertoires of the IgM, IgD and IgT in spleen of homozygous isogenic rainbow trout (Onchorhynchus mykiss) before, and after challenge with a rhabdovirus, the Viral Hemorrhagic Septicemia Virus (VHSV), using CDR3-length spectratyping and pyrosequencing of immunoglobulin (Ig) transcripts. In healthy fish, we observed distinct repertoires for IgM, IgD and IgT, respectively, with a few amplified μ and τ junctions, suggesting the presence of IgM- and IgT-secreting cells in the spleen. In infected animals, we detected complex and highly diverse IgM responses involving all VH subgroups, and dominated by a few large public and private clones. A lower number of robust clonal responses involving only a few VH were detected for the mucosal IgT, indicating that both IgM+ and IgT+ spleen B cells responded to systemic infection but at different degrees. In contrast, the IgD response to the infection was faint. Although fish IgD and IgT present different structural features and evolutionary origin compared to mammalian IgD and IgA, respectively, their implication in the B-cell response evokes these mouse and human counterparts. Thus, it appears that the general properties of antibody responses were already in place in common ancestors of fish and mammals, and were globally conserved during evolution with possible functional convergences., Author Summary In humans and mice, a broad and robust antibody response is the most effective system of defense against pathogens. While all vertebrates but the most ancient use an antibody system, it remains unknown if the clonal selection rules are conserved across vertebrates. Using high-throughput sequencing and antibody repertoire analysis, we show in teleost fish that virus infection induces a typical antibody response with clonal expansions. In spleen, the antibody response mainly consists in IgM, which is found across vertebrates, with a significant contribution of the fish-specific mucosal antibody IgT. IgD does not contribute as already observed in mammals. Although antibody molecules can be of different nature and structure across species, the general features of adaptive immunity were already in place in common ancestors of fish and mammals, and globally conserved during evolution while the anatomy and physiology of the immune system diverged.

Immunogenetics is the science that studies the genetics of the immune system and immune responses. Owing to the complexity and diversity of the immune repertoire, immunogenetics represents one of the greatest challenges for data interpretation: a large biological expertise, a considerable effort of standardization and the elaboration of an efficient system for the management of the related knowledge were required. IMGT®, the international ImMunoGeneTics information system® (http://www.imgt.org) has reached that goal through the building of a unique ontology, IMGT-ONTOLOGY, which represents the first ontology for the formal representation of knowledge in immunogenetics and immunoinformatics. IMGT-ONTOLOGY manages the immunogenetics knowledge through diverse facets that rely on the seven axioms of the Formal IMGT-ONTOLOGY or IMGT-Kaleidoscope: "IDENTIFICATION", "DESCRIPTION", "CLASSIFICATION", "NUMEROTATION", "LOCALIZATION", "ORIENTATION" and "OBTENTION". The concepts of identification, description, classification and numerotation generated from the axioms led to the elaboration of the IMGT® standards that constitute the IMGT Scientific chart: IMGT® standardized keywords (concepts of identification), IMGT® standardized labels (concepts of description), IMGT® standardized gene and allele nomenclature (concepts of classification) and IMGT unique numbering and IMGT Collier de Perles (concepts of numerotation). IMGT-ONTOLOGY has become the global reference in immunogenetics and immunoinformatics for the knowledge representation of immunoglobulins (IG) or antibodies, T cell receptors (TR) and major histocompatibility (MH) proteins of humans and other vertebrates, proteins of the immunoglobulin superfamily (IgSF) and MH superfamily (MhSF), related proteins of the immune system (RPI) of vertebrates and invertebrates, therapeutic monoclonal antibodies (mAbs), fusion proteins for immune applications (FPIA) and composite proteins for clinical application (CPCA).

IMGT/V-QUEST is the highly customized and integrated online IMGT(®) tool for the standardized analysis of the immunoglobulin (IG) or antibody and T cell receptor (TR) rearranged nucleotide sequences. The analysis of these antigen receptors represents a crucial challenge for the study of the adaptive immune response in normal and disease-related situations. The expressed IG and TR repertoires represent a potential of 10(12) IG and 10(12) TR per individual. This huge diversity results from mechanisms that occur at the DNA level during the IG and TR molecular synthesis. These mechanisms include the combinatorial rearrangements of the variable (V), diversity (D) and joining (J) genes, the N-diversity (deletion and addition at random of nucleotides during the V-(D)-J rearrangement) and, for IG, somatic hypermutations. IMGT/V-QUEST identifies the V, D, J genes and alleles by alignment with the germline IG and TR gene and allele sequences of the IMGT reference directory. The tool describes the V-REGION mutations and identifies the hot spot positions in the closest germline V gene. IMGT/V-QUEST integrates IMGT/JunctionAnalysis for a detailed analysis of the V-J and V-D-J junctions and IMGT/Automat for a complete annotation of the sequences and also provides IMGT Collier de Perles. IMGT/HighV-QUEST, the high-throughput version of IMGT/V-QUEST, implemented to answer the needs of deep sequencing data analysis from Next Generation Sequencing (NGS), allows the analysis of thousands of IG and TR sequences in a single run. IMGT/V-QUEST and IMGT/HighV-QUEST are available at the IMGT(®) Home page, http://www.imgt.org.

INTRODUCTIONIMGT/JunctionAnalysis is the online IMGT tool for the detailed and standardized analysis of the junctions between the variable (V), diversity (D), and joining (J) genes (V-J and V-D-J junctions) of the rearranged immunoglobulin (IG) or antibody and T cell receptor (TR) variable domains. The V-(D)-J junctions comprise the rearranged CDR3-IMGT and its anchors 2nd-CYS 104 and J-PHE or J-TRP 118. The diversity of the junctions that determines the antigen receptor specificity results from complex molecular mechanisms that occur at the DNA level during the IG and TR synthesis and create combinatorial diversity, N-diversity and, for IG, somatic hypermutations. The annotation of V-J or V-D-J junctions in rearranged IG and TR sequences represents a huge challenge due to its uniqueness and complexity. IMGT/JunctionAnalysis has been a major breakthrough by providing, for the first time, a very detailed and accurate analysis of the junctions. The tool, whose use is described here, identifies the D genes in the IGH, TRB, and TRD junctions, the trimmed nucleotides (nt) at the end of the genes which recombine, and the palindromic P regions in the absence of gene trimming. It delimits the N regions that result from the N-diversity, calculates the ratio of G+C nucleotides in the N regions, and evaluates the number of somatic hypermutations for each gene within the junction.

IMGT, the international ImMunoGeneTics information system (http://www. imgt.org) (Lefranc et al. 2009), was created in 1989 at Montpellier, France (CNRS and Universite Montpellier 2), to standardize the immunogenetics data and to manage the huge diversity of the antigen receptors, immunoglobulins (IG) or antibodies and T cell receptors (TR) (Lefranc and Lefranc 2001a, b). IMGT is the international reference in immunogenetics and immunoinformatics, and its standards have been approved by the World Health Organization–International Union of Immunological Societies (WHO–IUIS) Nomenclature Committee (Lefranc 2007, 2008). It provides a common access to standardized and integrated data from genome, proteome, genetics and three-dimensional (3D) structures (Lefranc et al. 2005a). IMGT comprises six databases (for sequences, genes and 3D structures), 15 online tools and Web resources (more than 10,000 HTML pages) (Lefranc et al. 2009) (Fig. 2.1). The accuracy and the consistency of the IMGT data are based on IMGT-ONTOLOGY, the first ontology for immunogenetics and immunoinformatics (Giudicelli and Lefranc 1999; Lefranc et al. 2004; Duroux et al. 2008). IMGT provides the informatics frame and knowledge environment for a standardized analysis of the antibody sequences and 3D structures, in the context of antibody engineering (single chain Fragment variable (scFv), phage displays, combinatorial libraries) and antibody humanization (chimeric, humanized and human antibodies).

The immunoglobulin (IG) and T cell receptor (TR) major loci span about 6 Megabases (Mb) of the human genome on chromosomes 2, 7, 14 and 22, and 9 Mb in mouse on chromosomes 6, 12, 13, 14 and 16. There are seven major loci: three IG loci (IGH, IGK, IGL) and four TR loci (TRA, TRB, TRG, TRD), with a distinct repartition of the variable (V), diversity (D), joining (J) and constant (C) genes. The human genome comprises a total number of 608-665 IG and TR genes (371-422 IG and 237-243 TR), depending on the haplotypes, per haploid genome ^1, 2^ of which 531-588 genes are located in the major loci (distributed in 369-418 V, 32 D, 105-109 J and 25-29 C genes). There are also 77 orphons (68 IG and 9 TR) including two processed IG genes, outside the major loci. The number of functional IG and TR genes is 308-356 (136-171 IG and 172-185 TR) per haploid genome. The mouse genome comprises an approximate number of 876 IG and TR genes (624 IG and 252 TR). All these genomic data are available in the IMGT® gene database, IMGT/GENE-DB ^3^. The major contribution of IMGT/GENE-DB has been to establish, for the first time, a standardized nomenclature of the IG and TR genes and alleles of humans and other vertebrates. In April 2009, IMGT/GENE-DB manages 1999 genes and 3026 alleles. [1] Lefranc M.-P. and Lefranc G., The Immunoglobulin FactsBook, Academic Press, London, 458 pages (2001).[2] Lefranc M.-P. and Lefranc G., The T cell receptor FactsBook, Academic Press, London, 398 pages (2001).[3] Giudicelli V. et al. Nucleic Acids Res., 33, D256-261 (2005).

B cells residing the marginal zone (MZ) provide a first line of defense against blood borne pathogens, producing the greater part of circulating natural antibodies conferring protection against infection. Dysregulated homeostasis and function of MZ B cells has been implicated in a wide range of B lymphoproliferations, encompassing the distinct MZ lymphomas recognized by the WHO Classification, the related provisional entities and even chronic lymphocytic leukemia (CLL), for which a MZ derivation has been proposed. Here, taking advantage of a large multi-institutional series, we aimed at obtaining insight into the ontogenetic relationship of MZ lymphoproliferations, related entities and CLL through cross-comparison of their B cell receptor immunoglobulin (BcR IG) gene repertoires. Our sequence dataset included 3660 unique IGHV-IGHD-IGHJ gene rearrangement sequences from our collaborative centers and/or public databases derived from: (1) MZ lymphomas: splenic (SMZL), n=379; nodal (NMZL), n=37; extranodal (ENMZL), n=95; (2) provisional entities of postulated MZ origin, including splenic diffuse red pulp lymphoma (SDRL, n=16) and clonal B cell lymphocytosis of MZ origin (CBL-MZ, n=60); (3) persistent polyclonal B cell lymphocytosis (PPBL), n=286 (from 2 cases); (4) MZ cells isolated from six spleen specimens free of neoplastic cells at histological inspection (non-malignant MZ), obtained at surgery for cancer, n=489; (5) autoimmune conditions, n=1243; (6) various types of normal B cells, n=1055. The most pronounced IG gene repertoire skewing was observed in SMZL with the IGHV1-2*04 gene accounting for 26% of cases. Restrictions, though less striking, were also identified in the other MZ lymphomas as well: (i) the IGHV4-34 gene predominated in NMZL (14.3%); and, (ii) the IGHV1-69 gene predominated in ENMZL (14.6%), albeit with significantly different distribution depending on the primary site of involvement, ranging from 38% in salivary ENMZL to 11% in gastric ENMZL to 4% in ocular adnexa ENMZL (p Disclosures Ghia: Janssen Pharmaceuticals: Research Funding.

IMGT/LIGM-DB is the IMGT comprehensive database of immunoglobulin (IG) and T cell receptor (TR) nucleotide sequences from human and other vertebrate species. It was created in 1989 by LIGM, Montpellier, France and is the oldest and the largest database of IMGT. IMGT/LIGM-DB includes all germline (non-rearranged) and rearranged IG and TR genomic DNA (gDNA) and complementary DNA (cDNA) sequences published in generalist databases. IMGT/LIGM-DB allows searches from the Web interface according to biological and immunogenetic criteria through five distinct modules depending on the user interest. For a given entry, nine types of display are available including the IMGT flat file, the translation of the coding regions and the analysis by the IMGT/V-QUEST tool. IMGT/LIGM-DB distributes expertly annotated sequences. The annotations hugely enhance the quality and the accuracy of the distributed detailed information. They include the sequence identification, the gene and allele classification, the constitutive and specific motif description, the codon and amino acid numbering, and the sequence obtaining information, according to the main concepts of IMGT-ONTOLOGY. They represent the main source of IG and TR gene and allele knowledge stored in IMGT/GENE-DB and in the IMGT reference directory. IMGT/LIGM-DB is freely available at http://imgt.cines.fr.

IMGT/GENE-DB is the comprehensive IMGT genome database for immunoglobulin (IG) and T cell receptor (TR) genes from human and mouse, and, in development, from other vertebrates. IMGT/GENE-DB is the international reference for the IG and TR gene nomenclature and works in close collaboration with the HUGO Nomenclature Committee, Mouse Genome Database and genome committees for other species. IMGT/GENE-DB allows a search of IG and TR genes by locus, group and subgroup, which are CLASSIFICATION concepts of IMGT-ONTOLOGY. Short cuts allow the retrieval gene information by gene name or clone name. Direct links with configurable URL give access to information usable by humans or programs. An IMGT/GENE-DB entry displays accurate gene data related to genome (gene localization), allelic polymorphisms (number of alleles, IMGT reference sequences, functionality, etc.) gene expression (known cDNAs), proteins and structures (Protein displays, IMGT Colliers de Perles). It provides internal links to the IMGT sequence databases and to the IMGT Repertoire Web resources, and external links to genome and generalist sequence databases. IMGT/GENE-DB manages the IMGT reference directory used by the IMGT tools for IG and TR gene and allele comparison and assignment, and by the IMGT databases for gene data annotation. IMGT/GENE-DB is freely available at http://imgt.cines.fr.

The human IGHV4-34 gene encodes antibodies which are intrinsically autoreactive when the VH domain is unmutated. Therefore, B cells expressing IGHV4-34 B-cell receptor immunoglobulins (BcR IG) are normally under close scrutiny in order to avoid unwanted autoreactivity, especially against DNA. The IGHV4-34 gene is frequently utilized in chronic lymphocytic leukemia (CLL), where, typically, it shows a high load of somatic hypermutation (SHM). We have previously reported distinctive SHM patterns amongst IGHV4-34 CLL, especially for subsets with stereotyped BcR IG. However, although a large number of cases (~2000) was previously studied, since even the largest subsets account for only ~3% of CLL, meaningful conclusions could not be reached for smaller subsets. Here we revisit this issue in a series of 16,528 CLL cases and focus on IGHV4-34 expressing subsets: #4 (IGHV4-34/IGHD5-18/IGHJ6 | 156 cases, 0.9%); #11 (IGHV4-34/IGHD3-10/IGHJ4 | 16 cases, 0.1%); #16 (IGHV4-34/IGHD2-15/IGHJ6 | 41 cases, 0.25%); #29 (IGHV4-34/IGHD: unassignable/IGHJ3 | 39 cases, 0.24%); and #201 (IGHV4-34/IGHD: unassignable/IGHJ3 | 43 cases 0.26%). Focusing on codons 27-104 within the VH domain (from CDR1-IMGT to FR3-IMGT), we calculated the sequence distance between subsets and the corresponding IGHV4-34 germline sequence based on a pairwise qualitative and quantitative comparison of the respective amino acid composition. The minimum distance calculated, and hence the greatest identity, was observed between subsets #4 and #16, both concerning IgG-switched cases (IgG-CLL), which is notable given the overall rarity of IgG-CLL. In contrast, the maximum distance, implying the least identity, was between subsets #16 and #201, the latter concerning IgM/D-CLL. Extreme variations between subsets were noted in codons spanning the entire VH domain. This result is consistent with our finding of a subset-biased distribution of mutations over the VH domain. More specifically, while subsets #11, #16, #29 and #201 had a lower frequency of mutations within VH CDR1 compared to VH CDR2, the exact opposite was seen in subset #4, with 40% of mutations in VH CDR1 versus 27% in VH CDR2. In addition, subsets #4, #11, #16 and #29 had a similar distribution of mutations in VH FR2 and VH FR3, in contrast to subset #201 that showed a preference for VH FR3 over VH FR2. Consequently, we noted that certain positions were targeted in a subset-specific manner e.g. codon 28 in VH CDR1 was heavily targeted in subsets #4 (68.6%) and #16 (87.8%), with most cases carrying an acidic amino acid (AA) introduced by SHM, glycine to glutamic acid, G>E: 51.3% for subset #4 and 78% for subset #16. The high prevalence of acidic AA introduced by SHM in these subsets is notable considering the electropositive nature of their VH CDR3 (especially of subset #4), strongly recalling edited anti-DNA antibodies. Interestingly, the G>E change was identified at a much lower frequency in other IGHV4-34 subsets: 18.75% for subset #11; 2.6% for subset #29; 7% for subset #201, all of which carried electronegative VH CDR3. Further, we noted that certain positions were heavily targeted in all subsets e.g. 56-86% targeting for SHM at codon 92 in VH FR3 where serine is encoded by the agc triplet, the ”hottest of hotspots”. This result could be viewed as sequence- rather than subset-dependent and linked to the molecular features of this codon, which is supported by the low targeting of codon 93 (0-6%), also encoding serine by the tct triplet. Other positions were targeted in all subsets but at vastly different frequencies e.g. codon 64 was targeted in 37.8% in subset #4 rising to 100% in subset #29. Finally, positions heavily targeted by SHM in certain subsets were unmutated in other subsets e.g. codon 36 in VH CDR1 remained unmutated in subset #16, in contrast 76.9% of subset #29 were mutated at this position resulting in an AA change. In conclusion, we document different spectra of SHM and AA changes between stereotyped IGHV4-34 CLL subsets. The finding of subset-biased, recurrent AA changes at certain codons indicates that the respective progenitor cells may have responded in a specific manner to the selecting antigen(s), despite expressing the same IGHV gene, indicating a functional purpose for these modifications. This is exemplified by the molecular characteristics of the recurrent AA changes in subset #4, thereby offering interesting pathogenetic hints. Disclosures No relevant conflicts of interest to declare.

Human anti-thyroid peroxidase (TPO) autoantibodies (aAb) are generated during autoimmune thyroid diseases (AITD). Within recent years, increasing knowledge of the TPO-specific aAb repertoire, gained mainly by the use of combinatorial library methodology, has led to the cloning and sequencing of around 180 human anti-TPO aAb. Analysis of the immunoglobulin (Ig) variable (V) genes encoding the TPO aAb in the ImMunoGeneTics database (IMGT) (http://imgt.cines.fr) reveals major features of the TPO-directed aAb repertoire during AITD. Heavy chain VH domains of TPO-specific aAb from Graves' disease patients preferentially use D proximal IGHV1 genes, whereas those from Hashimoto's thyroiditis are characterized more frequently by IGHV3 genes, mainly located in the middle of the IGH locus. A large proportion of the anti-TPO heavy chain VH domains is obtained following a VDJ recombination process that uses inverted D genes. J distal IGKV1 and IGLV1 genes are predominantly used in TPO aAb. In contrast to the numerous somatic hypermutations in the TPO-specific heavy chains, there is only limited amino acid replacement in most of the TPO-specific light chains, particularly in those encoded by J proximal IGLV or IGKV genes, suggesting that a defect in receptor editing can occur during aAb generation in AITD. Among the predominant IGHV1 or IGKV1 TPO aAb, conserved somatic mutations are the hallmark of the TPO aAb repertoire. The aim of this review is to provide new insights into aAb generation against TPO, a major autoantigen involved in AITD.