Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and ...are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.
We have established a highly efficient 96-well format based strategy to characterize the expressed murine antibody repertoire by combining immunoglobulin (Ig) gene cloning with antibody expression ...and reactivity profiling at the single cell level. Individual mouse B lineage cells are isolated based on defined surface marker expression patterns by fluorescence-activated cell sorting (FACS) and corresponding full-length Ig heavy (H) and Ig light (L) chain variable (V) region gene transcripts are amplified by RT-PCR. Cloning of the amplified products into eukaryotic expression vectors enables the
in vitro production of monoclonal antibodies with antigen specificities identical to the initial B cell antigen receptors. IgH and IgL chain gene sequence information is obtained as part of the cloning procedure and can be directly linked to reactivity profiles of the recombinant antibodies. In summary, our RT-PCR based strategy to generate recombinant monoclonal antibodies from single mouse B cells allows the highly efficient and unbiased characterization of the expressed murine antibody repertoire by sequence analysis and parallel antibody reactivity testing.
Analysis of immunoglobulin (Ig) repertoires aims to comprehend Ig diversity with the goal of predicting humoral immune responses in the context of infection, vaccination, autoimmunity, and ...malignancies. The first next-generation sequencing (NGS) analyses of bulk B cell populations dramatically advanced sampling depth over previous low-throughput single-cell-based protocols, albeit at the expense of accuracy and loss of chain-pairing information. In recent years the field has substantially differentiated, with bulk analyses becoming more accurate while single-cell approaches have gained in throughput. Additionally, new platforms striving to combine high throughput and chain pairing have been developed as well as various computational tools for analysis. Here we review the developments of the past 4–5 years and discuss the open challenges.
Single‐cell PCR and sequencing of full‐length Ig heavy (Igh) and Igk and Igl light chain genes is a powerful tool to measure the diversity of antibody repertoires and allows the functional assessment ...of B‐cell responses through direct Ig gene cloning and the generation of recombinant mAbs. However, the current methodology is not high‐throughput compatible. Here we developed a two‐dimensional bar‐coded primer matrix to combine Igh and Igk/Igl chain gene single‐cell PCR with next‐generation sequencing for the parallel analysis of the antibody repertoire of over 46 000 individual B cells. Our approach provides full‐length Igh and corresponding Igk/Igl chain gene‐sequence information and permits the accurate correction of sequencing errors by consensus building. The use of indexed cell sorting for the isolation of single B cells enables the integration of flow cytometry and Ig gene sequence information. The strategy is fully compatible with established protocols for direct antibody gene cloning and expression and therefore advances over previously described high‐throughput approaches to assess antibody repertoires at the single‐cell level.
Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and ...full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaired. We show at the single cell level that natural Pf infection induces the development of classical memory B cells (CM) and atypical memory B cells (AtM) that produce broadly neutralizing antibodies against blood stage Pf parasites. CM and AtM contribute to anti-Pf serum IgG production, but only AtM show signs of active antibody secretion. AtM and CM were also different in their IgG gene repertoire, suggesting that they develop from different precursors. The findings provide direct evidence that natural Pf infection leads to the development of protective memory B cell antibody responses and suggest that constant immune activation rather than impaired memory function leads to the accumulation of AtM in malaria. Understanding the memory B cell response to natural Pf infection may be key to the development of a malaria vaccine that induces long-lived protection.
Abstract
High-throughput sequencing of the adaptive immune receptor repertoire (AIRR-seq) is providing unprecedented insights into the immune response to disease and into the development of immune ...disorders. The accurate interpretation of AIRR-seq data depends on the existence of comprehensive germline gene reference sets. Current sets are known to be incomplete and unrepresentative of the degree of polymorphism and diversity in human and animal populations. A key issue is the complexity of the genomic regions in which they lie, which, because of the presence of multiple repeats, insertions and deletions, have not proved tractable with short-read whole genome sequencing. Recently, tools and methods for inferring such gene sequences from AIRR-seq datasets have become available, and a community approach has been developed for the expert review and publication of such inferences. Here, we present OGRDB, the Open Germline Receptor Database (https://ogrdb.airr-community.org), a public resource for the submission, review and publication of previously unknown receptor germline sequences together with supporting evidence.
Cardiovascular disease (CVD) is the leading cause of mortality in the world, with most CVD-related deaths resulting from myocardial infarction or stroke. The main underlying cause of thrombosis and ...cardiovascular events is atherosclerosis, an inflammatory disease that can remain asymptomatic for long periods. There is an urgent need for therapeutic and diagnostic options in this area. Atherosclerotic plaques contain autoantibodies
, and there is a connection between atherosclerosis and autoimmunity
. However, the immunogenic trigger and the effects of the autoantibody response during atherosclerosis are not well understood
. Here we performed high-throughput single-cell analysis of the atherosclerosis-associated antibody repertoire. Antibody gene sequencing of more than 1,700 B cells from atherogenic Ldlr
and control mice identified 56 antibodies expressed by in-vivo-expanded clones of B lymphocytes in the context of atherosclerosis. One-third of the expanded antibodies were reactive against atherosclerotic plaques, indicating that various antigens in the lesion can trigger antibody responses. Deep proteomics analysis identified ALDH4A1, a mitochondrial dehydrogenase involved in proline metabolism, as a target antigen of one of these autoantibodies, A12. ALDH4A1 distribution is altered during atherosclerosis, and circulating ALDH4A1 is increased in mice and humans with atherosclerosis, supporting the potential use of ALDH4A1 as a disease biomarker. Infusion of A12 antibodies into Ldlr
mice delayed plaque formation and reduced circulating free cholesterol and LDL, suggesting that anti-ALDH4A1 antibodies can protect against atherosclerosis progression and might have therapeutic potential in CVD.
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.
The sequencing of immunoglobulin (Ig) transcripts from single B cells yields essential information about Ig heavy:light chain pairing, which is lost in conventional bulk sequencing experiments. The ...previously limited throughput of single-cell approaches has recently been overcome by the introduction of multiple next-generation sequencing (NGS)-based platforms. Furthermore, single-cell techniques allow the assignment of additional data types (e.g. cell surface marker expression), which are crucial for biological interpretation. However, the currently available computational tools are not designed to handle single-cell data and do not provide integral solutions for linking of sequence data to other biological data.
Here we introduce sciReptor, a flexible toolkit for the processing and analysis of antigen receptor repertoire sequencing data at single-cell level. The software combines bioinformatics tools for immunoglobulin sequence annotation with a relational database, where raw data and analysis results are stored and linked. sciReptor supports attribution of additional data categories such as cell surface marker expression or immunological metadata. Furthermore, it comprises a quality control module as well as basic repertoire visualization tools.
sciReptor is a flexible framework for standardized sequence analysis of antigen receptor repertoires on single-cell level. The relational database allows easy data sharing and downstream analyses as well as immediate comparisons between different data sets.
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