June 1, 2021  |  

Allelic specificity of immunoglobulin heavy chain ([email protected]) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing

Oncogenic fusion of IGH-DUX4 has recently been reported as a hallmark that defines a B-ALL subtype present in up to 7% of adolescents and young adults B-ALL. The translocation of DUX4 into IGH results in aberrant activation of DUX4 by hijacking the intronic IGH enhancer (Eµ). How IGH-DUX4 translocation interplays with IGH allelic exclusion was never been explored. We investigated this in Nalm6 B-ALL cell line, using long-read (PacBio Iso-Seq method and 10X Chromium WGS), short-read (Illumina total stranded RNA and WGS), epigenome (H3K27ac ChIP-seq, ATAC-seq) and 3-D genome (Hi-C, H3K27ac HiChIP, Capture-C).

April 21, 2020  |  

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

September 22, 2019  |  

Next-generation approaches to advancing eco-immunogenomic research in critically endangered primates.

High-throughput sequencing platforms are generating massive amounts of genomic data from nonmodel species, and these data sets are valuable resources that can be mined to advance a number of research areas. An example is the growing amount of transcriptome data that allow for examination of gene expression in nonmodel species. Here, we show how publicly available transcriptome data from nonmodel primates can be used to design novel research focused on immunogenomics. We mined transcriptome data from the world’s most endangered group of primates, the lemurs of Madagascar, for sequences corresponding to immunoglobulins. Our results confirmed homology between strepsirrhine and haplorrhine primate immunoglobulins and allowed for high-throughput sequencing of expressed antibodies (Ig-seq) in Coquerel’s sifaka (Propithecus coquereli). Using both Pacific Biosciences RS and Ion Torrent PGM sequencing, we performed Ig-seq on two individuals of Coquerel’s sifaka. We generated over 150 000 sequences of expressed antibodies, allowing for molecular characterization of the antigen-binding region. Our analyses suggest that similar VDJ expression patterns exist across all primates, with sequences closely related to the human VH 3 immunoglobulin family being heavily represented in sifaka antibodies. Moreover, the antigen-binding region of sifaka antibodies exhibited similar amino acid variation with respect to haplorrhine primates. Our study represents the first attempt to characterize sequence diversity of the expressed antibody repertoire in a species of lemur. We anticipate that methods similar to ours will provide the framework for investigating the adaptive immune response in wild populations of other nonmodel organisms and can be used to advance the burgeoning field of eco-immunology. © 2014 John Wiley & Sons Ltd.

September 22, 2019  |  

Novel exons and splice variants in the human antibody heavy chain identified by single cell and single molecule sequencing.

Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

September 22, 2019  |  

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding.

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo.

September 22, 2019  |  

Transcriptome sequencing reveals thousands of novel long non-coding RNAs in B cell lymphoma.

Gene profiling of diffuse large B cell lymphoma (DLBCL) has revealed broad gene expression deregulation compared to normal B cells. While many studies have interrogated well known and annotated genes in DLBCL, none have yet performed a systematic analysis to uncover novel unannotated long non-coding RNAs (lncRNA) in DLBCL. In this study we sought to uncover these lncRNAs by examining RNA-seq data from primary DLBCL tumors and performed supporting analysis to identify potential role of these lncRNAs in DLBCL.We performed a systematic analysis of novel lncRNAs from the poly-adenylated transcriptome of 116 primary DLBCL samples. RNA-seq data were processed using de novo transcript assembly pipeline to discover novel lncRNAs in DLBCL. Systematic functional, mutational, cross-species, and co-expression analyses using numerous bioinformatics tools and statistical analysis were performed to characterize these novel lncRNAs.We identified 2,632 novel, multi-exonic lncRNAs expressed in more than one tumor, two-thirds of which are not expressed in normal B cells. Long read single molecule sequencing supports the splicing structure of many of these lncRNAs. More than one-third of novel lncRNAs are differentially expressed between the two major DLBCL subtypes, ABC and GCB. Novel lncRNAs are enriched at DLBCL super-enhancers, with a fraction of them conserved between human and dog lymphomas. We see transposable elements (TE) overlap in the exonic regions; particularly significant in the last exon of the novel lncRNAs suggest potential usage of cryptic TE polyadenylation signals. We identified highly co-expressed protein coding genes for at least 88 % of the novel lncRNAs. Functional enrichment analysis of co-expressed genes predicts a potential function for about half of novel lncRNAs. Finally, systematic structural analysis of candidate point mutations (SNVs) suggests that such mutations frequently stabilize lncRNA structures instead of destabilizing them.Discovery of these 2,632 novel lncRNAs in DLBCL significantly expands the lymphoma transcriptome and our analysis identifies potential roles of these lncRNAs in lymphomagenesis and/or tumor maintenance. For further studies, these novel lncRNAs also provide an abundant source of new targets for antisense oligonucleotide pharmacology, including shared targets between human and dog lymphomas.

September 22, 2019  |  

The antibody loci of the domestic goat (Capra hircus).

The domestic goat (Capra hircus) is an important ruminant species both as a source of antibody-based reagents for research and biomedical applications and as an economically important animal for agriculture, particularly for developing nations that maintain most of the global goat population. Characterization of the loci encoding the goat immune repertoire would be highly beneficial for both vaccine and immune reagent development. However, in goat and other species whose reference genomes were generated using short-read sequencing technologies, the immune loci are poorly assembled as a result of their repetitive nature. Our recent construction of a long-read goat genome assembly (ARS1) has facilitated characterization of all three antibody loci with high confidence and comparative analysis to cattle. We observed broad similarity of goat and cattle antibody-encoding loci but with notable differences that likely influence formation of the functional antibody repertoire. The goat heavy-chain locus is restricted to only four functional and nearly identical IGHV genes, in contrast to the ten observed in cattle. Repertoire analysis indicates that light-chain usage is more balanced in goats, with greater representation of kappa light chains (~ 20-30%) compared to that in cattle (~ 5%). The present study represents the first characterization of the goat antibody loci and will help inform future investigations of their antibody responses to disease and vaccination.

July 19, 2019  |  

Germinal center centroblasts transition to a centrocyte phenotype according to a timed program and depend on the dark zone for effective selection.

Germinal center (GC) B cells cycle between the dark zone (DZ) and light zone (LZ) during antibody affinity maturation. Whether this movement is necessary for GC function has not been tested. Here we show that CXCR4-deficient GC B cells, which are restricted to the LZ, are gradually outcompeted by WT cells indicating an essential role for DZ access. Remarkably, the transition between DZ centroblast and LZ centrocyte phenotypes occurred independently of positioning. However, CXCR4-deficient cells carried fewer mutations and were overrepresented in the CD73(+) memory compartment. These findings are consistent with a model where GC B cells change from DZ to LZ phenotype according to a timed cellular program but suggest that spatial separation of DZ cells facilitates more effective rounds of mutation and selection. Finally, we identify a network of DZ CXCL12-expressing reticular cells that likely support DZ functions. Copyright © 2013 Elsevier Inc. All rights reserved.

July 19, 2019  |  

SMRT Sequencing for parallel analysis of multiple targets and accurate SNP phasing.

Single-molecule real-time (SMRT) sequencing generates much longer reads than other widely used next-generation (next-gen) sequencing methods, but its application to whole genome/exome analysis has been limited. Here, we describe the use of SMRT sequencing coupled with barcoding to simultaneously analyze one or a small number of genomic targets derived from multiple sources. In the budding yeast system, SMRT sequencing was used to analyze strand-exchange intermediates generated during mitotic recombination and to analyze genetic changes in a forward mutation assay. The general barcoding-SMRT approach was then extended to diffuse large B-cell lymphoma primary tumors and cell lines, where detected changes agreed with prior Illumina exome sequencing. A distinct advantage afforded by SMRT sequencing over other next-gen methods is that it immediately provides the linkage relationships between SNPs in the target segment sequenced. The strength of our approach for mutation/recombination studies (as well as linkage identification) derives from its inherent computational simplicity coupled with a lack of reliance on sophisticated statistical analyses. Copyright © 2015 Guo et al.

July 19, 2019  |  

Highly efficient CRISPR/Cas9-mediated cloning and functional characterization of gastric cancer-derived Epstein-Barr virus strains.

The Epstein-Barr virus (EBV) is etiologically linked to approximately 10% of gastric cancers, in which viral genomes are maintained as multicopy episomes. EBV-positive gastric cancer cells are incompetent for progeny virus production, making viral DNA cloning extremely difficult. Here we describe a highly efficient strategy for obtaining bacterial artificial chromosome (BAC) clones of EBV episomes by utilizing a CRISPR/Cas9-mediated strand break of the viral genome and subsequent homology-directed repair. EBV strains maintained in two gastric cancer cell lines (SNU719 and YCCEL1) were cloned, and their complete viral genome sequences were determined. Infectious viruses of gastric cancer cell-derived EBVs were reconstituted, and the viruses established stable latent infections in immortalized keratinocytes. While Ras oncoprotein overexpression caused massive vacuolar degeneration and cell death in control keratinocytes, EBV-infected keratinocytes survived in the presence of Ras expression. These results implicate EBV infection in predisposing epithelial cells to malignant transformation by inducing resistance to oncogene-induced cell death.Recent progress in DNA-sequencing technology has accelerated EBV whole-genome sequencing, and the repertoire of sequenced EBV genomes is increasing progressively. Accordingly, the presence of EBV variant strains that may be relevant to EBV-associated diseases has begun to attract interest. Clearly, the determination of additional disease-associated viral genome sequences will facilitate the identification of any disease-specific EBV variants. We found that CRISPR/Cas9-mediated cleavage of EBV episomal DNA enabled the cloning of disease-associated viral strains with unprecedented efficiency. As a proof of concept, two gastric cancer cell-derived EBV strains were cloned, and the infection of epithelial cells with reconstituted viruses provided important clues about the mechanism of EBV-mediated epithelial carcinogenesis. This experimental system should contribute to establishing the relationship between viral genome variation and EBV-associated diseases. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

July 19, 2019  |  

ARTISAN PCR: rapid identification of full-length immunoglobulin rearrangements without primer binding bias.

B cells recognize specific antigens by their membrane-bound B-cell receptor (BCR). Functional BCR genes are assembled in pre-B cells by recombination of the variable (V), diversity (D) and joining (J) genes [V(D)J recombination]. When B cells participate in germinal centre reactions, non-templated point mutations are introduced into BCR genes by somatic hypermutation (SHM) (Rajewsky, 1996). V(D)J recombination and SHM create virtually unlimited BCR repertoires.

July 19, 2019  |  

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

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.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.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.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.

July 7, 2019  |  

In-depth determination and analysis of the human paired heavy- and light-chain antibody repertoire.

High-throughput immune repertoire sequencing has emerged as a critical step in the understanding of adaptive responses following infection or vaccination or in autoimmunity. However, determination of native antibody variable heavy-light pairs (VH-VL pairs) remains a major challenge, and no technologies exist to adequately interrogate the >1 × 10(6) B cells in typical specimens. We developed a low-cost, single-cell, emulsion-based technology for sequencing antibody VH-VL repertoires from >2 × 10(6) B cells per experiment with demonstrated pairing precision >97%. A simple flow-focusing apparatus was used to sequester single B cells into emulsion droplets containing lysis buffer and magnetic beads for mRNA capture; subsequent emulsion RT-PCR generated VH-VL amplicons for next-generation sequencing. Massive VH-VL repertoire analyses of three human donors provided new immunological insights including (i) the identity, frequency and pairing propensity of shared, or ‘public’, VL genes, (ii) the detection of allelic inclusion (an implicated autoimmune mechanism) in healthy individuals and (iii) the occurrence of antibodies with features, in terms of gene usage and CDR3 length, associated with broadly neutralizing antibodies to rapidly evolving viruses such as HIV-1 and influenza.

July 7, 2019  |  

Proteomic analysis of Pemphigus autoantibodies indicates a larger, more diverse, and more dynamic repertoire than determined by B cell genetics.

In autoantibody-mediated diseases such as pemphigus, serum antibodies lead to disease. Genetic analysis of B cells has allowed characterization of antibody repertoires in such diseases but would be complemented by proteomic analysis of serum autoantibodies. Here, we show using proteomic analysis that the serum autoantibody repertoire in pemphigus is much more polyclonal than that found by genetic studies of B cells. In addition, many B cells encode pemphigus autoantibodies that are not secreted into the serum. Heavy chain variable gene usage of serum autoantibodies is not shared among patients, implying targeting of the coded proteins will not be a useful therapeutic strategy. Analysis of autoantibodies in individual patients over several years indicates that many antibody clones persist but the proportion of each changes. These studies indicate a dynamic and diverse autoantibody response not revealed by genetic studies and explain why similar overall autoantibody titers may give variable disease activity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

July 7, 2019  |  

Antibodyomics: bioinformatics technologies for understanding B-cell immunity to HIV-1.

Numerous antibodies have been identified from HIV-1-infected donors that neutralize diverse strains of HIV-1. These antibodies may provide the basis for a B cell-mediated HIV-1 vaccine. However, it has been unclear how to elicit similar antibodies by vaccination. To address this issue, we have undertaken an informatics-based approach to understand the genetic and immunologic processes controlling the development of HIV-1-neutralizing antibodies. As DNA sequencing comprises the fastest growing database of biological information, we focused on incorporating next-generation sequencing of B-cell transcripts to determine the origin, maturation pathway, and prevalence of broadly neutralizing antibody lineages (Antibodyomics1, 2, 4, and 6). We also incorporated large-scale robotic analyses of serum neutralization to identify and quantify neutralizing antibodies in donor cohorts (Antibodyomics3). Statistical analyses furnish another layer of insight (Antibodyomics5), with physical characteristics of antibodies and their targets through molecular dynamics simulations (Antibodyomics7) and free energy perturbation analyses (Antibodyomics8) providing information-rich output. Functional interrogation of individual antibodies (Antibodyomics9) and synthetic antibody libraries (Antibodyomics10) also yields multi-dimensional data by which to understand and improve antibodies. Antibodyomics, described here, thus comprise resolution-enhancing tools, which collectively embody an information-driven discovery engine aimed toward the development of effective B cell-based vaccines.© 2017 The Authors. Immunological Reviews published by John Wiley & Sons Ltd.

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