Enhancers within the Ig V Gene Region Orchestrate Chromatin Topology and Regulate V Gene Rearrangement Frequency to Shape the B Cell Receptor Repertoire Specificities.

Effective Ab-mediated responses depend on a highly diverse Ab repertoire with the ability to bind a wide range of epitopes in disease-causing agents. The generation of this repertoire depends on the somatic recombination of the variable (V), diversity (D), and joining (J) genes in the Ig loci of developing B cells. It has been known for some time that individual V, D, and J gene segments rearrange at different frequencies, but the mechanisms behind this unequal V gene usage have not been well understood. However, recent work has revealed that newly described enhancers scattered throughout the V gene-containing portion of the Ig loci regulate the V gene recombination frequency in a regional manner. Deletion of three of these enhancers revealed that these elements exert many layers of control during V(D)J recombination, including long-range chromatin interactions, epigenetic milieu, chromatin accessibility, and compartmentalization.

The evolutionary and functional significance of germline immunoglobulin gene variation.

The recombination between immunoglobulin (IG) gene segments determines an individual's naïve antibody repertoire and, consequently, (auto)antigen recognition. Emerging evidence suggests that mammalian IG germline variation impacts humoral immune responses associated with vaccination, infection, and autoimmunity - from the molecular level of epitope specificity, up to profound changes in the architecture of antibody repertoires. These links between IG germline variants and immunophenotype raise the question on the evolutionary causes and consequences of diversity within IG loci. We discuss why the extreme diversity in IG loci remains a mystery, why resolving this is important for the design of more effective vaccines and therapeutics, and how recent evidence from multiple lines of inquiry may help us do so.

Immunoglobulins genes in Neoceratodus forsteri and Protopterus annectens explain the origin of the immunoglobulins of the animals that passed ashore.

Sarcopterygian fishes are a taxon of bony fishes. They include lungfish and coelacanths (six species of lungfish and two species of coelacanths). Evolutionary adaptations arose with these fish, such as the appearance of lungs and paired lobed fins that help them move over the bottom of the sea. In the Devonian period, they came ashore, and tetrapods (amphibians, reptiles, and mammals) arose from them. Within immunology, they can teach us about the emergence of immunoglobulins D, A/X, and Y already present in amphibians. We have studied the genes of the immunoglobulins in the fish Sarcopterygii Neoceratodus forsteri and Protopterus annectens. In the first fish, we find that several loci for the constant chains of immunoglobulins are distributed across 4 chromosomes. We have found four genes for IgM, a gene for IgW and a gene for IgN. In the second, we find one locus with genes for IgN and IgM and another with one gene for IgW. With these sequences, together with those obtained in other publications, we have been able to study the possible evolution and emergence of immunoglobulin classes. We conclude that there are two evolutionary lineages, one focused on IgM and very conservative, and the other focused on IgW, which allows high variability. In the case of the animals that went to land, their IgD is formed only by domains whose origin is in the W lineage. IgA/X and IgY are unique since they arose from the recombination between the two evolutionary lineagess (M and W). In both IgA/X and IgY, the CH1 and CH2 domains come from domains whose origin is the W lineage, while their CH3 and CH4 derive from the M lineage.

Next-Generation Sequencing-Based Clonality Detection of Immunoglobulin Gene Rearrangements in B-Cell Lymphoma.

Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma's that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis.

cfDNA-Based NGS IG Analysis in Lymphoma.

Liquid biopsy is a novel diagnostic approach at first developed to characterize the molecular profile of solid tumors by analyzing body fluids. For cancer patients, it represents a noninvasive way to monitor the status of the solid tumor with respect to representative biomarkers. There is growing interest in the utilization of circulating tumor DNA (ctDNA) analysis also in the diagnostic and prognostic fields of lymphomas. Clonal immunoglobulin (IG) gene rearrangements are fingerprints of the respective lymphoid malignancy and thus are highly suited as specific molecular targets for minimal residual disease (MRD) detection. Tracing of the clonal IG rearrangement patterns in ctDNA pool during treatment can be used for MRD assessment in B-cell lymphomas. Here, we describe a reproducible next-generation sequencing assay to identify and characterize clonal IG gene rearrangements for MRD detection in cell-free DNA.

HMCES protects immunoglobulin genes specifically from deletions during somatic hypermutation.

Somatic hypermutation (SHM) produces point mutations in immunoglobulin (Ig) genes in B cells when uracils created by the activation-induced deaminase are processed in a mutagenic manner by enzymes of the base excision repair (BER) and mismatch repair (MMR) pathways. Such uracil processing creates DNA strand breaks and is susceptible to the generation of deleterious deletions. Here, we demonstrate that the DNA repair factor HMCES strongly suppresses deletions without significantly affecting other parameters of SHM in mouse and human B cells, thereby facilitating the production of antigen-specific antibodies. The deletion-prone repair pathway suppressed by HMCES operates downstream from the uracil glycosylase UNG and is mediated by the combined action of BER factor APE2 and MMR factors MSH2, MSH6, and EXO1. HMCES's ability to shield against deletions during SHM requires its capacity to form covalent cross-links with abasic sites, in sharp contrast to its DNA end-joining role in class switch recombination but analogous to its genome-stabilizing role during DNA replication. Our findings lead to a novel model for the protection of Ig gene integrity during SHM in which abasic site cross-linking by HMCES intercedes at a critical juncture during processing of vulnerable gapped DNA intermediates by BER and MMR enzymes.

Promoter Proximity Defines Mutation Window for VH and VΚ Genes Rearranged to Different J Genes.

Somatic hypermutation induced by activation-induced deaminase (AID) occurs at high densities between the Ig V gene promoter and intronic enhancer, which encompasses DNA encoding the rearranged V gene exon and J intron. It has been proposed that proximity between the promoter and enhancer defines the boundaries of mutation in V regions. However, depending on the J gene used, the distance between the promoter and enhancer is quite variable and may result in differential targeting around the V gene. To examine the effect of distance in mutation accumulation, we sequenced 320 clones containing different endogenous rearranged V genes in the IgH and Igκ loci from Peyer's patch B cells of mice. Clones were grouped by their use of different J genes. Distances between the V gene and enhancer ranged from ∼2.3 kb of intron DNA for rearrangements using J1, ∼2.0 kb for rearrangements using J2, ∼1.6 kb for rearrangements using J3 (H) or 4 (κ), and 1.1 kb for rearrangements using J4 (H) or 5 (κ). Strikingly, >90% of intron mutations occurred within 1 kb downstream of the J gene for both H and κ clones, regardless of which J gene was used. Thus, there is no evidence that the intron sequence or enhancer plays a role in determining the extent of mutation. The results indicate that V region intron mutations are targeted by their proximity to the promoter, suggesting they result from AID interactions with RNA polymerase II over a 1-kb region.

Somatic Diversification of Rearranged Antibody Gene Segments by Intra- and Interchromosomal Templated Mutagenesis.

The ability of the humoral immune system to generate Abs capable of specifically binding a myriad of Ags is critically dependent on the somatic hypermutation program. This program induces both templated mutations (i.e., gene conversion) and untemplated mutations. In humans, somatic hypermutation is widely believed to result in untemplated point mutations. In this study, we demonstrate detection of large-scale templated events that occur in human memory B cells and circulating plasmablasts. We find that such mutations are templated intrachromosomally from IGHV genes and interchromosomally from IGHV pseudogenes as well as other homologous regions unrelated to IGHV genes. These same donor regions are used in multiple individuals, and they predominantly originate from chromosomes 14, 15, and 16. In addition, we find that exogenous sequences placed at the IgH locus, such as LAIR1, undergo templated mutagenesis and that homology appears to be the major determinant for donor choice. Furthermore, we find that donor tracts originate from areas in proximity with open chromatin, which are transcriptionally active, and are found in spatial proximity with the IgH locus during the germinal center reaction. These donor sequences are inserted into the Ig gene segment in association with overlapping activation-induced cytidine deaminase hotspots. Taken together, these studies suggest that diversity generated during the germinal center response is driven by untemplated point mutations as well as templated mutagenesis using local and distant regions of the genome.

The spliceosome component Usp39 controls B cell development by regulating immunoglobulin gene rearrangement.

The spliceosome is a large ribonucleoprotein complex responsible for pre-mRNA splicing and genome stability maintenance. Disruption of the spliceosome activity may lead to developmental disorders and tumorigenesis. However, the physiological role that the spliceosome plays in B cell development and function is still poorly defined. Here, we demonstrate that ubiquitin-specific peptidase 39 (Usp39), a spliceosome component of the U4/U6.U5 tri-snRNP complex, is essential for B cell development. Ablation of Usp39 in B cell lineage blocks pre-pro-B to pro-B cell transition in the bone marrow, leading to a profound reduction of mature B cells in the periphery. We show that Usp39 specifically regulates immunoglobulin gene rearrangement in a spliceosome-dependent manner, which involves modulating chromatin interactions at the Igh locus. Moreover, our results indicate that Usp39 deletion reduces the pre-malignant B cells in Eµ-Myc transgenic mice and significantly improves their survival.

HomA and HomB, outer membrane proteins of Helicobacter pylori down-regulate activation-induced cytidine deaminase (AID) and Ig switch germline transcription and thereby affect class switch recombination (CSR) of Ig genes in human B-cells.

H. pylori is one of the major causes of chronic gastritis, peptic ulcer disease (PUD), gastric mucosa-associated lymphoid tissue lymphoma (MALT) and gastric carcinoma. H. pylori toxin VacA is responsible for host cell apoptosis, whereas CagA is known to aberrantly induce expression of activation-induced cytidine deaminase (AID) in gastric epithelial cells that causes mutations in oncogenes and tumour suppressor genes, leading to the transformation of normal cells into cancerous cells. Although, a significant amount of research has been conducted to understand the role of bacterial factors modulating deregulated host cell pathways, the interaction between H. pylori and immune cells of the marginal zone and its consequences are still not well understood. HomB and HomA, outer membrane proteins (OMPs) from H. pylori, which assist in the adhesion of bacteria to host cells, are found to be associated with H. pylori virulent strains and promote inflammation. Interestingly, we observed that the interaction of HomB/HomA OMPs with B-cells transiently downregulates AID expression and Ig switch germline transcription. Downregulation of AID leads to impairment of class switch recombination (CSR), resulting in significantly reduced switching to IgG and IgA antibodies. Besides, we examined the immune-suppressive response of B-cells and observed that the cells stimulated with HomA/B show upregulation in the levels of IL10, IL35, as well as PDL1, a T-cell inhibition marker. Our study suggests the potential role of OMPs in immune response modulation strategies used by the pathogen to evade the immune response. These results provide a better understanding of H. pylori pathogenesis and assist in identifying novel targets for therapy.

Limitations of lymphoblastoid cell lines for establishing genetic reference datasets in the immunoglobulin loci.

Lymphoblastoid cell lines (LCLs) have been critical to establishing genetic resources for biomedical science. They have been used extensively to study human genetic diversity, genome function, and inform the development of tools and methodologies for augmenting disease genetics research. While the validity of variant callsets from LCLs has been demonstrated for most of the genome, previous work has shown that DNA extracted from LCLs is modified by V(D)J recombination within the immunoglobulin (IG) loci, regions that harbor antibody genes critical to immune system function. However, the impacts of V(D)J on short read sequencing data generated from LCLs has not been extensively investigated. In this study, we used LCL-derived short read sequencing data from the 1000 Genomes Project (n = 2,504) to identify signatures of V(D)J recombination. Our analyses revealed sample-level impacts of V(D)J recombination that varied depending on the degree of inferred monoclonality. We showed that V(D)J associated somatic deletions impacted genotyping accuracy, leading to adulterated population-level estimates of allele frequency and linkage disequilibrium. These findings illuminate limitations of using LCLs and short read data for building genetic resources in the IG loci, with implications for interpreting previous disease association studies in these regions.

A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain.

The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.

Mutational mechanisms shaping the coding and noncoding genome of germinal center derived B-cell lymphomas.

B cells have the unique property to somatically alter their immunoglobulin (IG) genes by V(D)J recombination, somatic hypermutation (SHM) and class-switch recombination (CSR). Aberrant targeting of these mechanisms is implicated in lymphomagenesis, but the mutational processes are poorly understood. By performing whole genome and transcriptome sequencing of 181 germinal center derived B-cell lymphomas (gcBCL) we identified distinct mutational signatures linked to SHM and CSR. We show that not only SHM, but presumably also CSR causes off-target mutations in non-IG genes. Kataegis clusters with high mutational density mainly affected early replicating regions and were enriched for SHM- and CSR-mediated off-target mutations. Moreover, they often co-occurred in loci physically interacting in the nucleus, suggesting that mutation hotspots promote increased mutation targeting of spatially co-localized loci (termed hypermutation by proxy). Only around 1% of somatic small variants were in protein coding sequences, but in about half of the driver genes, a contribution of B-cell specific mutational processes to their mutations was found. The B-cell-specific mutational processes contribute to both lymphoma initiation and intratumoral heterogeneity. Overall, we demonstrate that mutational processes involved in the development of gcBCL are more complex than previously appreciated, and that B cell-specific mutational processes contribute via diverse mechanisms to lymphomagenesis.

Diverse immunoglobulin gene usage and convergent epitope targeting in neutralizing antibody responses to SARS-CoV-2.

It is unclear whether individuals with enormous diversity in B cell receptor repertoires are consistently able to mount effective antibody responses against SARS-CoV-2. We analyzed antibody responses in a cohort of 55 convalescent patients and isolated 54 potent neutralizing monoclonal antibodies (mAbs). While most of the mAbs target the angiotensin-converting enzyme 2 (ACE2) binding surface on the receptor binding domain (RBD) of SARS-CoV-2 spike protein, mAb 47D1 binds only to one side of the receptor binding surface on the RBD. Neutralization by 47D1 is achieved independent of interfering RBD-ACE2 binding. A crystal structure of the mAb-RBD complex shows that the IF motif at the tip of 47D1 CDR H2 interacts with a hydrophobic pocket in the RBD. Diverse immunoglobulin gene usage and convergent epitope targeting characterize neutralizing antibody responses to SARS-CoV-2, suggesting that vaccines that effectively present the receptor binding site on the RBD will likely elicit neutralizing antibody responses in a large fraction of the population.

Gouania willdenowi is a teleost fish without immunoglobulin genes.

Immunoglobulin (Ig) genes encode antibodies in jawed vertebrates. They are essential elements of the adaptive immune response. Ig exists in soluble form or as part of the B cell membrane antigen receptor (BCR). Studies of Ig genes in fish genomes reveal the absence of Ig genes in Gouania willdenowi by deletion of the entire Ig locus from the canonical chromosomal region. The genes coding for integral BCR proteins, CD79a and CD79b, are also absent. Genes exist for T α/ß lymphocyte receptors but not for the T γ/δ receptors. The results of the genomic analysis are independently corroborated with RNA-Seq transcriptomes from other Gobiesocidae species. From the transcriptome studies, Ig is also absent from these other Gobiesocidae species, Acyrtus sp. and Tomicodon sp. Present evidence suggests that Ig is missing from all species of the Gobiesocidae family.

Immunoglobulins in teleosts.

Immunoglobulins are glycoproteins which are produced as membrane-bound receptors on B-cells or in a secreted form, known as antibodies. In teleosts, three immunoglobulin isotypes, IgM, IgT, and IgD, are present, each comprising two identical heavy and two identical light polypeptide chains. The basic mechanisms for generation of immunoglobulin diversity are similar in teleosts and higher vertebrates. The B-cell pre-immune repertoire is diversified by VDJ recombination, junctional flexibility, addition of nucleotides, and combinatorial association of light and heavy chains, while the post-immune repertoire undergoes somatic hypermutation during clonal expansion. Typically, the teleost immunoglobulin heavy chain gene complex has a modified translocon arrangement where the Dτ-Jτ-Cτ cluster of IgT is generally located between the variable heavy chain (VH) region and the Dµ/δ-Jµ/δ-Cµ-Cδ gene segments, or within the set of VH gene segments. However, multiple genome duplication and deletion events and loss of some individual genes through evolution has complicated the IgH gene organization. The IgH gene arrangement allows the expression of either IgT or IgM/IgD. Alternative splicing is responsible for the regulation of IgM/IgD expression and the secreted versus transmembrane forms of IgT, IgD, and IgM. The overall structure of IgM and IgT is usually conserved across species, whereas IgD has a large variety of structures. IgM is the main effector molecule in both systemic and mucosal immunity and shows a broad range of concentrations in different teleost species. Although IgM is usually present in higher concentrations under normal conditions, IgT is considered the main mucosal Ig.

An apoptosis-dependent checkpoint for autoimmunity in memory B and plasma cells.

B lymphocytes acquire self-reactivity as an unavoidable byproduct of antibody gene diversification in the bone marrow and in germinal centers (GCs). Autoreactive B cells emerging from the bone marrow are silenced in a series of well-defined checkpoints, but less is known about how self-reactivity that develops by somatic mutation in GCs is controlled. Here, we report the existence of an apoptosis-dependent tolerance checkpoint in post-GC B cells. Whereas defective GC B cell apoptosis has no measurable effect on autoantibody development, disruption of post-GC apoptosis results in accumulation of autoreactive memory B cells and plasma cells, antinuclear antibody production, and autoimmunity. The data presented shed light on mechanisms that regulate immune tolerance and the development of autoantibodies.

Immature Immunoglobulin Gene Rearrangements Are Recurrent in B Precursor Adult Acute Lymphoblastic Leukemia Carrying TP53 Molecular Alterations.

Here, we describe the immunoglobulin and T cell receptor (Ig/TCR) molecular rearrangements identified as a leukemic clone hallmark for minimal residual disease assessment in relation to TP53 mutational status in 171 Ph-negative Acute Lymphoblastic Leukemia (ALL) adult patients at diagnosis. The presence of a TP53 alterations, which represents a marker of poor prognosis, was strictly correlated with an immature DH/JH rearrangement of the immunoglobulin receptor (p < 0.0001). Furthermore, TP53-mutated patients were classified as pro-B ALL more frequently than their wild-type counterpart (46% vs. 25%, p = 0.05). Although the reasons for the co-presence of immature Ig rearrangements and TP53 mutation need to be clarified, this can suggest that the alteration in TP53 is acquired at an early stage of B-cell maturation or even at the level of pre-leukemic transformation.

AncesTree: An interactive immunoglobulin lineage tree visualizer.

High-throughput sequencing of human immunoglobulin genes allows analysis of antibody repertoires and the reconstruction of clonal lineage evolution. The study of antibodies (Abs) affinity maturation is of specific interest to understand the generation of Abs with high affinity or broadly neutralizing activities. Moreover, phylogenic analysis enables the identification of the key somatic mutations required to achieve optimal antigen binding. The Immcantation framework provides a start-to-finish set of analytical methods for high-throughput adaptive immune receptor repertoire sequencing (AIRR-Seq; Rep-Seq) data. Furthermore, Immcantation's Change-O package has developed IgPhyML, an algorithm designed to build specifically immunoglobulin (Ig) phylogenic trees. Meanwhile Phylip, an algorithm that has been originally developed for applications in ecology and macroevolution, can also be used for the phylogenic reconstruction of antibodies maturation pathway. To complement Ig lineages made by IgPhyML or Dnaml (Phylip), we developed AncesTree, a graphic user interface (GUI) that aims to give researchers the opportunity to interactively explore antibodies clonal evolution. AncesTree displays interactive immunoglobulins phylogenic tree, Ig related mutations and sequence alignments using additional information coming from specialized antibody tools. The GUI is a Java standalone application allowing interaction with Ig tree that can run under Windows, Linux and Mac OS.

Lack of Evidence for a Substantial Rate of Templated Mutagenesis in B Cell Diversification.

BCR sequences diversify through mutations introduced by purpose-built cellular machinery. A recent paper has concluded that a "templated mutagenesis" process is a major contributor to somatic hypermutation and therefore Ig diversification in mice and humans. In this proposed process, mutations in the Ig locus are introduced by copying short segments from other Ig genes. If true, this would overturn decades of research on B cell diversification and would require a complete rewrite of computational methods to analyze B cell data for these species. In this paper, we re-evaluate the templated mutagenesis hypothesis. By applying the original inferential method using potential donor templates absent from B cell genomes, we obtain estimates of the methods' false positive rates. We find false positive rates of templated mutagenesis in murine and human Ig loci that are similar to or even higher than the original rate inferences, and by considering the bases used in substitution, we find evidence that if templated mutagenesis occurs, it is at a low rate. We also show that the statistically significant results in the original paper can easily result from a slight misspecification of the null model.