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.

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.

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.

Phenotypic and Ig Repertoire Analyses Indicate a Common Origin of IgD-CD27- Double Negative B Cells in Healthy Individuals and Multiple Sclerosis Patients.

IgD-CD27- double negative (DN) B cells with proinflammatory characteristics are abnormally elevated in a proportion of multiple sclerosis (MS) patients. In this study, the origin and selection characteristics of DN B cells were studied in MS patients and healthy controls (HC). Expression of developmental markers on peripheral blood DN, IgD-CD27+ class-switched memory (CSM) and IgD+CD27- naive B cells of HC (n = 48) and MS patients (n = 96) was determined by flow cytometry. High-throughput adaptive immune receptor repertoire sequencing was performed on peripheral blood DN and CSM B cells of HC and MS patients (n = 3 each). DN B cells from HC and MS patients showed similar phenotypic and Ig repertoire characteristics. Phenotypic analysis indicated a mature state of DN B cells by low CD5, CD10, and CD38 expression. However, the frequency of CD95+ and IgA+ cells was lower in DN versus CSM B cells. DN B cells are Ag experienced, as shown by somatic hypermutation of their Ig genes in adaptive immune receptor repertoire sequencing, although they showed a lower mutation load than CSM B cells. Shared clones were found between DN and CSM B cells, although >95% of the clones were unique to each population, and differences in V(D)J usage and CDR3 physicochemical properties were found. Thus, DN B cells arise in HC and MS patients via a common developmental pathway that is probably linked to immune aging. However, DN and CSM B cells develop through unique differentiation pathways, with most DN B cells representing an earlier maturation state.

Signalling circuits that direct early B-cell development.

In mammals, the B-cell lineage arises from pluripotent progenitors in the bone marrow. During their development, B-cells undergo lineage specification and commitment, followed by expansion and selection. These processes are mediated by regulated changes in gene expression programmes, rearrangements of immunoglobulin (Ig) genes, and well-timed rounds of proliferation and apoptosis. Many of these processes are initiated by environmental factors including cytokines, chemokines, and cell-cell contacts. Developing B-cells process these environmental cues into stage-specific functions via signalling pathways including the PI3K, MAPK, or JAK-STAT pathway. The cytokines FLT3-Ligand and c-Kit-Ligand are important for the early expansion of the B-cell precursors at different developmental stages and conditions. Interleukin 7 is essential for commitment to the B-cell lineage and for orchestrating the Ig recombination machinery. After rearrangement of the immunoglobulin heavy chain, proliferation and apoptosis, and thus selection, are mediated by the clonal pre-B-cell receptor, and, following light chain rearrangement, by the B-cell receptor.

Identification of Two Nonrearranging IgSF Genes in Chicken Reveals a Novel Family of Putative Remnants of an Antigen Receptor Precursor.

In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane proteins, and their recombinant products formed a monomer and a disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel gene family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of the immune system of jawed vertebrates.

Considerations for monitoring minimal residual disease using immunoglobulin clonality in patients with precursor B-cell lymphoblastic leukemia.

BACKGROUND: Minimal residual disease (MRD) monitoring is a powerful tool to predict the risk of relapse. Herein, we present an MRD monitoring strategy for B-cell lymphoblastic leukemia (B-ALL) using high-throughput sequencing (HTS) of immunoglobulin (Ig) clonality before implementation into routine practice. METHODS: We selected 74 bone marrow (BM) specimens from 47 patients who were diagnosed with B-ALL. Ig clonality was analyzed using both fragment analysis and HTS. The performance of Ig clonality was evaluated through comparison of the results from real-time quantitative polymerase chain reaction (qPCR) of leukemia-specific fusion transcripts and flow cytometry. RESULTS: IGH clonality was observed in all patients, and the sum of clonal burden varied (9.47%-96.77%). IGK clonality was identified in 70% of patients and availed in cases with low IGH clonal burden. The total IGH clonal burden was significantly correlated with the proportion of leukemic blasts, leukemia-specific fusion transcripts, and flow cytometry. We recognized the different responses of each clone and emerging clones originating from the trace of Ig rearrangement presented in the initial specimen. IGH clonal burden after chemotherapy represented patient outcomes well. IGH assay also provided information of repertoire diversity of IGH rearrangement. CONCLUSION: The Ig clonality assay via HTS will be a promising tool for MRD monitoring of B-ALL through an adequate strategy to identify and monitor individual clones and determine repertoire diversity.

Immunoglobulin heavy variable (IgHV) gene mutation and micro-RNA expression in Burkitt's lymphoma at Moi Teaching and Referral Hospital in Western Kenya.

INTRODUCTION: Burkitt's lymphoma (BL) is a virus associated childhood B-cell cancer common in Eastern Africa. Continued survival of B-cells in germinal centres depend on expression of high affinity immunoglobulins (Ig) to complementary antigens by somatic hypermutation of Ig genes. Cellular microRNAs, non-coding RNAs have been reported to play role in cell cycle regulation. Both viral antigen dependent mutation and micro-RNA expression maybe involved in BL pathogenesis. OBJECTIVE: To describe immunoglobulin heavy variable (IgHV) rearrangement and micro-RNA expressions in BL tumours. METHODS: Genomic DNA were extracted and purified from BL tissue blocks at Moi Teaching and Referral Hospital, before amplification using IgHV consensus primers and sequencing. The sequences were then aligned with germline alleles in IMGT/V-QUEST® database. Total RNA extracted from tissue blocks and cell lines were used to determine relative expression of hsamiR-34a and hsa-miR-127. RESULTS: In all tumours, allele alignment scores and number of mutations range were 89.2-93.2%, 15-24 respectively. The range of IgHV amino acid changes were higher in EBER-1+ (15-25) than EBER-1- (9-15). In MYC+ tumours, the relative expression were: hsa-miR-127(2.09);hsa-miR-34a (2.8) and MYC- hsa-miR-127 (1.2), hsa-miR-34a (1.0). CONCLUSION: B-cell in BL contained somatic mutated IgHV gene and upregulated cellular microRNAs with possible pathogenetic role(s).

Identification of a Transcriptionally Forward α Gene and Two υ Genes within the Pigeon (Columba livia) IgH Gene Locus.

Compared with mammals, the bird Ig genetic system relies on gene conversion to create an Ab repertoire, with inversion of the IgA-encoding gene and very few cases of Ig subclass diversification. Although gene conversion has been studied intensively, class-switch recombination, a mechanism by which the IgH C region is exchanged, has rarely been investigated in birds. In this study, based on the published genome of pigeon (Columba livia) and high-throughput transcriptome sequencing of immune-related tissues, we identified a transcriptionally forward α gene and found that the pigeon IgH gene locus is arranged as µ-α-υ1-υ2. In this article, we show that both DNA deletion and inversion may result from IgA and IgY class switching, and similar junction patterns were observed for both types of class-switch recombination. We also identified two subclasses of υ genes in pigeon, which share low sequence identity. Phylogenetic analysis suggests that divergence of the two pigeon υ genes occurred during the early stage of bird evolution. The data obtained in this study provide new insight into class-switch recombination and Ig gene evolution in birds.

Massively Parallel Sequencing of Peritoneal and Splenic B Cell Repertoires Highlights Unique Properties of B-1 Cell Antibodies.

B-1 cells are a unique subset of B cells that are positively selected for expressing autoreactive BCRs. We isolated RNA from peritoneal (B-1a, B-1b, B-2) and splenic (B-1a, marginal zone, follicular) B cells from C57BL/6 mice and used 5'-RACE to amplify the IgH V region using massively parallel sequencing. By analyzing 379,000 functional transcripts, we demonstrate that B-1a cells use a distinct and restricted repertoire. All B-1 cell subsets, especially peritoneal B-1a cells, had a high proportion of sequences without N additions, suggesting predominantly prenatal development. Their transcripts differed markedly and uniquely contained VH11 and VH12 genes, which were rearranged only with a restricted selection of D and J genes, unlike other V genes. Compared to peritoneal B-1a, the peritoneal B-1b repertoire was larger, had little overlap with B-1a, and most sequences contained N additions. Similarly, the splenic B-1a repertoire differed from peritoneal B-1a sequences, having more unique sequences and more frequent N additions, suggesting influx of B-1a cells into the spleen from nonperitoneal sites. Two CDR3s, previously described as Abs to bromelain-treated RBCs, comprised 43% of peritoneal B-1a sequences. We show that a single-chain variable fragment designed after the most prevalent B-1a sequence bound oxidation-specific epitopes such as the phosphocholine of oxidized phospholipids. In summary, we provide the IgH V region library of six murine B cell subsets, including, to our knowledge for the first time, a comparison between B-1a and B-1b cells, and we highlight qualities of B-1 cell Abs that indicate unique selection processes.

Molecular analysis of immunoglobulin variable genes supports a germinal center experienced normal counterpart in primary cutaneous diffuse large B-cell lymphoma, leg-type.

BACKGROUND: Immunophenotype of primary cutaneous diffuse large B-cell lymphoma, leg-type (PCLBCL-LT) suggests a germinal center-experienced B lymphocyte (BCL2+ MUM1+ BCL6+/-). OBJECTIVES: As maturation history of B-cell is "imprinted" during B-cell development on the immunoglobulin gene sequence, we studied the structure and sequence of the variable part of the genes (IGHV, IGLV, IGKV), immunoglobulin surface expression and features of class switching in order to determine the PCLBCL-LT cell of origin. METHODS: Clonality analysis with BIOMED2 protocol and VH leader primers was done on DNA extracted from frozen skin biopsies on retrospective samples from 14 patients. The clonal DNA IGHV sequence of the tumor was aligned and compared with the closest germline sequence and homology percentage was calculated. Superantigen binding sites were studied. Features of selection pressure were evaluated with the multinomial Lossos model. RESULTS: A functional monoclonal sequence was observed in 14 cases as determined for IGHV (10), IGLV (2) or IGKV (3). IGV mutation rates were high (>5%) in all cases but one (median:15.5%), with superantigen binding sites conservation. Features of selection pressure were identified in 11/12 interpretable cases, more frequently negative (75%) than positive (25%). Intraclonal variation was detected in 3 of 8 tumor specimens with a low rate of mutations. Surface immunoglobulin was an IgM in 12/12 cases. FISH analysis of IGHM locus, deleted during class switching, showed heterozygous IGHM gene deletion in half of cases. The genomic PCR analysis confirmed the deletions within the switch µ region. IGV sequences were highly mutated but functional, with negative features of selection pressure suggesting one or more germinal center passage(s) with somatic hypermutation, but superantigen (SpA) binding sites conservation. Genetic features of class switch were observed, but on the non functional allele and co-existing with primary isotype IgM expression. CONCLUSION: These data suggest that cell-of origin is germinal center experienced and superantigen driven selected B-cell, in a stage between germinal center B-cell and plasma cell.

The Human Thymus Is Enriched for Autoreactive B Cells.

The human thymus has been shown to host B cells, which have been implicated in presentation of autoantigens for negative selection of T cell progenitors. Although these Ags are thought to be taken up through their surface Igs, data on thymic Ig gene repertoires are limited and reactivity to autoantigens has not been demonstrated. We therefore studied the Ig gene repertoires and reactivity to autoantigens of single-sorted B cells from pediatric thymus, and compared these with mature B cells from fetal and pediatric bone marrow. Nearly all B cells in thymus were mature and displayed an Ig gene repertoire that was similar to pediatric bone marrow. Fetal mature B cells predominantly used proximal V, D, and J genes, and their Abs were highly reactive to dsDNA. In contrast, thymic B cells were enriched for autoreactive clones that showed increased specificity to peptide autoantigens. Thus, most B cells in the thymus are resident rather than developing, and are enriched for autoantigen binding. These features support current models for a role of thymic B cells in presentation of autoantigens to developing T cells during negative selection.

Diversification of the Primary Antibody Repertoire by AID-Mediated Gene Conversion.

Gene conversion, mediated by activation-induced cytidine deaminase (AID), has been found to contribute to generation of the primary antibody repertoire in several vertebrate species. Generation of the primary antibody repertoire by gene conversion of immunoglobulin (Ig) genes occurs primarily in gut-associated lymphoid tissues (GALT) and is best described in chicken and rabbit. Here, we discuss current knowledge of the mechanism of gene conversion as well as the contribution of the microbiota in promoting gene conversion of Ig genes. Finally, we propose that the antibody diversification strategy used in GALT species, such as chicken and rabbit, is conserved in a subset of human and mouse B cells.

Activation-Induced Cytidine Deaminase Expression in Human B Cell Precursors Is Essential for Central B Cell Tolerance.

Activation-induced cytidine deaminase (AID), the enzyme-mediating class-switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes, is essential for the removal of developing autoreactive B cells. How AID mediates central B cell tolerance remains unknown. We report that AID enzymes were produced in a discrete population of immature B cells that expressed recombination-activating gene 2 (RAG2), suggesting that they undergo secondary recombination to edit autoreactive antibodies. However, most AID+ immature B cells lacked anti-apoptotic MCL-1 and were deleted by apoptosis. AID inhibition using lentiviral-encoded short hairpin (sh)RNA in B cells developing in humanized mice resulted in a failure to remove autoreactive clones. Hence, B cell intrinsic AID expression mediates central B cell tolerance potentially through its RAG-coupled genotoxic activity in self-reactive immature B cells.

Association of VH4-59 Antibody Variable Gene Usage with Recognition of an Immunodominant Epitope on the HIV-1 Gag Protein.

The human antibody response against HIV-1 infection recognizes diverse antigenic subunits of the virion, and includes a high level of antibodies to the Gag protein. We report here the isolation and characterization of a subset of Gag-specific human monoclonal antibodies (mAbs) that were prevalent in the antibody repertoire of an HIV-infected individual. Several lineages of Gag-specifc mAbs were encoded by a single antibody heavy chain variable region, VH4-59, and a representative antibody from this group designated mAb 3E4 recognized a linear epitope on the globular head of the p17 subunit of Gag. We found no evidence that mAb 3E4 exhibited any function in laboratory studies aimed at elucidating the immunologic activity, including assays for neutralization, Ab-dependent cell-mediated virus inhibition, or enhanced T cell reactivity caused by Gag-3E4 complexes. The findings suggest this immunodominant epitope in Gag protein, which is associated with VH4-59 germline gene usage, may induce a high level of B cells that encode binding but non-functional antibodies that occupy significant repertoire space following HIV infection. The studies define an additional specific molecular mechanism in the immune distraction activity of the HIV virion.

Violation of an evolutionarily conserved immunoglobulin diversity gene sequence preference promotes production of dsDNA-specific IgG antibodies.

Variability in the developing antibody repertoire is focused on the third complementarity determining region of the H chain (CDR-H3), which lies at the center of the antigen binding site where it often plays a decisive role in antigen binding. The power of VDJ recombination and N nucleotide addition has led to the common conception that the sequence of CDR-H3 is unrestricted in its variability and random in its composition. Under this view, the immune response is solely controlled by somatic positive and negative clonal selection mechanisms that act on individual B cells to promote production of protective antibodies and prevent the production of self-reactive antibodies. This concept of a repertoire of random antigen binding sites is inconsistent with the observation that diversity (DH) gene segment sequence content by reading frame (RF) is evolutionarily conserved, creating biases in the prevalence and distribution of individual amino acids in CDR-H3. For example, arginine, which is often found in the CDR-H3 of dsDNA binding autoantibodies, is under-represented in the commonly used DH RFs rearranged by deletion, but is a frequent component of rarely used inverted RF1 (iRF1), which is rearranged by inversion. To determine the effect of altering this germline bias in DH gene segment sequence on autoantibody production, we generated mice that by genetic manipulation are forced to utilize an iRF1 sequence encoding two arginines. Over a one year period we collected serial serum samples from these unimmunized, specific pathogen-free mice and found that more than one-fifth of them contained elevated levels of dsDNA-binding IgG, but not IgM; whereas mice with a wild type DH sequence did not. Thus, germline bias against the use of arginine enriched DH sequence helps to reduce the likelihood of producing self-reactive antibodies.