Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua).

BACKGROUND: The Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod's immune response on a molecular level. RESULTS: A comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5'RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes. CONCLUSIONS: This study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Distinct groups of autoantigens as drivers of ocular adnexal MALT lymphoma pathogenesis.

Chronic B-cell receptor signals incited by cognate antigens are believed to play a crucial role in the pathogenesis of mucosa-associated lymphoid tissue lymphomas. We have explored the immunoglobulin variable regions (IGHV) expressed by 124 ocular adnexal MALT lymphomas (OAML) and tested the in vitro reactivity of recombinant IgM derived from 23 OAMLs. Six of 124 OAMLs (5%) were found to express a high-affinity stereotyped rheumatoid factor. OAMLs have a biased IGHV4-34 usage, which confers intrinsic super auto-antigen reactivity with poly-N-acetyllactosamine (NAL) epitopes, present on cell surface glycoproteins of erythrocytes and B cells. Twenty-one OAMLs (17%) expressed IGHV4-34-encoded B-cell receptors. Five of the 23 recombinant OAML IgMs expressed IGHV4-34, four of which bound to the linear NAL i epitope expressed on B cells but not to the branched NAL I epitope on erythrocytes. One non-IGHV4-34-encoded OAML IgM was also reactive with B cells. Interestingly, three of the 23 OAML IgMs (13%) specifically reacted with proteins of U1-/U-snRNP complexes, which have been implicated as cognate-antigens in various autoimmune diseases such as systemic lupus erythematosus and mixed connective tissue disease. The findings indicate that local autoimmune reactions are instrumental in the pathogenesis of a substantial fraction of OAMLs.

Interpretable deep learning reveals the role of an E-box motif in suppressing somatic hypermutation of AGCT motifs within human immunoglobulin variable regions.

Introduction: Somatic hypermutation (SHM) of immunoglobulin variable (V) regions by activation induced deaminase (AID) is essential for robust, long-term humoral immunity against pathogen and vaccine antigens. AID mutates cytosines preferentially within WRCH motifs (where W=A or T, R=A or G and H=A, C or T). However, it has been consistently observed that the mutability of WRCH motifs varies substantially, with large variations in mutation frequency even between multiple occurrences of the same motif within a single V region. This has led to the notion that the immediate sequence context of WRCH motifs contributes to mutability. Recent studies have highlighted the potential role of local DNA sequence features in promoting mutagenesis of AGCT, a commonly mutated WRCH motif. Intriguingly, AGCT motifs closer to 5' ends of V regions, within the framework 1 (FW1) sub-region1, mutate less frequently, suggesting an SHM-suppressing sequence context. Methods: Here, we systematically examined the basis of AGCT positional biases in human SHM datasets with DeepSHM, a machine-learning model designed to predict SHM patterns. This was combined with integrated gradients, an interpretability method, to interrogate the basis of DeepSHM predictions. Results: DeepSHM predicted the observed positional differences in mutation frequencies at AGCT motifs with high accuracy. For the conserved, lowly mutating AGCT motifs in FW1, integrated gradients predicted a large negative contribution of 5'C and 3'G flanking residues, suggesting that a CAGCTG context in this location was suppressive for SHM. CAGCTG is the recognition motif for E-box transcription factors, including E2A, which has been implicated in SHM. Indeed, we found a strong, inverse relationship between E-box motif fidelity and mutation frequency. Moreover, E2A was found to associate with the V region locale in two human B cell lines. Finally, analysis of human SHM datasets revealed that naturally occurring mutations in the 3'G flanking residues, which effectively ablate the E-box motif, were associated with a significantly increased rate of AGCT mutation. Discussion: Our results suggest an antagonistic relationship between mutation frequency and the binding of E-box factors like E2A at specific AGCT motif contexts and, therefore, highlight a new, suppressive mechanism regulating local SHM patterns in human V regions.

Unmutated IGHV at diagnosis in patients with early stage CLL independently predicts for shorter follow-up time to first treatment (TTFT).

The mutational status of the IGHV gene is routinely assessed in patients with chronic lymphocytic leukaemia (CLL), since it is both prognostic of clinical outcome and predictive of response to treatment. This study evaluates the IGHV mutational status, assessed in newly diagnosed CLL patients, as a stand-alone predictor of time to first treatment (TTFT). We analysed the data of 236 CLL patients, diagnosed at our centre between January 2004 and September 2020, with a minimum follow-up period of 3.0 years, Binet A-B and Rai 0-II stages. IGHV was unmutated in 38.1 % and mutated in 61.9 % of cases. The univariate analysis showed a statistically significant difference (p < 0.001) in TTFT based on unmutated (85.2 % at 14 years, 95 % CI = 63.3-94.5 %) or mutated (41.3 % at 14 years, 95 % CI = 29.5-51.8 %) and the need for treatment at 1, 3 and 5 years was of 20.0 % vs 4.1 % (p < 0.001), 42.7 % vs 11.4 % (p < 0.001) and 55.8 % vs 20.0 % (p < 0.001) in unmutated and mutated IGHV patients, respectively. Multivariate analysis confirmed that unmutated IGHV status negatively affects TTFT (p < 0.001), in addition to high-risk genomic aberration (p = 0.025), Rai stage I (p = 0.007) and II (p-value < 0.001). The difference in TTFT based on unmutated or mutated IGHV status remains statistically significant also when considering the subgroups by the genomic aberrations and Rai stages. Our findings suggest that, with the single analysis of the IGHV mutational status at CLL diagnosis, along with clinical and laboratory data, and without karyotype and TP53 data, clinicians will have prognostic and predictive indications for the first clinical treatment and appropriate follow-up of patients.

Large-scale data mining of four billion human antibody variable regions reveals convergence between therapeutic and natural antibodies that constrains search space for biologics drug discovery.

The naïve human antibody repertoire has theoretical access to an estimated > 1015 antibodies. Identifying subsets of this prohibitively large space where therapeutically relevant antibodies may be found is useful for development of these agents. It was previously demonstrated that, despite the immense sequence space, different individuals can produce the same antibodies. It was also shown that therapeutic antibodies, which typically follow seemingly unnatural development processes, can arise independently naturally. To check for biases in how the sequence space is explored, we data mined public repositories to identify 220 bioprojects with a combined seven billion reads. Of these, we created a subset of human bioprojects that we make available as the AbNGS database (https://naturalantibody.com/ngs/). AbNGS contains 135 bioprojects with four billion productive human heavy variable region sequences and 385 million unique complementarity-determining region (CDR)-H3s. We find that 270,000 (0.07% of 385 million) unique CDR-H3s are highly public in that they occur in at least five of 135 bioprojects. Of 700 unique therapeutic CDR-H3, a total of 6% has direct matches in the small set of 270,000. This observation extends to a match between CDR-H3 and V-gene call as well. Thus, the subspace of shared ('public') CDR-H3s shows utility for serving as a starting point for therapeutic antibody design.

Improved detection of tryptic immunoglobulin variable region peptides by chromatographic and gas-phase fractionation techniques.

The polyclonal repertoire of circulating antibodies potentially holds valuable information about an individual's humoral immune state. While bottom-up proteomics is well suited for serum proteomics, the vast number of antibodies and dynamic range of serum challenge this analysis. To acquire the serum proteome more comprehensively, we incorporated high-field asymmetric waveform ion-mobility spectrometry (FAIMS) or two-dimensional chromatography into standard trypsin-based bottom-up proteomics. Thereby, the number of variable region (VR)-related spectra increased 1.7-fold with FAIMS and 10-fold with chromatography fractionation. To match antibody VRs to spectra, we combined de novo searching and BLAST alignment. Validation of this approach showed that, as peptide length increased, the de novo accuracy decreased and BLAST performance increased. Through in silico calculations on antibody repository sequences, we determined the uniqueness of tryptic VR peptides and their suitability as antibody surrogate. Approximately one-third of these peptides were unique, and about one-third of all antibodies contained at least one unique peptide.

Molecular basis for differential Igk versus Igh V(D)J joining mechanisms.

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease1. RAG orchestrates Igh V(D)J recombination upon capturing a JH-RSS within the JH-RSS-based recombination centre1-3 (RC). JH-RSS orientation programmes RAG to scan upstream D- and VH-containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion4-7. During Igh scanning, RAG robustly utilizes only D-RSSs or VH-RSSs in convergent (deletional) orientation with JH-RSSs4-7. However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters8, inconsistent with linear scanning2. Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements9,10, which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element11. Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to VH locus inversion7, DJH inversion abrogated VH-to-DJH joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh, we assayed Vκ-to-JH and D-to-Jκ rearrangements in hybrid Igh-Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk-RSSs to promote robust diffusional joining compared with Igh-RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating VH joining by RAG-scanning impediments.

Diversification of the VH3-53 immunoglobulin gene segment by somatic hypermutation results in neutralization of SARS-CoV-2 virus variants.

COVID-19 induces re-circulating long-lived memory B cells (MBC) that, upon re-encounter with the pathogen, are induced to mount immunoglobulin responses. During convalescence, antibodies are subjected to affinity maturation, which enhances the antibody binding strength and generates new specificities that neutralize virus variants. Here, we performed a single-cell RNA sequencing analysis of spike-specific B cells from a SARS-CoV-2 convalescent subject. After COVID-19 vaccination, matured infection-induced MBC underwent recall and differentiated into plasmablasts. Furthermore, the transcriptomic profiles of newly activated B cells transiently shifted toward the ones of atypical and CXCR3+ B cells and several B-cell clonotypes massively expanded. We expressed monoclonal antibodies (mAbs) from all B-cell clones from the largest clonotype that used the VH3-53 gene segment. The in vitro analysis revealed that some somatic hypermutations enhanced the neutralization breadth of mAbs in a putatively stochastic manner. Thus, somatic hypermutation of B-cell clonotypes generates an anticipatory memory that can neutralize new virus variants.

[Biological characteristics and clinical significance of stereotyped B-cell receptor in chronic lymphocytic leukemia].

Chronic lymphocytic leukemia (CLL) is the most common leukemia in the Western adults, although the incidence of CLL is relatively low in Asian populations. However, with the aging population, the incidence of CLL is increasing in China. The interaction between CLL cells and the microenvironment plays a crucial role in the recognition of antigens by the B-cell receptor immunoglobulin (BCR IG). The mutational status of the immunoglobulin heavy variable region (IGHV) is a classical prognostic marker for CLL. Over 40% of CLL patients exhibit biased usage of IGHV and highly similar amino acid sequences in the heavy complementarity-determining region 3 (HCDR3), known as the BCR stereotypy. Different subgroups of stereotyped BCR exhibit distinct biological and clinical features. Among them, subset #2 with mutated IGHV and poor prognosis, as well as the subset #8 with a high risk of Richter transformation, have been recommended by the European Research Initiative on CLL to be included in clinical reports on IGHV mutational status. This review summarizes the definition, distribution, biological characteristics, and clinical significance of clonality patterns of the BCR in CLL.

Prognostic impact of genetic abnormalities in 536 first-line chronic lymphocytic leukaemia patients without 17p deletion treated with chemoimmunotherapy in two prospective trials: Focus on IGHV-mutated subgroups (a FILO study).

The potential prognostic influence of genetic aberrations on chronic lymphocytic leukaemia (CLL) can vary based on various factors, such as the immunoglobulin heavy variable (IGHV) status. We conducted an integrative analysis on genetic abnormalities identified through cytogenetics and targeted next-generation sequencing in 536 CLL patients receiving first-line chemo(immuno)therapies (CIT) as part of two prospective trials. We evaluated the prognostic implications of the main abnormalities, with specific attention to their relative impact according to IGHV status. In the entire cohort, unmutated (UM)-IGHV, complex karyotype, del(11q) and ATM mutations correlated significantly with shorter progression-free survival (PFS). Focusing on the subset of mutated IGHV (M-IGHV) patients, univariate analysis showed that complex karyotype, del(11q), SF3B1 and SAMHD1 mutations were associated with significant lower PFS. The prognostic influence varied based on the patient's IGHV status, as these abnormalities did not affect outcomes in the UM-IGHV subgroup. TP53 mutations had no significant impact on outcomes in the M-IGHV subgroup. Our findings highlight the diverse prognostic influence of genetic aberrations depending on the IGHV status in symptomatic CLL patients receiving first-line CIT. The prognosis of gene mutations and cytogenetic abnormalities needs to be investigated with a compartmentalized methodology, taking into account the IGVH status of patients receiving first-line BTK and/or BCL2 inhibitors.

Characteristics of chronic lymphocytic leukemia in Japan: Comprehensive analysis of the CLLRSG-01 study.

Chronic lymphocytic leukemia (CLL) is rare in Japan. We conducted the nationwide, prospective observational study CLLRSG-01 to clarify the current state of CLL in Japan and to make accurate international comparisons by preparing naturally air-dried smears like those used in other countries. Of the 201 untreated patients enrolled and evaluated, 119 were diagnosed with CLL and 82 with non-CLL mature B-cell neoplasms, based on the WHO classification. Of the 119 CLL patients, 90 were classified as typical and 29 as atypical according to FAB classification morphology, with the proportion of atypical CLL consistent with reports from other countries. Immunophenotypic analyses by flow cytometry showed that 55% of Japanese CLL patients had a Matutes score of 4 or higher, which is lower than the rate of about 90% in Europeans. Mutated IGHV was identified in 80% of Japanese CLL patients, which is a higher rate than in Western patients. The most frequent IGHV gene was VH3-30 (15%), followed by VH3-23 (12%) and VH4-34 (10%). VH1-69, the most common gene in Western countries, was identified in only one patient. These results indicate that the pattern of immunophenotypes and IGHV gene usage in Japanese CLL patients differs from that in Western patients.

Analysis of Mutational Status of IGHV, and Cytokine Polymorphisms as Prognostic Factors in Chronic Lymphocytic Leukemia: The Romanian Experience.

The aim of the current study was to assess the associations between genetic risk factors (such as the mutational status of the IGHV gene and polymorphisms of the IL-10 and TNF-α genes) and CLL risk, prognosis, and overall survival. Another goal of this study was to evaluate the multivariate effect of the combination of multiple genetic risk factors (mutational status of the IGHV gene, somatic mutations, DNA CNVs, and cytokine SNPs) on the clinical characteristics and survival of patients. A total of 125 CLL patients and 239 healthy controls were included for comparative SNP analysis. IL-10 (rs1800896 and rs1800872) and TNF-α (rs361525 and rs1800750) SNPs and haplotypes were not associated with CLL risk. The absence of hypermutation in the IGHV gene was shown to be of important prognostic value, being associated with short OS. Further individual risk factors for short OS were an age above 65 years at diagnosis and the presence of somatic mutations and/or CNVs. In our multivariable analysis, the presence of somatic mutations and the IL-10 rs1800872 variant allele, and the association of CNVs with the IL-10 rs1800896 variant allele, were identified as risk factors for short OS. Moreover, the OS in unmutated IGHV patients was additionally affected (decreased) by the presence of CNVs and/or somatic mutations. Similarly, IL-10 rs1800896 modulated the OS in unmutated IGHV patients with CNVs.

Polyreactivity of antibodies from different B-cell subpopulations is determined by distinct sequence patterns of variable region.

An antibody molecule that can bind to multiple distinct antigens is defined as polyreactive. In the present study, we performed statistical analyses to assess sequence correlates of polyreactivity of >600 antibodies cloned from different B-cell types of healthy humans. The data revealed several sequence patterns of variable regions of heavy and light immunoglobulin chains that determine polyreactivity. The most prominent identified patterns were increased number of basic amino acid residues, reduced frequency of acidic residues, increased number of aromatic and hydrophobic residues, and longer length of CDR L1. Importantly, our study revealed that antibodies isolated from different B-cell populations used distinct sequence patterns (or combinations of them) for polyreactive antigen binding. Furthermore, we combined the data from sequence analyses with molecular modeling of selected polyreactive antibodies and demonstrated that human antibodies can use multiple pathways for achieving antigen-binding promiscuity. These data reconcile some contradictions in the literature regarding the determinants of antibody polyreactivity. Moreover, our study demonstrates that the mechanism of polyreactivity of antibodies evolves during immune response and might be tailored to specific functional properties of different B-cell compartments. Finally, these data can be of use for efforts in the development and engineering of therapeutic antibodies.

Human IgM-expressing memory B cells.

A hallmark of T cell dependent (TD) humoral immune responses is the generation of long-lived memory B cells. The generation of these cells occurs primarily in the germinal center (GC) reaction, where antigen-activated B cells undergo affinity maturation as a major consequence of the combined processes of proliferation, somatic hypermutation of their immunoglobulin V (IgV) region genes, and selection for improved affinity of their B-cell antigen receptors. As many B cells also undergo class-switching to IgG or IgA in these TD responses, there was traditionally a focus on class-switched memory B cells in both murine and human studies on memory B cells. However, it has become clear that there is also a large subset of IgM-expressing memory B cells, which have important phenotypic and functional similarities but also differences to class-switched memory B cells. There is an ongoing discussion about the origin of distinct subsets of human IgM+ B cells with somatically mutated IgV genes. We argue here that the vast majority of human IgM-expressing B cells with somatically mutated IgV genes in adults is indeed derived from GC reactions, even though a generation of some mostly lowly mutated IgM+ B cells from other differentiation pathways, mainly in early life, may exist.

Conformational features and interaction mechanisms of VH H antibodies with ß-hairpin CDR3: A case of Nb8-HigB2 interaction.

The ß-hairpin conformation is regarded as an important basic motif to form and regulate protein-protein interactions. Single-domain VH H antibodies are potential therapeutic and diagnostic tools, and the third complementarity-determining regions of the heavy chains (CDR3s) of these antibodies are critical for antigen recognition. Although the sequences and conformations of the CDR3s are diverse, CDR3s sometimes adopt ß-hairpin conformations. However, characteristic features and interaction mechanisms of ß-hairpin CDR3s remain to be fully elucidated. In this study, we investigated the molecular recognition of the anti-HigB2 VH H antibody Nb8, which has a CDR3 that forms a ß-hairpin conformation. The interaction was analyzed by evaluation of alanine-scanning mutants, molecular dynamics simulations, and hydrogen/deuterium exchange mass spectrometry. These experiments demonstrated that positions 93 and 94 (Chothia numbering) in framework region 3, which is right outside CDR3 by definition, play pivotal roles in maintaining structural stability and binding properties of Nb8. These findings will facilitate the design and optimization of single-domain antibodies.

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.

Design and deep learning of synthetic B-cell-specific promoters.

Synthetic biology and deep learning synergistically revolutionize our ability for decoding and recoding DNA regulatory grammar. The B-cell-specific transcriptional regulation is intricate, and unlock the potential of B-cell-specific promoters as synthetic elements is important for B-cell engineering. Here, we designed and pooled synthesized 23 640 B-cell-specific promoters that exhibit larger sequence space, B-cell-specific expression, and enable diverse transcriptional patterns in B-cells. By MPRA (Massively parallel reporter assays), we deciphered the sequence features that regulate promoter transcriptional, including motifs and motif syntax (their combination and distance). Finally, we built and trained a deep learning model capable of predicting the transcriptional strength of the immunoglobulin V gene promoter directly from sequence. Prediction of thousands of promoter variants identified in the global human population shows that polymorphisms in promoters influence the transcription of immunoglobulin V genes, which may contribute to individual differences in adaptive humoral immune responses. Our work helps to decipher the transcription mechanism in immunoglobulin genes and offers thousands of non-similar promoters for B-cell engineering.

Genetic markers of chronic lymphocytic leukemia: a retrospective study of 312 patients from a reference center in Lebanon.

Aim: Chronic lymphocytic leukemia (CLL) is a highly heterogenous hemopathy. Genetic stratification of CLL patients has important prognostic and therapeutic values - mainly immunoglobulin heavy chain variable region gene (IGHV) mutational status and the presence of cytogenetic abnormalities. The genetics of CLL in Lebanon is scarcely described in the literature. Patients & methods: In this work, we studied the genetic biomarkers of 312 Lebanese CLL patients. Results: Prominent IGHV genes were IGHV4-34, IGHV1-69 and IGHV3-30; and CLL #1 and #5 presented major subsets. Some similarities as well as major differences were highlighted when comparing our data with previously published data. Conclusion: The distribution of IGHV alleles in our series differed from previously described distributions, suggesting involvement of antigenic selection and regional variables in CLL pathogenesis.