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Humanized mice are limited in terms of modeling human immunity, particularly with regards to antibody responses. Here we constructed a humanized (THX) mouse by grafting non-γ-irradiated, genetically myeloablated KitW-41J mutant immunodeficient pups with human cord blood CD34+ cells, followed by 17β-estradiol conditioning to promote immune cell differentiation. THX mice reconstitute a human lymphoid and myeloid immune system, including marginal zone B cells, germinal center B cells, follicular helper T cells and neutrophils, and develop well-formed lymph nodes and intestinal lymphoid tissue, including Peyers patches, and human thymic epithelial cells. These mice have diverse human B cell and T cell antigen receptor repertoires and can mount mature T cell-dependent and T cell-independent antibody responses, entailing somatic hypermutation, class-switch recombination, and plasma cell and memory B cell differentiation. Upon flagellin or a Pfizer-BioNTech coronavirus disease 2019 (COVID-19) mRNA vaccination, THX mice mount neutralizing antibody responses to Salmonella or severe acute respiratory syndrome coronavirus 2 Spike S1 receptor-binding domain, with blood incretion of human cytokines, including APRIL, BAFF, TGF-β, IL-4 and IFN-γ, all at physiological levels. These mice can also develop lupus autoimmunity after pristane injection. By leveraging estrogen activity to support human immune cell differentiation and maturation of antibody responses, THX mice provide a platform to study the human immune system and to develop human vaccines and therapeutics.

Poultry immunoglobulin genes undergo diversification through homologous recombination (HR) and somatic hypermutation (SHM). Most animals share a similar system in immunoglobulin diversification, with the rare exception that human and murine immunoglobulin genes diversify through V(D)J recombination. Poultry possesses only one functional variable gene for each immunoglobulin heavy (HC) and light chains (LC), with clusters of nonproductive pseudogenes upstream. During the B cell development, the functional variable gene is overwritten by sequences from the pseudo-variable genes via a process known as gene conversion (GC), a kind of HR. Point mutations caused in the functional variable gene also contribute to immunoglobulin diversification. This review discusses the latest findings on the molecular mechanisms of antibody gene diversification in poultry, using chickens as a model. Additionally, it will outline how these basic research findings have recently been applied especially in the medical field. [ABSTRACT FROM AUTHOR]

Simple Summary: It is known that both B cells and myeloblasts from acute myeloid leukemia (AML) express immunoglobulin (Ig). However, the difference between Ig from myeloblasts and B cells has not been explored; therefore, the function and significance of AML-Ig are not well known. Here, we performed 5′ RACE-related PCR coupled with PacBio sequencing to analyze the Ig repertoire of Chinese AML patients. Myeloblasts expressed all five classes of IgH, especially Igγ, with a high expression frequency. Compared with B-Ig in the same AML patient, AML-Ig showed different biased V(D)J usages and mutation patterns. More importantly, mutations of AML-Ig frequently occurred at the sites of post-translational modification. In summary, our results showed that AML-Ig had different sequence characteristics than those of B-Ig and that the unique V(D)J usages of AML-Ig may serve as a novel biomarker for personalized minimal/measurable residual disease monitoring and therapeutic targets in AML patients. It is common knowledge that immunoglobulin (Ig) is produced by B lymphocytes and mainly functions as an antibody. However, it has been shown recently that myeloblasts from acute myeloid leukemia (AML) could also express Ig and that AML-Ig played a role in leukemogenesis and AML progression. The difference between Ig from myeloblasts and B cells has not been explored. Studying the characteristics of the Ig repertoire in myeloblasts and B cells will be helpful to understand the function and significance of AML-Ig. We performed 5′ RACE-related PCR coupled with PacBio sequencing to analyze the Ig repertoire in myeloblasts and B cells from Chinese AML patients. Myeloblasts expressed all five classes of IgH, especially Igγ, with a high expression frequency. Compared with B-Ig in the same patient, AML-Ig showed different biased V(D)J usages and mutation patterns. In addition, the CDR3 length distribution of AML-Ig was significantly different from those of B-Ig. More importantly, mutations of AML-IgH, especially Igμ, Igα, and Igδ, were different from that of B-IgH in each AML patient, and the mutations frequently occurred at the sites of post-translational modification. AML-Ig has distinct characteristics of variable regions and mutations, which may have implications for disease monitoring and personalized therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

[Display omitted] Ataxia telangiectasia (AT) is characterized by cerebellar ataxia, telangiectasia, immunodeficiency, and increased cancer susceptibility and is caused by mutations in the ataxia telangiectasia mutated (ATM) gene. The immunodeficiency comprises predominantly immunoglobulin deficiency, mainly IgA and IgG 2 , with a variable severity. So far, the exact mechanisms underlying the immunoglobulin deficiency, especially the variable severity, remain unelucidated. We characterized the clinical impact of immunoglobulin deficiencies in AT and elucidated their mechanisms in AT. We analyzed long-term immunoglobulin levels, immunophenotyping, and survival time in our cohort (n = 87, median age 16 years; maximum 64 years). Somatic hypermutation and class-switch junctions in B cells were analyzed by next-generation sequencing. Furthermore, an in vitro class-switching induction assay was performed, followed by RNA sequencing, to assess the effect of ATM inhibition. Only the hyper-IgM AT phenotype significantly worsened survival time, while IgA or IgG 2 deficiencies did not. The immunoglobulin levels showed predominantly decreased IgG 2 and IgA. Moreover, flow cytometric analysis demonstrated reduced naive B and T lymphocytes and a deficiency of class-switched IgG 2 and IgA memory B cells. Somatic hypermutation frequencies were lowered in IgA- and IgG 2 -deficient patients, indicating hampered germinal center reaction. In addition, the microhomology of switch junctions was elongated, suggesting alternative end joining during class-switch DNA repair. The in vitro class switching and proliferation were negatively affected by ATM inhibition. RNA sequencing analysis showed that ATM inhibitor influenced expression of germinal center reaction genes. Immunoglobulin deficiency in AT is caused by disturbed development of class-switched memory B cells. ATM deficiency affects both germinal center reaction and choice of DNA-repair pathway in class switching. [ABSTRACT FROM AUTHOR]

Human herpesvirus 8 (HHV8)-associated diseases include Kaposi sarcoma (KS), multicentric Castleman disease (MCD), germinotropic lymphoproliferative disorder (GLPD), Kaposi sarcoma inflammatory cytokine syndrome (KICS), HHV8-positive diffuse large B-cell lymphoma (HHV8+ DLBCL), primary effusion lymphoma (PEL), and extra-cavitary PEL (ECPEL). We report the case of a human immunodeficiency virus (HIV)-negative male treated for cutaneous KS, who developed generalized lymphadenopathy, hepatosplenomegaly, pleural and abdominal effusions, renal insufficiency, and pancytopenia. The excised lymph node showed features of concomitant involvement by micro-KS and MCD, with aggregates of HHV8+, Epstein Barr virus (EBV)-negative, IgM+, and lambda+ plasmablasts reminiscent of microlymphoma. Molecular investigations revealed a somatically hypermutated (SHM) monoclonal rearrangement of the immunoglobulin heavy chain (IGH), accounting for 4% of the B-cell population of the lymph node. Mutational analyses identified a pathogenic variant of KMT2D and variants of unknown significance in KMT2D, FOXO1, ARID1A, and KMT2A. The patient died shortly after surgery. The histological features (HHV8+, EBV−, IgM+, Lambda+, MCD+), integrated with the molecular findings (monoclonal IGH, SHM+, KMT2D mutated), supported the diagnosis of a monoclonal HHV8+ microlymphoma, with features intermediate between an incipient HHV8+ DLBCL and an EBV-negative ECPEL highlighting the challenges in the accurate classification of HHV8-driven lymphoid proliferations. [ABSTRACT FROM AUTHOR]

B cell receptors (BCRs) denote antigen specificity, while corresponding cell subsets indicate B cell functionality. Since each B cell uniquely encodes this combination, physical isolation and subsequent processing of individual B cells become indispensable to identify both attributes. However, this approach accompanies high costs and inevitable information loss, hindering high-throughput investigation of B cell populations. Here, we present BCR-SORT, a deep learning model that predicts cell subsets from their corresponding BCR sequences by leveraging B cell activation and maturation signatures encoded within BCR sequences. Subsequently, BCR-SORT is demonstrated to improve reconstruction of BCR phylogenetic trees, and reproduce results consistent with those verified using physical isolation-based methods or prior knowledge. Notably, when applied to BCR sequences from COVID-19 vaccine recipients, it revealed inter-individual heterogeneity of evolutionary trajectories towards Omicron-binding memory B cells. Overall, BCR-SORT offers great potential to improve our understanding of B cell responses. [ABSTRACT FROM AUTHOR]

Mouse models have revealed an intrinsic nonrandom hypermutation profile within antibody-coding exons. The somatic mutator deaminase protein, activation-induced cytidine deaminase (AID), favors DNA motifs that lie in a flexible configuration; both micro- and mesoscale preferences can shape in vivo somatic hypermutation profiles. DNA mechanics play an essential role in generating beneficial mutations in antigen-contacting residues of coding sequences. This cis -mechanical feature is conserved in antibody genes from species that use somatic hypermutation as a major diversification strategy. DNA mechanics can affect mutagenesis in AID off targets, contributing to lymphomagenesis. During antibody affinity maturation, beneficial mutations preferentially occur in DNA regions encoding antigen-contacting amino acids. The nonrandomness of predisposed mutations has long been a mystery in immunology. Mouse models indicate a mysterious cis element in DNA sequences that contributes to sequence-dependent DNA flexibility, with the latter being selected during the evolution of adaptive immunity. This is relevant because coding sequences can play a noncoding role through DNA mechanics, a code which might harbor hidden genetic information. Antibody-coding genes accumulate somatic mutations to achieve antibody affinity maturation. Genetic dissection using various mouse models has shown that intrinsic hypermutations occur preferentially and are predisposed in the DNA region encoding antigen-contacting residues. The molecular basis of nonrandom/preferential mutations is a long-sought question in the field. Here, we summarize recent findings on how single-strand (ss)DNA flexibility facilitates activation-induced cytidine deaminase (AID) activity and fine-tunes the mutation rates at a mesoscale within the antibody variable domain exon. We propose that antibody coding sequences are selected based on mutability during the evolution of adaptive immunity and that DNA mechanics play a noncoding role in the genome. The mechanics code may also determine other cellular DNA metabolism processes, which awaits future investigation. [ABSTRACT FROM AUTHOR]

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders. [ABSTRACT FROM AUTHOR]

Abstract Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma. A major mutagenic process in DLBCL is aberrant somatic hypermutation (aSHM) by activation-induced cytidine deaminase (AID), which occurs preferentially at RCH/TW sequence motifs proximal to transcription start sites. Splice sequences are highly conserved, rich in RCH/TW motifs, and recurrently mutated in DLBCL. Therefore, we hypothesized that aSHM may cause recurrent splicing mutations in DLBCL. In a meta-cohort of > 1,800 DLBCLs, we found that 77.5% of splicing mutations in 29 recurrently mutated genes followed aSHM patterns. In addition, in whole-genome sequencing (WGS) data from 153 DLBCLs, proximal mutations in splice sequences, especially in donors, were significantly enriched in RCH/TW motifs (p 2,000; p 6,300). Comparing sequencing data from mouse models with and without AID activity showed that the splice donor sequences were the top genomic feature enriched in AID-induced mutations (p

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma. A major mutagenic process in DLBCL is aberrant somatic hypermutation (aSHM) by activation-induced cytidine deaminase (AID), which occurs preferentially at RCH/TW sequence motifs proximal to transcription start sites. Splice sequences are highly conserved, rich in RCH/TW motifs, and recurrently mutated in DLBCL. Therefore, we hypothesized that aSHM may cause recurrent splicing mutations in DLBCL. In a meta-cohort of > 1,800 DLBCLs, we found that 77.5% of splicing mutations in 29 recurrently mutated genes followed aSHM patterns. In addition, in whole-genome sequencing (WGS) data from 153 DLBCLs, proximal mutations in splice sequences, especially in donors, were significantly enriched in RCH/TW motifs (p < 0.01). We validated this enrichment in two additional DLBCL cohorts (N > 2,000; p < 0.0001) and confirmed its absence in 12 cancer types without aSHM (N > 6,300). Comparing sequencing data from mouse models with and without AID activity showed that the splice donor sequences were the top genomic feature enriched in AID-induced mutations (p < 0.0001). Finally, we observed that most AID-related splice site mutations are clonal within a sample, indicating that aSHM may cause early loss-of-function events in lymphomagenesis. Overall, these findings support that AID causes an overrepresentation of clonal splicing mutations in DLBCL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ABSTRACTAdult females of reproductive age develop greater antibody responses to inactivated influenza vaccines (IIV) than males. How sex, age, and sex steroid concentrations impact B cells and durability of IIV-induced immunity and protection over 4 months post-vaccination (mpv) was analyzed. Vaccinated adult females had greater germinal center B cell and plasmablast frequencies in lymphoid tissues, higher neutralizing antibody responses 1–4 mpv, and better protection against live H1N1 challenge than adult males. Aged mice, regardless of sex, had reduced B cell frequencies, less durable antibody responses, and inferior protection after challenge than adult mice, which correlated with diminished estradiol among aged females. To confirm that greater IIV-induced immunity was caused by sex hormones, four core genotype (FCG) mice were used, in which the testes-determining gene, Sry, was deleted from chromosome Y (ChrY) and transferred to Chr3 to separate gonadal sex (i.e., ovaries or testes) from sex chromosome complement (i.e., XX or XY complement). Vaccinated, gonadal female FCG mice (XXF and XYF) had greater numbers of B cells, higher antiviral antibody titers, and reduced pulmonary virus titers following live H1N1 challenge than gonadal FCG males (XYM and XXM). To establish that lower estradiol concentrations cause diminished immunity, adult and aged females received either a placebo or estradiol replacement therapy prior to IIV. Estradiol replacement significantly increased IIV-induced antibody responses and reduced morbidity after the H1N1 challenge among aged females. These data highlight that estradiol is a targetable mechanism mediating greater humoral immunity following vaccination among adult females.IMPORTANCEFemales of reproductive ages develop greater antibody responses to influenza vaccines than males. We hypothesized that female-biased immunity and protection against influenza were mediated by estradiol signaling in B cells. Using diverse mouse models ranging from advanced-age mice to transgenic mice that separate sex steroids from sex chromosome complement, those mice with greater concentrations of estradiol consistently had greater numbers of antibody-producing B cells in lymphoid tissue, higher antiviral antibody titers, and greater protection against live influenza virus challenge. Treatment of aged female mice with estradiol enhanced vaccine-induced immunity and protection against disease, suggesting that estradiol signaling in B cells is critical for improved vaccine outcomes in females.

It is common knowledge that immunoglobulin (Ig) is produced by B lymphocytes and mainly functions as an antibody. However, it has been shown recently that myeloblasts from acute myeloid leukemia (AML) could also express Ig and that AML-Ig played a role in leukemogenesis and AML progression. The difference between Ig from myeloblasts and B cells has not been explored. Studying the characteristics of the Ig repertoire in myeloblasts and B cells will be helpful to understand the function and significance of AML-Ig. We performed 5′ RACE-related PCR coupled with PacBio sequencing to analyze the Ig repertoire in myeloblasts and B cells from Chinese AML patients. Myeloblasts expressed all five classes of IgH, especially Igγ, with a high expression frequency. Compared with B-Ig in the same patient, AML-Ig showed different biased V(D)J usages and mutation patterns. In addition, the CDR3 length distribution of AML-Ig was significantly different from those of B-Ig. More importantly, mutations of AML-IgH, especially Igμ, Igα, and Igδ, were different from that of B-IgH in each AML patient, and the mutations frequently occurred at the sites of post-translational modification. AML-Ig has distinct characteristics of variable regions and mutations, which may have implications for disease monitoring and personalized therapy.

Objective·To screen the human-mouse chimeric antibodies that neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and analyse the key amino acid site changes in their light chain.Methods·Antibody heavy chain gene-humanized mice were immunized with the spike protein of SARS-CoV-2. Enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay were performed to analyse the antibody titer in serum level. Flow cytometry was used to sort plasma cells and germinal center (GC) B cells from the mouse spleen and lymph nodes, and the proportions of different B cells were analysed. RNA was extracted to construct a mouse-origin antibody light chain gene library, which was paired with the knocked-in human-origin antibody heavy chain gene for antibody expression. ELISA was employed to screen for high-affinity chimeric antibodies. Amino acid changes in the light chain of high-affinity chimeric antibodies were analysed by comparing them with mouse-origin germline antibody light chain genes. Chimeric antibodies with single amino acid changes in the light chain were constructed and expressed, and their affinity and neutralization abilities were tested through ELISA and pseudovirus neutralization experiments. By comparing the affinity and neutralization abilities of the chimeric antibodies with or without single amino acid changes in the light chain, the key single amino acid change was analysed.Results·Compared to unimmunized mice, the immunized mice showed an increased titer of specific antibodies against SARS-CoV-2 on the serum level, along with an elevated proportion of plasma cells and GC B cells in the spleen and lymph nodes. ELISA showed that a high-affinity chimeric antibody was screened from GC B cells in the mouse spleen. The light chain of the antibody had a 76th serine-to-isoleucine substitution and a 98th phenylalanine deletion. Another ELISA showed that the chimeric antibody exhibited high affinity, the antibody without amino acid change on the light chain and the antibody with only the 98th phenylalanine deletion showed low affinity, and the antibody with only the 76th serine-to-isoleucine substitution demonstrated intermediate affinity between the chimeric antibody and the antibody without amino acid change on the light chain. Pseudovirus neutralization experiments revealed that the chimeric antibody had a half-maximal inhibitory concentration (IC50) of 0.995 ng/μL; the antibody with only the 76th serine-to-isoleucine substitution had an IC50 of 1.724 ng/μL, indicating a slight decrease in neutralization ability; the antibody with only the 98th phenylalanine deletion had an IC50 of 71.05 ng/μL; the antibody without amino acid change on the light chain had an IC50 of 42.06 ng/μL, suggesting weak neutralization ability.Conclusion·For the screened human-mouse chimeric antibodies that neutralize SARS-CoV-2 in this study, the key amino acid change determining the neutralization ability of the chimeric antibody is the 76th serine-to-isoleucine substitution.

Secondary immunoglobulin diversification by somatic hypermutation and class switch recombination in B cells is instrumental for an adequate adaptive humoral immune response. These genetic events may, however, also introduce aberrations into other cellular genes and thereby cause B cell malignancies. While the basic mechanism of somatic hypermutation and class switch recombination is now well understood, their regulation and in particular the mechanism of their specific targeting to immunoglobulin genes is still rather mysterious. In this review, we summarize the current knowledge on the mechanism and regulation of secondary immunoglobulin diversification and discuss known mechanisms of physiological targeting to immunoglobulin genes and mistargeting to other cellular genes. We summarize open questions in the field and provide an outlook on future research. [ABSTRACT FROM AUTHOR]

Introduction: Normal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma. Methods: We microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes. Results: The CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity. Discussion: We conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma. [ABSTRACT FROM AUTHOR]

The adaptive immune system of jawed vertebrates generates a highly diverse repertoire of antibodies to meet the antigenic challenges of a constantly evolving biological ecosystem. Most of the diversity is generated by two mechanisms: V(D)J gene recombination and somatic hypermutation (SHM). SHM introduces changes in the variable domain of antibodies, mostly in the regions that form the paratope, yielding antibodies with higher antigen binding affinity. However, antigen recognition is only possible if the antibody folds into a stable functional conformation. Therefore, a key force determining the survival of B cell clones undergoing somatic hypermutation is the ability of the mutated heavy and light chains to efficiently fold and assemble into a functional antibody. The antibody is the structural context where the selection of the somatic mutations occurs, and where both the heavy and light chains benefit from protectivemechanisms that counteract the potentially deleterious impact of the changes. However, in patients with monoclonal gammopathies, the proliferating plasma cell clone may overproduce the light chain, which is then secreted into the bloodstream. This places the light chain out of the protective context provided by the quaternary structure of the antibody, increasing the risk of misfolding and aggregation due to destabilizing somatic mutations. Light chain-derived (AL) amyloidosis, light chain deposition disease (LCDD), Fanconi syndrome, and myeloma (cast) nephropathy are a diverse group of diseases derived from the pathologic aggregation of light chains, in which somatic mutations are recognized to play a role. In this review, we address the mechanisms by which somatic mutations promote the misfolding and pathological aggregation of the light chains, with an emphasis on AL amyloidosis. We also analyze the contribution of the variable domain (VL) gene segments and somatic mutations on light chain cytotoxicity, organ tropism, and structure of the AL fibrils. Finally, we analyze the most recent advances in the development of computational algorithms to predict the role of somatic mutations in the cardiotoxicity of amyloidogenic light chains and discuss the challenges and perspectives that this approach faces. [ABSTRACT FROM AUTHOR]

Circulating antibody-secreting cells are present in the peripheral blood of healthy individuals reflecting the continued activity of the humoral immune system. Antibody-secreting cells typically express CD27. Here we describe and characterize a small population of antibody-secreting class switched CD19+CD43+ B cells that lack expression of CD27 in the peripheral blood of healthy subjects. In this study, we characterized CD27-CD43+ cells. We demonstrate that class-switched CD27-CD43+ B cells possess characteristics of conventional plasmablasts as they spontaneously secrete antibodies, are morphologically similar to antibody-secreting cells, show downregulation of B cell differentiation markers, and have a gene expression profile related to conventional plasmablasts. Despite these similarities, we observed differences in IgA and IgG subclass distribution, expression of homing markers, replication history, frequency of somatic hypermutation, immunoglobulin repertoire, gene expression related to Toll-like receptors, cytokines, and cytokine receptors, and antibody response to vaccination. Their frequency is altered in immunemediated disorders. Conclusion: we characterized CD27-CD43+ cells as antibody-secreting cells with differences in function and homing potential as compared to conventional CD27+ antibody-secreting cells. [ABSTRACT FROM AUTHOR]

We have employed mathematical modeling techniques to construct a comprehensive framework for elucidating the intricate response mechanisms of the immune system, facilitating a deeper understanding of B-cell clonal deletion and somatic hypermutation. Our improved model introduces innovative mechanisms that shed light on positive and negative selection processes during T-cell and B-cell development. Notably, clonal deletion is attributed to the attenuated immune stimulation exerted by self-antigens with high binding affinities, rendering them less effective in eliciting subsequent B-cell maturation and differentiation. Secondly, our refined model places particular emphasis on the crucial role played by somatic hypermutation in modulating the immune system's functionality. Through extensive investigation, we have determined that somatic hypermutation not only expedites the production of highly specific antibodies pivotal in combating microbial infections but also serves as a regulatory mechanism to dampen autoimmunity and enhance self-tolerance within the organism. Lastly, our model advances the understanding of the implications of antibody in vivo evolution in the overall process of organismal aging. With the progression of time, the age-associated amplification of autoimmune activity becomes apparent. While somatic hypermutation effectively delays this process, mitigating the levels of autoimmune response, it falls short of reversing this trajectory entirely. In conclusion, our advanced mathematical model offers a comprehensive and scholarly approach to comprehend the intricacies of the immune system. By encompassing novel mechanisms for selection, emphasizing the functional role of somatic hypermutation, and illuminating the consequences of in vivo antibody evolution, our model expands the current understanding of immune responses and their implications in aging. [ABSTRACT FROM AUTHOR]

Objective ·To screen the human-mouse chimeric antibodies that neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and analyse the key amino acid site changes in their light chain. Methods ·Antibody heavy chain gene-humanized mice were immunized with the spike protein of SARS-CoV-2. Enzyme-linked immunosorbent assay (ELISA) and pseudovirus neutralization assay were performed to analyse the antibody titer in serum level. Flow cytometry was used to sort plasma cells and germinal center (GC) B cells from the mouse spleen and lymph nodes, and the proportions of different B cells were analysed. RNA was extracted to construct a mouse-origin antibody light chain gene library, which was paired with the knocked-in human-origin antibody heavy chain gene for antibody expression. ELISA was employed to screen for high-affinity chimeric antibodies. Amino acid changes in the light chain of high-affinity chimeric antibodies were analysed by comparing them with mouse-origin germline antibody light chain genes. Chimeric antibodies with single amino acid changes in the light chain were constructed and expressed, and their affinity and neutralization abilities were tested through ELISA and pseudovirus neutralization experiments. By comparing the affinity and neutralization abilities of the chimeric antibodies with or without single amino acid changes in the light chain, the key single amino acid change was analysed. Results ·Compared to unimmunized mice, the immunized mice showed an increased titer of specific antibodies against SARS-CoV-2 on the serum level, along with an elevated proportion of plasma cells and GC B cells in the spleen and lymph nodes. ELISA showed that a high-affinity chimeric antibody was screened from GC B cells in the mouse spleen. The light chain of the antibody had a 76th serine-to-isoleucine substitution and a 98th phenylalanine deletion. Another ELISA showed that the chimeric antibody exhibited high affinity, the antibody without amino acid change on the light chain and the antibody with only the 98th phenylalanine deletion showed low affinity, and the antibody with only the 76th serine-to-isoleucine substitution demonstrated intermediate affinity between the chimeric antibody and the antibody without amino acid change on the light chain. Pseudovirus neutralization experiments revealed that the chimeric antibody had a half-maximal inhibitory concentration (IC50) of 0.995 ng/μL; the antibody with only the 76th serine-to-isoleucine substitution had an IC50 of 1.724 ng/μL, indicating a slight decrease in neutralization ability; the antibody with only the 98th phenylalanine deletion had an IC50 of 71.05 ng/μL; the antibody without amino acid change on the light chain had an IC50 of 42.06 ng/μL, suggesting weak neutralization ability. Conclusion ·For the screened human-mouse chimeric antibodies that neutralize SARS-CoV-2 in this study, the key amino acid change determining the neutralization ability of the chimeric antibody is the 76th serine-to-isoleucine substitution. [ABSTRACT FROM AUTHOR]

In the fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), monoclonal antibodies (mAbs) serve as key strategies for the rapid prevention and treatment of COVID-19. However, analysis to fully characterize functional SARS-CoV-2 mAbs is still needed. In this study, by interrogating 1,695 published or patented mAbs of human origin and validated SARS-CoV-2-binding potency, we found a highly preferential usage of IGHV3-53/3-66 germline genes that was then revealed as a distinct selectivity of SARS-CoV-2-induced humoral immunity across other coronaviruses. Moreover, among the rare somatic hypermutations, we identified a novel mutation signature of F27 to I, L, or V with high frequency, which was located in the CDR1 region of the heavy chain among IGHV3-53/3-66-encoded antibodies. This convergent mutation contributed to improving SARS-CoV-2 binding affinity and may advance our knowledge of the humoral immunity to SARS-CoV-2.

Introduction: The success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance. Methods: We report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls. Results: In contrast to previous studies, our approach successfully stratifies noninfected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients. Discussion: These features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges. [ABSTRACT FROM AUTHOR]

The majority of neutralizing antibodies (NAbs) against SARS-CoV-2 recognize the receptor-binding domain (RBD) of the spike (S) protein. As an escaping strategy, the RBD of the virus is highly variable, evolving mutations to thwart a natural immune response or vaccination. Targeting non-RBD regions of the S protein thus provides a viable alternative to generating potential, robust NAbs. Using a pre-pandemic combinatorial antibody library of 1011, through an alternate negative and positive screening strategy, 11 non-RBD-targeting antibodies are identified. Amongst one NAb that binds specifically to the N-terminal domain of the S protein, SA3, shows mutually non-exclusive binding of the angiotensin-converting enzyme 2 receptor with the S protein. SA3 appears to be insensitive to the conformational change and to interact with both the "open" and "closed" configurations of the trimeric S protein. SA3 shows compatible neutralization as S-E6, an RBD-targeting NAb, against the wild type and variant of concern (VOC) B.1.351 (Beta) of the SARS-CoV-2 pseudo virus. More importantly, the combination of SA3 with S-E6 is synergistic and recovers from the 10-fold loss in neutralization efficacy against the VOC B.1.351 pseudo virus. [ABSTRACT FROM AUTHOR]

Antibody phage display is a key technology for the discovery and development of target-specific monoclonal antibodies (mAbs) for use in research, diagnostics, and therapy. The construction of a high-quality antibody library, with larger and more diverse antibody repertoires, is essential for the successful development of phage display-derived mAbs. In this study, a large human combinatorial single-chain variable fragment library (1.5 × 1011 colonies) was constructed from Epstein–Barr virus-infected human peripheral blood mononuclear cells stimulated with a combination of two of the activators of human B cells, the Toll-like receptor 7/8 agonist R848 and interleukin-2. Next-generation sequencing analysis with approximately 1.9 × 106 and 2.7 × 106 full-length sequences of heavy chain variable (VH) and κ light chain variable (Vκ) domains, respectively, revealed that the library consists of unique VH (approximately 94%) and Vκ (approximately 91%) sequences with greater diversity than germline sequences. Lastly, multiple unique mAbs with high affinity and broad cross-species reactivity could be isolated from the library against two therapeutically relevant target antigens, validating the library quality. These findings suggest that the novel antibody library we have developed may be useful for the rapid development of target-specific phage display-derived recombinant human mAbs for use in therapeutic and diagnostic applications. [ABSTRACT FROM AUTHOR]

Adaptive immunity systems found in different organisms fall into two major types. Prokaryotes possess CRISPR-Cas systems that recognize former invaders using memorized (captured) pieces of their DNA as pathogen signatures. Mammals possess a vast repertoire of antibodies and T-cell receptor variants generated in advance. In this second type of adaptive immunity, a pathogen presentation to the immune system specifically activates the cells that express matching antibodies or receptors. These cells proliferate to fight the infection and form the immune memory. The principle of preemptive production of diverse defense proteins for future use can hypothetically take place in microbes too. We propose a hypothesis that prokaryotes employ diversity-generating retroelements to prepare defense proteins against yet-unknown invaders. In this study, we test this hypothesis with the methods of bioinformatics and identify several candidate defense systems based on diversity-generating retroelements. [ABSTRACT FROM AUTHOR]

IntroductionNormal CD30+ B cells represent a distinct B-cell differentiation stage with features of strong activation. We lack an in depth understanding of these cells, because they are not present in peripheral blood and are typically very rare in reactive lymphoid organs. CD30+ B cells have been discussed as a potential precursor population for the malignant CD30+ Hodgkin and Reed-Sternberg cells in classical Hodgkin lymphoma. As CD30+ B cells can be more numerous in some cases of reactive lymphadenitis, we aimed to characterize these CD30+ B cells in terms of their differentiation stage and clonal composition, also as a means to clarify whether such CD30+ B-cell populations may represent potential precursor lesions of Hodgkin lymphoma.MethodsWe microdissected single CD30+ B cells from tissue sections of eight reactive lymph nodes with substantial numbers of such cells and sequenced their rearranged immunoglobulin (Ig) heavy chain V region (IGHV) genes.ResultsThe CD30+ B cells were polyclonal B cells in all instances, and they not only encompass post-germinal center (GC) B cells with mutated IGHV genes, but also include a substantial fraction of pre-germinal center B cells with unmutated IGHV genes. In five of the lymph nodes, mostly small clonal expansions were detected among the CD30+ B cells. Most of the expanded clones carried somatically mutated IGHV genes and about half of the mutated clones showed intraclonal diversity.DiscussionWe conclude that in human reactive lymph nodes with relatively many CD30+ B cells, these cells are a heterogenous population of polyclonal B cells encompassing activated pre-GC B cells as well as GC and post-GC B cells, with some clonal expansions. Because of their polyclonality and frequent pre-GC differentiation stage, there is no indication that such cell-rich CD30+ B-cell populations represent precursor lesions of Hodgkin lymphoma.

The adaptive immune system of jawed vertebrates generates a highly diverse repertoire of antibodies to meet the antigenic challenges of a constantly evolving biological ecosystem. Most of the diversity is generated by two mechanisms: V(D)J gene recombination and somatic hypermutation (SHM). SHM introduces changes in the variable domain of antibodies, mostly in the regions that form the paratope, yielding antibodies with higher antigen binding affinity. However, antigen recognition is only possible if the antibody folds into a stable functional conformation. Therefore, a key force determining the survival of B cell clones undergoing somatic hypermutation is the ability of the mutated heavy and light chains to efficiently fold and assemble into a functional antibody. The antibody is the structural context where the selection of the somatic mutations occurs, and where both the heavy and light chains benefit from protective mechanisms that counteract the potentially deleterious impact of the changes. However, in patients with monoclonal gammopathies, the proliferating plasma cell clone may overproduce the light chain, which is then secreted into the bloodstream. This places the light chain out of the protective context provided by the quaternary structure of the antibody, increasing the risk of misfolding and aggregation due to destabilizing somatic mutations. Light chain-derived (AL) amyloidosis, light chain deposition disease (LCDD), Fanconi syndrome, and myeloma (cast) nephropathy are a diverse group of diseases derived from the pathologic aggregation of light chains, in which somatic mutations are recognized to play a role. In this review, we address the mechanisms by which somatic mutations promote the misfolding and pathological aggregation of the light chains, with an emphasis on AL amyloidosis. We also analyze the contribution of the variable domain (VL) gene segments and somatic mutations on light chain cytotoxicity, organ tropism, and structure of the AL fibrils. Finally, we analyze the most recent advances in the development of computational algorithms to predict the role of somatic mutations in the cardiotoxicity of amyloidogenic light chains and discuss the challenges and perspectives that this approach faces.

Circulating antibody-secreting cells are present in the peripheral blood of healthy individuals reflecting the continued activity of the humoral immune system. Antibody-secreting cells typically express CD27. Here we describe and characterize a small population of antibody-secreting class switched CD19+CD43+ B cells that lack expression of CD27 in the peripheral blood of healthy subjects. In this study, we characterized CD27-CD43+ cells. We demonstrate that class-switched CD27-CD43+ B cells possess characteristics of conventional plasmablasts as they spontaneously secrete antibodies, are morphologically similar to antibody-secreting cells, show downregulation of B cell differentiation markers, and have a gene expression profile related to conventional plasmablasts. Despite these similarities, we observed differences in IgA and IgG subclass distribution, expression of homing markers, replication history, frequency of somatic hypermutation, immunoglobulin repertoire, gene expression related to Toll-like receptors, cytokines, and cytokine receptors, and antibody response to vaccination. Their frequency is altered in immune-mediated disorders.Conclusionwe characterized CD27-CD43+ cells as antibody-secreting cells with differences in function and homing potential as compared to conventional CD27+ antibody-secreting cells.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation of immunoglobulin (Ig) gene variable regions and class switch recombination (CSR) of Ig heavy chain constant regions. Two decades of intensive research has greatly expanded our knowledge of how AID functions in peripheral B cells to optimize antibody responses against infections, while maintaining tight regulation of AID to restrain its activity to protect B cell genomic integrity. The many exciting recent advances in the field include: 1) the first description of AID’s molecular structure, 2) remarkable advances in high throughput approaches that precisely track AID targeting genome-wide, and 3) the discovery that the cohesion-mediate loop extrusion mechanism [initially discovered in V(D)J recombination studies] also governs AID-medicated CSR. These advances have significantly advanced our understanding of AID’s biochemical properties in vitro and AID’s function and regulation in vivo. This mini review will discuss these recent discoveries and outline the challenges and questions that remain to be addressed.

Antibodies are important immune molecules that are elicited by B cells to protect our bodies during viral infections or vaccinations. In humans, the antibody repertoire is diversified by programmed DNA lesion processes to ensure specific and high affinity binding to various antigens. Broadly neutralizing antibodies (bnAbs) are antibodies that have strong neutralizing activities against different variants of a virus. bnAbs such as anti-HIV bnAbs often have special characteristics including insertions and deletions, long complementarity determining region 3 (CDR3), and high frequencies of mutations, often at improbable sites of the variable regions. These unique features are rare mutational outcomes that are acquired during antibody diversification processes. In this review, we will discuss possible mechanisms that generate these rare antibody mutational outcomes. The understanding of the mechanisms that generate these rare mutational outcomes during antibody diversification will have implications in vaccine design strategies to elicit bnAbs.

IntroductionThe success of the human body in fighting SARS-CoV2 infection relies on lymphocytes and their antigen receptors. Identifying and characterizing clinically relevant receptors is of utmost importance.MethodsWe report here the application of a machine learning approach, utilizing B cell receptor repertoire sequencing data from severely and mildly infected individuals with SARS-CoV2 compared with uninfected controls.ResultsIn contrast to previous studies, our approach successfully stratifies non-infected from infected individuals, as well as disease level of severity. The features that drive this classification are based on somatic hypermutation patterns, and point to alterations in the somatic hypermutation process in COVID-19 patients.DiscussionThese features may be used to build and adapt therapeutic strategies to COVID-19, in particular to quantitatively assess potential diagnostic and therapeutic antibodies. These results constitute a proof of concept for future epidemiological challenges.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries. However, CLL is relatively rare in Asia; its genetic features are rarely studied. Here, we aimed to genetically characterize Korean CLL patients and to elucidate the genetic and clinical associations based on data obtained from 113 patients at a single Korean institute. We used next-generation sequencing to explore the multi-gene mutational data and immunoglobulin heavy chain variable gene clonality with somatic hypermutation (SHM). MYD88 (28.3%), including L265P (11.5%) and V217F (13.3%), was the most frequently mutated gene, followed by KMT2D (6.2%), NOTCH1 (5.3%), SF3B1 (5.3%), and TP53 (4.4%). MYD88-mutated CLL was characterized by SHM and atypical immunophenotype with fewer cytogenetic abnormalities. The 5-year time to treatment (TTT) of the overall cohort was 49.8% ± 8.2% (mean ± standard deviation) and the 5-year overall survival was 86.2% ± 5.8%. Patients with SHM, isolated del(13q), TP53-wild type, and NOTCH1-wild type showed better results than those without these conditions. In the subgroup analyses, patients with SHM and L265P presented shorter TTT than patients with SHM but not L265P. In contrast, V217F was associated with a higher SHM percentage and showed a favorable prognosis. Our study revealed the distinct characteristics of Korean CLL patients with high frequencies of MYD88 mutations and their clinical relevance. [ABSTRACT FROM AUTHOR]

Recent research has revealed substantial interindividual variation in immunoglobulin (IG) loci as well as associations between germline IG genotypes and the humoral response. It is unknown how population- and species-level germline variation at IG loci is maintained or which are the mechanisms by which such variation leads to functional differences in the evolved B cell repertoire. Characterizing these mechanisms may be important for the design of vaccines and other therapeutics. Resolving this mystery includes studying the interplay of evolutionary processes at two different levels of biological organization: within individuals and within populations. Substantial human inter-individual genetic variation at germline immunoglobulin (antibody) loci has been recently reported, in addition to associations between such variation and adaptive humoral responses. Certain hypotheses suggest that evolution may play a role in shaping germline IG loci and antibody repertoires, for which emerging evidence supports (or not) these hypotheses. There is a need to establish better evolutionary models that might better inform and could contribute to the development of candidate vaccines/therapeutics. 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. [ABSTRACT FROM AUTHOR]

DNA polymerase eta (Pol η) is a Y-family polymerase and the product of the POLH gene. Autosomal recessive inheritance of POLH mutations is the cause of the xeroderma pigmentosum variant, a cancer predisposition syndrome. This review summarizes mounting evidence for expanded Pol η cellular functions in addition to DNA lesion bypass that are critical for maintaining genome stability. In vitro, Pol η displays efficient DNA synthesis through difficult-to-replicate sequences, catalyzes D-loop extensions, and utilizes RNA–DNA hybrid templates. Human Pol η is constitutively present at the replication fork. In response to replication stress, Pol η is upregulated at the transcriptional and protein levels, and post-translational modifications regulate its localization to chromatin. Numerous studies show that Pol η is required for efficient common fragile site replication and stability. Additionally, Pol η can be recruited to stalled replication forks through protein–protein interactions, suggesting a broader role in replication fork recovery. During somatic hypermutations, Pol η is recruited by mismatch repair proteins and is essential for VH gene A:T basepair mutagenesis. Within the global context of repeat-dense genomes, the recruitment of Pol η to perform specialized functions during replication could promote genome stability by interrupting pure repeat arrays with base substitutions. Alternatively, not engaging Pol η in genome duplication is costly, as the absence of Pol η leads to incomplete replication and increased chromosomal instability. [ABSTRACT FROM AUTHOR]

Les voies qui régulent la formation et le maintien des centres germinatifs sont très souvent impliquées dans le processus de transformation maligne des clones B. La plupart des lymphomes dérivent des LB du centre germinatif et post-centre germinatif qui ont subi les étapes d'hypermutation somatique et de recombinaison de classe. Ainsi, l'expansion clonale B anormale s'accompagne souvent de production de quantité variable d'immunoglobulines monoclonales qui, par leurs caractéristiques physico-chimiques, peuvent établir des interactions moléculaires cellulaires rénales et/ou extra-rénales qui aboutissent à des entités anatomopathologiques bien définies. Toutes les structures rénales peuvent être atteintes. Les mécanismes qui mènent à la toxicité rénale des immunoglobulines monoclonales sont multiples et complexes. Certains sont bien connus et d'autres le sont moins. La sélection aléatoire de certains gènes de la lignée germinale, la structure primaire de l'immunoglobuline, ses modifications structurelles post-traductionnelles et ses interactions avec le micro-environnement rénal sont les déterminants majeurs des dépôts rénaux. D'une manière générale, les immunoglobulines pathologiques génèrent une toxicité rénale par deux mécanismes distincts : (i) soit par excès de production qui surpasse les capacités normales de catabolisme des cellules épithéliales tubulaires proximales, occasionnant ainsi une arrivée significative de chaînes légères libres dans les tubules distaux dont l'environnement physico-chimique peut être propice à la précipitation des chaînes légères libres ; (ii) soit par leurs caractéristiques structurales, indépendamment de leur taux sérique, qui les prédisposent à donner une atteinte rénale bien précise. L'objectif de ce manuscrit est de faire une large revue de la littérature en partant des mécanismes moléculaires de l'expansion des clones B à la toxicité rénale des immunoglobulines pathologiques. Germinal center regulation pathways are often involved in lymphomagenesis and myelomagenesis. Most of the lymphomas (and multiple myeloma) derive from post-germinal center B-cells that have undergone somatic hypermutation and class switch recombination. Hence, B-cell clonal expansion can be responsible for the presence of a monoclonal component (immunoglobulin) of variable titer which, owing to physicochemical properties, can provoke pathologically defined entities of diseases. These diseases can affect any functional part of the kidney, by multiple mechanisms, either well known or not. The presence of renal deposition is influenced by germinal gene involved, immunoglobulin primary structure, post-translational modifications and microenvironmental interactions. The two ways immunoglobulin can cause kidney toxicity are (i) an excess of production (overcoming catabolism power by proximal tubule epithelial cells) with an excess of free light chains within the distal tubules and a subsequent risk of precipitation due to local physicochemical properties; (ii) by structural characteristics that predispose immunoglobulin to a renal disease (whatever their titer). The purpose of this manuscript is to review literature concerning the pathophysiology of renal toxicities of clonal immunoglobulin, from molecular B-cell expansion mechanisms to immunoglobulin renal toxicity. [ABSTRACT FROM AUTHOR]

• AID, Chromatin remodelers, and Epigenetics (ACE) play a crucial role in antibody diversity. • Chromatin dynamics modulate AID expression and guide its targeting at the Ig loci. • AID-mediated epigenetic changes cause immune disorders, including B-cell lymphoma. • The epigenetic role of G9a makes it a promising target for melanoma therapy. Activation-induced cytidine deaminase (AID) is responsible for the initiation of somatic hypermutation (SHM) and class-switch recombination (CSR), which result in antibody affinity maturation and isotype switching, thus producing pathogen-specific antibodies. Chromatin dynamics and accessibility play a significant role in determining AID expression and its targeting. Chromatin remodelers contribute to the accessibility of the chromatin structure, thereby influencing the targeting of AID to Ig genes. Epigenetic modifications, including DNA methylation, histone modifications, and miRNA expression, profoundly impact the regulation of AID and chromatin remodelers targeting Ig genes. Additionally, epigenetic modifications lead to chromatin rearrangement and thereby can change AID expression levels and its preferential targeting to Ig genes. This interplay is symbolized as the ACE phenomenon encapsulates three interconnected aspects: AID, Chromatin remodelers, and Epigenetic modifications. This review emphasizes the importance of understanding the intricate relationship between these aspects to unlock the therapeutic potential of these molecular processes and molecules. [ABSTRACT FROM AUTHOR]

Memory B cells (MBCs) formed over the individual's lifetime constitute nearly half of the circulating B cell repertoire in humans. These pre-existing MBCs dominate recall responses to their cognate antigens, but how they respond to recognition of novel antigens is not well understood. Here, we tracked the origin and followed the differentiation paths of MBCs in the early anti-spike (S) response to mRNA vaccination in SARS-CoV-2-naive individuals on single-cell and monoclonal antibody levels. Pre-existing, highly mutated MBCs showed no signs of germinal center re-entry and rapidly developed into mature antibody-secreting cells (ASCs). By contrast, and despite similar levels of S reactivity, naive B cells showed strong signs of antibody affinity maturation before differentiating into MBCs and ASCs. Thus, pre-existing human MBCs differentiate into ASCs in response to novel antigens, but the quality of the humoral and cellular anti-S response improved through the clonal selection and affinity maturation of naive precursors. [Display omitted] • SARS-CoV-2 mRNA vaccination activates naive and pre-existing MBCs in naive individuals • S-reactive naive, but not pre-existing, MBCs show signs of strong affinity maturation • Despite low S reactivity, pre-existing MBCs dominate the early ASC response • High-affinity anti-S MBCs and ASCs develop from naive, not MBC, precursors Memory B cells dominate recall responses, but how they react to novel antigens is not well understood. Li and Obraztsova et al. show that MBCs with similar antigen reactivity as those of naive B cells rapidly differentiate into ASCs without signs of germinal center re-entry, whereas affinity maturation of naive B cells underlies improvements in humoral immunity to novel antigens. [ABSTRACT FROM AUTHOR]

The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (Ig) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.

Classification of patients with chronic lymphocytic leukemia (CLL) based on the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene has established predictive and prognostic relevance. The SHM status is assessed based on the number of mutations within the IG heavy variable domain sequence, albeit only over the rearranged IGHV gene excluding the variable heavy complementarity determining region 3 (VH CDR3). This may lead to an underestimation of the actual impact of SHM, in fact overlooking the most critical region for antigen-antibody interactions, i.e. the VH CDR3. Here we investigated whether SHM may be present within the VH CDR3 of cases bearing ‘truly unmutated’ IGHV genes (i.e. 100% germline identity across VH FR1-VH FR3) employing Next Generation Sequencing. We studied 16 patients bearing a ‘truly unmutated’ CLL clone assigned to stereotyped subsets #1 (n=12) and #6 (n=4). We report the existence of SHM within the germline-encoded 3’IGHV, IGHD, 5’IGHJ regions of the VH CDR3 in both the main IGHV-IGHD-IGHJ gene clonotype and its variants. Recurrent somatic mutations were identified between different patients of the same subset, supporting the notion that they represent true mutational events rather than technical artefacts; moreover, they were located adjacent to/within AID hotspots, pointing to SHM as the underlying mechanism. In conclusion, we provide immunogenetic evidence for intra-VH CDR3 variations, attributed to SHM, in CLL patients carrying ‘truly unmutated’ IGHV genes. Although the clinical implications of this observation remain to be defined, our findings offer a new perspective into the immunobiology of CLL, alluding to the operation of VH CDR3-restricted SHM in U-CLL.

DNA mutagenesis during antibody affinity maturation has potentially oncogenic or autoimmune outcomes if not tightly controlled as it is in mammalian germinal centers. Cold blooded vertebrates lack germinal centers, yet have a functional Ig gene mutator enzyme, Aicda. In fish there are clusters of Aicda+ cells encircled by pigmented ‘melano-macrophages’ and we test the hypothesis that these clusters are functionally analogous to germinal centers. Sequenced IgH VDJ repertoire libraries from individual isolated clusters showed evidence of B-cell clonal expansion and VDJ somatic hypermutation. Construction of Ig clonal lineage trees revealed that unlike surrounding lymphoid tissue, each cluster is dominated by a few B-cell VDJ clonotypes having hundreds of mutated variants. Recruitment of B-cells to the clusters appears to be ongoing, as there are additional Ig clones having smaller lineages. Finally, we show evidence for positive selection for replacement mutations in regions encoding the antigen contact loops, but not in the framework regions, consistent with functional antibody modification. Melano-macrophages appear to trap the Ag used for post-mutation B-cell selection, performing a role analogous to the follicular dendritic cells of mammalian germinal centers. These findings provide insights into the evolution of the affinity maturation process, the improvement of fish vaccines and possibly also the workings of atypical ectopic germinal centers generated in several human diseases.