IgM Repertoire Research Articles

Characteristics of the IgM Repertoires in the Peripheral Blood of Early Rheumatoid Arthritis Patients.

Rheumatoid arthritis is a common autoimmune disease, but little is known about the characteristics of the B cell repertoires in the peripheral blood. In this study, the peripheral IgM repertoires of early rheumatoid arthritis (ERA) patients were analyzed by high-throughput sequencing and bioinformatics analyses. Clonal expansion was observed in IgM repertoires of ERA patients. Interestingly, a subset of the dominant clones in ERA repertoires showed self- and poly-reactivity to several autoantigens. The clones were also present in IgM repertoires of healthy adults but they were not expanded, suggesting that may stem from the natural auto-reactive B cell repertoire. Additionally, the ERA repertoires exhibited a greater extent of somatic hypermutations, particularly in the ERA dominant clones, resulting in an enrichment of amino acids important for antigen-antibody interaction. The in-depth analysis of B-cell repertoires improved our knowledge of the IgM repertoires in early rheumatoid arthritis, offering potential insights into the disease's pathogenesis.

Chickens with a Truncated Light Chain Transgene Express Single-Domain H Chain-Only Antibodies.

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In invitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.

The common variable immunodeficiency IgM repertoire narrowly recognizes erythrocyte and platelet glycans

BackgroundAutoantibody-mediated cytopenias (AICs) regularly occur in profoundly IgG-deficient common variable immunodeficiency (CVID) patients. The isotypes, antigenic targets, and origin(s) of their disease-causing autoantibodies are unclear. ObjectiveTo determine reactivity, clonality and provenance of AIC-associated IgM autoantibodies in CVID patients. MethodsWe utilized glycan arrays, patient erythrocytes, and platelets to determine targets of CVID IgM autoantibodies. Glycan binding profiles were used to identify auto-reactive clones across B cell subsets, specifically circulating marginal zone-like (MZ) B cells, for sorting and IGH sequencing. The locations, transcriptomes and responses of tonsillar MZ B cells to different T helper cell subsets were determined by confocal microscopy, RNA-sequencing, and co-cultures, respectively. ResultsAutoreactive IgM coated erythrocytes and platelets from many CVID patients with AICs (CVID+AIC). On glycan arrays, CVID+AIC plasma IgM narrowly recognized erythrocytic i antigens and platelet i-related antigens and failed to bind hundreds of pathogen- and tumor-associated carbohydrates. Polyclonal i antigen-recognizing B-cell receptors were highly enriched among CVID+AIC circulating marginal zone (MZ) B cells. Within tonsillar tissues, MZ B cells secreted copious IgM when activated by the combination of IL-10 and IL-21 or when cultured with IL10/IL-21 secreting FOXP3-CD25hiTfh cells. In lymph nodes from immunocompetent controls, MZ B cells, plentiful FOXP3+ regulatory T cells, and rare FOXP3-CD25+ cells that represented likely CD25hiTfh cells, all localized outside of GCs. In CVID+AIC lymph nodes, cellular positions were similar but CD25hiTfh cells greatly outnumbered regulatory cells. ConclusionsOur findings indicate glycan-reactive IgM autoantibodies produced outside of GCs may contribute to the autoimmune pathogenesis of CVID.

Generation of a Naïve Human scFv Phage Display Library and Panning Selection.

Phage display antibody libraries have been successfully used as the essential tool to produce monoclonal antibodies against a plethora of targets ranging from diseases to native biologically important proteins as well as small molecules. It is well documented that diverse antibody genes are the major genetic source for the construction of a high-quality antibody library and selection of high-affinity antibodies. Naïve antibody libraries are derived using the IgM repertoire of healthy donors obtained from B-cells isolated from human peripheral blood mononuclear cell (PBMC). Single-chain fragment variable (scFv) is a routinely used format due to its smaller size and preference for phage display. The process involves the use of a two-step cloning method for library construction. The protocol also covers the biopanning process for target positive clone selection.

Hindlimb unloading, a physiological model of microgravity, modifies the murine bone marrow IgM repertoire in a similar manner as aging but less strongly

BackgroundThe spaceflight environment is an extreme environment that affects the immune system of approximately 50% of astronauts. With planned long-duration missions, such as the deployment of the Lunar Gateway and possible interplanetary missions, it is mandatory to determine how all components of the immune system are affected, which will allow the establishment of countermeasures to preserve astronaut health. However, despite being an important component of the immune system, antibody-mediated humoral immunity has rarely been investigated in the context of the effects of the space environment. It has previously been demonstrated that 30 days aboard the BION-M1 satellite and 21 days of hindlimb unloading (HU), a model classically used to mimic the effects of microgravity, decrease murine B lymphopoiesis. Furthermore, modifications in B lymphopoiesis reported in young mice subjected to 21 days of HU were shown to be similar to those observed in aged mice (18–22 months). Since the primary antibody repertoire composed of IgM is created by V(D) J recombination during B lymphopoiesis, the objective of this study was to assess the degree of similarity between changes in the bone marrow IgM repertoire and in the V(D)J recombination process in 2.5-month-old mice subjected to 21 days of HU and aged (18 months) mice.ResultsWe found that in 21 days, HU induced changes in the IgM repertoire that were approximately 3-fold less than those in aged mice, which is a rapid effect. Bone remodeling and epigenetics likely mediate these changes. Indeed, we previously demonstrated a significant decrease in tibial morphometric parameters from day 6 of HU and a progressive reduction in these parameters until day 21 of HU, and it has been shown that age and microgravity induce epigenetic changes.ConclusionThese data reveal novel immune changes that are akin to advanced aging and underline the importance of studying the effects of spaceflight on antibody-mediated humoral immunity.

Mice possess a more limited natural antihuman factor VIII antibody repertoire than humans that is produced disproportionately by marginal zone B cells

BackgroundOne-third of patients with severe hemophilia A develop neutralizing antibodies to the factor VIII (FVIII) protein in response to intravenous replacement therapy. Patients may also generate natural, nonneutralizing antibodies to FVIII before FVIII exposure. These patients are at increased risk of developing neutralizing antibodies to FVIII. However, natural anti-FVIII antibodies are also present in healthy human donors. ObjectivesTo further characterize the natural antihuman (h) FVIII antibody repertoire in mice and humans. MethodsAn in-house ELISA was developed using a purified polyclonal immunoglobulin (Ig) standard to quantify anti-hFVIII Ig in cell culture supernatant or plasma from mice (wild-type and FVIII-/-) and adult human donors. ResultsAll naïve wild-type and FVIII-/- mice, as well as healthy human donors, possess natural anti-hFVIII antibodies. Mice only have natural anti-hFVIII IgM, which is present in germ-free mice, suggesting that they are germline encoded. Although murine marginal zone B cells (MZBs) contribute 44% to all circulating natural IgM, they contribute disproportionately to the anti-hFVIII IgM repertoire (82%). This naturally occurring murine MZB-derived IgM is not B-domain specific and is reduced by intravenously administered hFVIII, suggesting that it may form immune complexes immediately upon hFVIII administration. Natural anti-hFVIII antibodies of IgG, IgM, and IgA isotypes can be detected in adult human donors. There were increased levels of B-domain-favoring anti-hFVIII IgG in 14% of healthy donors, which were markedly different from the rest of the “low-titer” population. ConclusionsThere is a preponderance of natural anti-hFVIII antibodies in both mice and healthy adult human donors.

Differentiation and traffic of IgM+ B cells between focal dark spots in skeletal muscle of Atlantic salmon, lymphoid and adipose tissues.

Focal dark spots (DS) in farmed Atlantic salmon fillets contain a significant number of B cells as revealed by the high abundance of immunoglobulin (Ig) transcripts in transcriptome data. The immune response in DS remains unknown while they represent a major problem in commercial aquaculture. Here, we characterized the diversity and clonal composition of B cells in DS. Sixteen gene markers of immune cells and antigen presentation were analyzed with RT-qPCR. All genes expression showed a positive correlation with DS area and intensity. The flatter the DS, the higher the expression of cd28, csfr, ctla, igt, and sigm, the lower expression of cd83 and btla, and the larger the cumulative frequency within DS. The expression of most of the analyzed immune genes, including three Ig types and markers of B cells was lower in DS than in the lymphatic organs, head kidney and spleen, but significantly higher compared to skeletal muscle. High levels of ctla4 and cd28 in DS might indicate the recruitment of T cells. Sequencing of IgM repertoire (Ig-seq) assessed migration of B cells by co-occurrence of identical CDR3 sequences in different tissues. The combination of gene expression and Ig-seq revealed the presence of several stages of B cell differentiation in DS. B cells at the earliest stage, with high ratio of membrane to secretory IgM (migm and sigm), showed minor Ig repertoire overlap with other tissues. Further differentiation stage (increased sigm to migm ratio and high expression of pax5 and cd79) was associated with active movement of B cells from DS towards lymphatic organs and visceral fat. Traffic and expression of immune genes decreased at later stages. These B cells could be involved in a response directed against viruses, pathogenic or opportunistic bacteria in DS. Seven of eight fish were positive for salmon alphavirus, and levels were higher in DS than in unstained muscle. PCR with universal primers to the 16S rRNA gene did not detect bacteria in DS. Although the evolution of DS most likely implies local exposure to antigens, neither this nor previous studies have found a necessary association between DS and pathogens or self-antigens.

Sequencing the B cell receptor repertoires of antibody-deficient individuals with and without infection susceptibility

PurposeMost individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT. MethodsWe sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT. ResultsCompared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations). ConclusionAntibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.

FLAIRR-Seq: A Method for Single-Molecule Resolution of Near Full-Length Antibody H Chain Repertoires.

Current Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using short-read sequencing strategies resolve expressed Ab transcripts with limited resolution of the C region. In this article, we present the near-full-length AIRR-seq (FLAIRR-seq) method that uses targeted amplification by 5' RACE, combined with single-molecule, real-time sequencing to generate highly accurate (99.99%) human Ab H chain transcripts. FLAIRR-seq was benchmarked by comparing H chain V (IGHV), D (IGHD), and J (IGHJ) gene usage, complementarity-determining region 3 length, and somatic hypermutation to matched datasets generated with standard 5' RACE AIRR-seq using short-read sequencing and full-length isoform sequencing. Together, these data demonstrate robust FLAIRR-seq performance using RNA samples derived from PBMCs, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving H chain gene features not documented in IMGT at the time of submission. FLAIRR-seq data provide, for the first time, to our knowledge, simultaneous single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk-expressed Ab repertoires to date.

Sequencing the B Cell Receptor Repertoires of Antibody-Deficient Individuals With and Without Infection Susceptibility

PurposeMost individuals with antibody deficiency (hypogammaglobulinemia) need immunoglobulin replacement therapy (IgG-RT) from healthy plasma donors to stay clear of infections. However, a small subset of hypogammaglobulinemic patients do not require this substitution therapy. We set out to investigate this clinical conundrum by asking whether the peripheral B cell receptor repertoires differ between antibody-deficient patients who do and do not need IgG-RT.MethodsWe sequenced and analyzed IgG and IgM heavy chain B cell receptor repertoires from peripheral blood mononuclear cells (PBMCs) isolated from patients with low serum IgG concentrations who did or did not require IgG-RT.ResultsCompared to the patients who did not need IgG-RT, those who needed IgG-RT had higher numbers of IgG antibody clones, higher IgM diversity, and less oligoclonal IgG and IgM repertoires. The patient cohorts had different heavy chain variable gene usage, and the patients who needed IgG-RT had elevated frequencies of IgG clones with higher germline identity (i.e., fewer somatic hypermutations).ConclusionAntibody-deficient patients with infection susceptibility who needed IgG-RT had more diverse peripheral antibody repertoires that were less diverged from germline and thus may not be as optimal for targeting pathogens, possibly contributing to infection susceptibility.

B-1 plasma cells require non-cognate CD4 T cell help to generate a unique repertoire of natural IgM.

Evolutionarily conserved, "natural" (n)IgM is broadly reactive to both self and foreign antigens. Its selective deficiency leads to increases in autoimmune diseases and infections. In mice, nIgM is secreted independent of microbial exposure to bone marrow (BM) and spleen B-1 cell-derived plasma cells (B-1PC), generating the majority of nIgM, or by B-1 cells that remain non-terminally differentiated (B-1sec). Thus, it has been assumed that the nIgM repertoire is broadly reflective of the repertoire of body cavity B-1 cells. Studies here reveal, however, that B-1PC generate a distinct, oligoclonal nIgM repertoire, characterized by short CDR3 variable immunoglobulin heavy chain regions, 7-8 amino acids in length, some public, many arising from convergent rearrangements, while specificities previously associated with nIgM were generated by a population of IgM-secreting B-1 (B-1sec). BM, but not spleen B-1PC, or B-1sec also required the presence of TCRαβ CD4 T cells for their development from fetal precursors. Together, the studies identify important previously unknown characteristics of the nIgM pool.

Construction of Naïve and Immune Human Fab Phage Display Library.

Phage display has been applied successfully for the rapid isolation of monoclonal antibodies against various targets including infectious diseases, autoantigens, cancer markers, and even small molecules. The main component in anyphage display experiment is the availabilityof an antibody library to carry out the selection process of target-specific antibodies through an iterative process termed as biopanning. To generate human antibody libraries, the antibody repertoire can be obtained from human peripheral blood mononuclear cell (PBMC) or directly from cell-sorted B-cell populations. The choice of antibody isotype is dictated by the nature ofthe library. Naïve libraries would utilize IgM repertoires, whereas the IgG repertoire is commonly used for immune libraries. Antibody genes are amplified through polymerase chain reaction (PCR) and paired in a combinatorial fashion to expand the diversity of the cloned library repertoire. The protocol here describes the use of a two-step cloning method that can be appliedfor the construction of eithera naïve or immune human antibody library in Fab format followed by the subsequent panning.

Age-dependent nasal immune responses in non-hospitalized bronchiolitis children.

Bronchiolitis in children is associated with significant rates of morbidity and mortality. Many studies have been performed using samples from hospitalized bronchiolitis patients, but little is known about the immunological responses from infants suffering from mild/moderate bronchiolitis that do not require hospitalization. We have studied a collection of nasal lavage fluid (NLF) samples from outpatient bronchiolitis children as a novel strategy to unravel local humoral and cellular responses, which are not fully characterized. The children were age-stratified in three groups, two of them (GI under 2-months, GII between 2-4 months) presenting a first episode of bronchiolitis, and GIII (between 4 months and 2 years) with recurrent respiratory infections. Here we show that elevated levels of pro-inflammatory cytokines (IL1β, IL6, TNFα, IL18, IL23), regulatory cytokines (IL10, IL17A) and IFNγ were found in the three bronchiolitis cohorts. However, little or no change was observed for IL33 and MCP1, at difference to previous results from bronchiolitis hospitalized patients. Furthermore, our results show a tendency to IL1β, IL6, IL18 and TNFα increased levels in children with mild pattern of symptom severity and in those in which non RSV respiratory virus were detected compared to RSV+ samples. By contrast, no such differences were found based on gender distribution. Bronchiolitis NLFs contained more IgM, IgG1, IgG3 IgG4 and IgA than NLF from their age-matched healthy controls. NLF from bronchiolitis children predominantly contained neutrophils, and also low frequency of monocytes and few CD4+ and CD8+ T cells. NLF from infants older than 4-months contained more intermediate monocytes and B cell subsets, including naïve and memory cells. BCR repertoire analysis of NLF samples showed a biased VH1 usage in IgM repertoires, with low levels of somatic hypermutation. Strikingly, algorithmic studies of the mutation profiles, denoted antigenic selection on IgA-NLF repertoires. Our results support the use of NLF samples to analyze immune responses and may have therapeutic implications.

Impact of dyslipidemia on the IgM repertoire in atherosclerotic mice

Background and Aims : Inflammation together with hypercholesterolemia is a key contributor in atherosclerosis. Importantly, IgM have been shown to play a protective role in atherosclerosis, and data from mice and epidemiological studies in humans demonstrate a negative correlation between IgM levels and atherosclerotic cardiovascular disease. Dyslipidemia has been shown to affect the IgM antibody levels in mouse models of atherosclerosis. Our aim is to investigate the impact of dyslipidemia on the IgM repertoire in the context of atherosclerosis.

Ontogeny of the B Cell Receptor Repertoire and Microbiome in Mice

The immune system matures throughout childhood to achieve full functionality in protecting our bodies against threats. The immune system has a strong reciprocal symbiosis with the host bacterial population and the two systems co-develop, shaping each other. Despite their fundamental role in health physiology, the ontogeny of these systems is poorly characterized. In this study, we investigated the development of the BCR repertoire by analyzing high-throughput sequencing of their receptors in several time points of young C57BL/6J mice. In parallel, we explored the development of the gut microbiome. We discovered that the gut IgA repertoires change from birth to adolescence, including an increase in CDR3 lengths and somatic hypermutation levels. This contrasts with the spleen IgM repertoires that remain stable and distinct from the IgA repertoires in the gut. We also discovered that large clones that germinate in the gut are initially confined to a specific gut compartment, then expand to nearby compartments and later on expand also to the spleen and remain there. Finally, we explored the associations between diversity indices of the B cell repertoires and the microbiome, as well as associations between bacterial and BCR clusters. Our results shed light on the ontogeny of the adaptive immune system and the microbiome, providing a baseline for future research.

Let’s talk about sex, baby: sex influences age-related changes in natural antibodies and natural antibody producing B-1a cells

Abstract Natural antibodies are primarily produced by B-1a cells and are essential for protection against Streptococcus pneumoniae. The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and IgM repertoire. Our investigation demonstrates the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum immunoglobulin, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC) nor PPS3-specific IgM. Interestingly, we observed aged females have an increase in the total number of B-1a cells, higher serum IL-5 levels, and a larger percentage of aged female B-1a cells expressed CD86 as compared to aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal and splenic PC+ B-1a cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female B-1a cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2. Together, these data indicate females maintain a more diverse and active B-1a cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease. Supported by grants from NIH NIAID (R01AI154539)

Restriction of the Global IgM Repertoire in Antiphospholipid Syndrome.

The typical anti-phospholipid antibodies (APLA) in the anti-phospholipid syndrome (APS) are reactive with the phospholipid-binding protein β2GPI as well as a growing list of other protein targets. The relation of APLA to natural antibodies and the fuzzy set of autoantigens involved provoked us to study the changes in the IgM repertoire in APS. To this end, peptides selected by serum IgM from a 7-residue linear peptide phage display library (PDL) were deep sequenced. The analysis was aided by a novel formal representation of the Igome (the mimotope set reflecting the IgM specificities) in the form of a sequence graph. The study involved women with APLA and habitual abortions (n=24) compared to age-matched clinically healthy pregnant women (n=20). Their pooled Igomes (297 028 mimotope sequences) were compared also to the global public repertoire Igome of pooled donor plasma IgM (n=2 796 484) and a set of 7-mer sequences found in the J regions of human immunoglobulins (n=4 433 252). The pooled Igome was represented as a graph connecting the sequences as similar as the mimotopes of the same monoclonal antibody. The criterion was based on previously published data. In the resulting graph, identifiable clusters of vertices were considered related to the footprints of overlapping antibody cross-reactivities. A subgraph based on the clusters with a significant differential expression of APS patients’ mimotopes contained predominantly specificities underrepresented in APS. The differentially expressed IgM footprints showed also an increased cross-reactivity with immunoglobulin J regions. The specificities underexpressed in APS had a higher correlation with public specificities than those overexpressed. The APS associated specificities were strongly related also to the human peptidome with 1 072 mimotope sequences found in 7 519 human proteins. These regions were characterized by low complexity. Thus, the IgM repertoire of the APS patients was found to be characterized by a significant reduction of certain public specificities found in the healthy controls with targets representing low complexity linear self-epitopes homologous to human antibody J regions.

Sex Influences Age-Related Changes in Natural Antibodies and CD5+ B-1 Cells.

Natural Abs are primarily produced by B-1 cells and are essential for protection against Streptococcus pneumoniae The incidence and mortality rate for pneumococcal infection increases dramatically after age 65, disproportionately affecting males in both human and murine systems. To date, there is a significant gap in our understanding of the relationship among sex, aging, natural IgM efficacy, and the natural IgM repertoire. Our investigation demonstrates that the protective capacity of serum IgM against pneumococcal infection is maintained in IgM obtained from aged female mice but absent in IgM from aged male mice. To understand this difference in protective capacity, we examined serum Ig, discovering that the protective change was not associated with shifts in levels of phosphorylcholine (PC)- or pneumococcal capsular polysaccharide serotype 3-specific IgM. Interestingly, we observed that aged females have an increase in the total number of CD5+ B-1 cells, higher serum IL-5 levels, and a larger percentage of aged female CD5+ B-1 cells that express CD86 as compared with aged males. Furthermore, single-cell IgM repertoire analysis from peritoneal PC+, splenic PC+, and bone marrow CD5+ B-1 cell subsets demonstrated greater diversity with age and a higher level of germline status in female mice than previously observed in studies of aged male mice. Aged female CD5+ B-1 cells also expressed higher levels of transcripts associated with cell activity and self-renewal, such as Nanog and Hmga2 Taken together, these data indicate that females maintain a more diverse and active CD5+ B-1 cell pool and natural IgM repertoire, which has implications for sex-related susceptibility to infection and disease.

Characterization of human IgM and IgG repertoires in individuals with chronic HIV-1 infection.

Advancements in high-throughput sequencing (HTS) of antibody repertoires (Ig-Seq) have unprecedentedly improved our ability to characterize the antibody repertoires on a large scale. However, currently, only a few studies explored the influence of chronic HIV-1 infection on human antibody repertoires and many of them reached contradictory conclusions, possibly limited by inadequate sequencing depth and throughput. To better understand how HIV-1 infection would impact humoral immune system, in this study, we systematically analyzed the differences between the IgM (HIV-IgM) and IgG (HIV-IgG) heavy chain repertoires of HIV-1 infected patients, as well as between antibody repertoires of HIV-1 patients and healthy donors (HH). Notably, the public unique clones accounted for only a negligible proportion between the HIV-IgM and HIV-IgG repertoires libraries, and the diversity of unique clones in HIV-IgG remarkably reduced. In aspect of somatic mutation rates of CDR1 and CDR2, the HIV-IgG repertoire was higher than HIV-IgM. Besides, the average length of CDR3 region in HIV-IgM was significant longer than that in the HH repertoire, presumably caused by the great number of novel VDJ rearrangement patterns, especially a massive use of IGHJ6. Moreover, some of the B cell clonotypes had numerous clones, and somatic variants were detected within the clonotype lineage in HIV-IgG, indicating HIV-1 neutralizing activities. The in-depth characterization of HIV-IgG and HIV-IgM repertoires enriches our knowledge in the profound effect of HIV-1 infection on human antibody repertoires and may have practical value for the discovery of therapeutic antibodies.

VDJ Gene Usage in IgM Repertoires of Rhesus and Cynomolgus Macaques.

Macaques are frequently used to evaluate candidate vaccines and to study infection-induced antibody responses, requiring an improved understanding of their naïve immunoglobulin (IG) repertoires. Baseline gene usage frequencies contextualize studies of antigen-specific immune responses, providing information about how easily one may stimulate a response with a particular VDJ recombination. Studies of human IgM repertoires have shown that IG VDJ gene frequencies vary several orders of magnitude between the most and least utilized genes in a manner that is consistent across many individuals but to date similar analyses are lacking for macaque IgM repertoires. Here, we quantified VDJ gene usage levels in unmutated IgM repertoires of 45 macaques, belonging to two species and four commonly used subgroups: Indian and Chinese origin rhesus macaques and Indonesian and Mauritian origin cynomolgus macaques. We show that VDJ gene frequencies differed greatly between the most and least used genes, with similar overall patterns observed in macaque subgroups and individuals. However, there were also clear differences affecting the use of specific V, D and J genes. Furthermore, in contrast to humans, macaques of both species utilized IGHV4 family genes to a much higher extent and showed evidence of evolutionary expansion of genes of this family. Finally, we used the results to inform the analysis of a broadly neutralizing HIV-1 antibody elicited in SHIV-infected rhesus macaques, RHA1.V2.01, which binds the apex of the Env trimer in a manner that mimics the binding mode of PGT145. We discuss the likelihood that similar antibodies could be elicited in different macaque subgroups.