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Germinal centers (GC) are microanatomical lymphoid structures where affinity-matured memory B cells and long-lived bone marrow plasma cells are primarily generated. It is unclear how the maturation of B cells within the GC impacts the breadth and durability of B cell responses to influenza vaccination in humans. We used fine needle aspiration of draining lymph nodes to longitudinally track antigen-specific GC B cell responses to seasonal influenza vaccination. Antigen-specific GC B cells persisted for at least 13 wk after vaccination in two out of seven individuals. Monoclonal antibodies (mAbs) derived from persisting GC B cell clones exhibit enhanced binding affinity and breadth to influenza hemagglutinin (HA) antigens compared with related GC clonotypes isolated earlier in the response. Structural studies of early and late GC-derived mAbs from one clonal lineage in complex with H1 and H5 HAs revealed an altered binding footprint. Our study shows that inducing sustained GC reactions after influenza vaccination in humans supports the maturation of responding B cells., (© 2024 McIntire et al.)

Abs are vital to human immune responses and are composed of genetically variable H and L chains. These structures are initially expressed as BCRs. BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated H and L chains, but advancements in single-cell sequencing now pair H and L chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired H and L chain sequences to build phylogenetic trees. We found that incorporating L chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree-building methods and persisted even when mixing bulk and single-cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some L chains were missing, such as when mixing single-cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for H and L chain gene partitions. Thus, we recommend using maximum likelihood methods with separate H and L chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Food allergy is caused by allergen-specific immunoglobulin E (IgE) antibodies, but little is known about the B cell memory of persistent IgE responses. Here, we describe, in human pediatric peanut allergy, a population of CD23 + IgG1 + memory B cells arising in type 2 immune responses that contain high-affinity peanut-specific clones and generate IgE-producing cells upon activation. The frequency of CD23 + IgG1 + memory B cells correlated with circulating concentrations of IgE in children with peanut allergy. A corresponding population of "type 2-marked" IgG1 + memory B cells was identified in single-cell RNA sequencing experiments. These cells differentially expressed interleukin-4 (IL-4)- and IL-13-regulated genes, such as FCER2 / CD23 + , IL4R , and germline IGHE , and carried highly mutated B cell receptors (BCRs). In children with high concentrations of serum peanut-specific IgE, high-affinity B cells that bind the main peanut allergen Ara h 2 mapped to the population of "type 2-marked" IgG1 + memory B cells and included clones with convergent BCRs across different individuals. Our findings indicate that CD23 + IgG1 + memory B cells transcribing germline IGHE are a unique memory population containing precursors of high-affinity pathogenic IgE-producing cells that are likely to be involved in the long-term persistence of peanut allergy.

The widespread availability of single-cell RNA sequencing (scRNA-seq) has led to the development of new methods for understanding immune responses. Single-cell transcriptome data can now be paired with B cell receptor (BCR) sequences. However, RNA from BCRs cannot be analyzed like most other genes because BCRs are genetically diverse within individuals. In humans, BCRs are shaped through recombination followed by mutation and selection for antigen binding. As these processes co-occur with cell division, B cells can be studied using phylogenetic trees representing the mutations within a clone. B cell trees can link experimental timepoints, tissues, or cellular subtypes. Here, we review the current state and potential of how B cell phylogenetics can be combined with single-cell data to understand immune responses., Competing Interests: Declaration of interests S.H.K. receives consulting fees from Peraton. K.B.H. receives consulting fees from Prellis Biologics., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Protective immunity following vaccination is sustained by long-lived antibody-secreting cells and resting memory B cells (MBCs). Responses to two-dose SARS-CoV-2 mRNA-1273 vaccination are evaluated longitudinally by multimodal single-cell analysis in three infection-naïve individuals. Integrated surface protein, transcriptomics, and B cell receptor (BCR) repertoire analysis of sorted plasmablasts and spike + (S-2P + ) and S-2P - B cells reveal clonal expansion and accumulating mutations among S-2P + cells. These cells are enriched in a cluster of immunoglobulin G-expressing MBCs and evolve along a bifurcated trajectory rooted in CXCR3 + MBCs. One branch leads to CD11c + atypical MBCs while the other develops from CD71 + activated precursors to resting MBCs, the dominant population at month 6. Among 12 evolving S-2P + clones, several are populated with plasmablasts at early timepoints as well as CD71 + activated and resting MBCs at later timepoints, and display intra- and/or inter-cohort BCR convergence. These relationships suggest a coordinated and predictable evolution of SARS-CoV-2 vaccine-generated MBCs., Competing Interests: Declaration of interests S.H.K. receives consulting fees from Peraton. K.B.H. receives consulting fees from Prellis Biologics., (Published by Elsevier Inc.)

Age-associated B cells (ABCs) are formed under inflammatory conditions and are considered a type of memory B cell (MBC) expressing the transcription factor T-bet. In SLE, ABC frequency is correlated with disease, and they are thought to be the source of autoantibody-secreting cells. However, in inflammatory conditions, whether autoreactive B cells can become resting MBCs is uncertain. Further, the phenotypic identity of ABCs and their relationship to other B cell subsets, such as plasmablasts, is unclear. Whether ABCs directly promote disease is untested. Here we report, in the MRL/lpr SLE model, unexpected heterogeneity among ABC-like cells for expression of the integrins CD11b and CD11c, T-bet, and memory or plasmablast markers. Transfer and labeling studies demonstrated that ABCs are dynamic, rapidly turning over. scRNA-seq identified B cell clones present in multiple subsets, revealing that ABCs can be plasmablast precursors or undergo cycles of reactivation. Deletion of CD11c-expressing B cells revealed a direct role for ABC-like B cells in lupus pathogenesis., (© 2023 Nickerson et al.)

T cell-B cell interaction is the key immune response to protect the host from severe viral infection. However, how T cells support B cells to exert protective humoral immunity in humans is not well understood. Here, we use COVID-19 as a model of acute viral infections and analyze CD4 + T cell subsets associated with plasmablast expansion and clinical outcome. Peripheral helper T cells (Tph cells; denoted as PD-1 high CXCR5 - CD4 + T cells) are significantly increased, as are plasmablasts. Tph cells exhibit "B cell help" signatures and induce plasmablast differentiation in vitro. Interestingly, expanded plasmablasts show increased CXCR3 expression, which is positively correlated with higher frequency of activated Tph cells and better clinical outcome. Mechanistically, Tph cells help B cell differentiation and produce more interferon γ (IFNγ), which induces CXCR3 expression on plasmablasts. These results elucidate a role for Tph cells in regulating protective B cell response during acute viral infection., Competing Interests: Declaration of interests D.A.H. has received research funding from Bristol-Myers Squibb, Novartis, Sanofi, and Genentech. He has been a consultant for Bayer Pharmaceuticals, Bristol Myers Squibb, Compass Therapeutics, EMD Serono, Genentech, Juno Therapeutics, Novartis Pharmaceuticals, Proclara Biosciences, Sage Therapeutics, and Sanofi Genzyme. Further information regarding funding is available at https://openpaymentsdata.cms.gov/physician/166753/general-payments. N.K. reports personal fees from Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Indalo, Theravance, LifeMax, Three Lake Partners, and RohBar in the last 36 months and Equity in Pliant. N.K. is also a recipient of a grant from Veracyte and non-financial support from Miragen. In addition, N.K. has patents on New Therapies in Pulmonary Fibrosis and ARDS (unlicensed) and Peripheral Blood Gene Expression as biomarkers in IPF (licensed to biotech), all outside the submitted work. S.H.K. receives consulting fees from Northrop Grumman. K.B.H. receives consulting fees from Prellis Biologics., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Multicellular life requires altruistic cooperation between cells. The adaptive immune system is a notable exception, wherein germinal center B cells compete vigorously for limiting positive selection signals. Studying primary human lymphomas and developing new mouse models, we found that mutations affecting BTG1 disrupt a critical immune gatekeeper mechanism that strictly limits B cell fitness during antibody affinity maturation. This mechanism converted germinal center B cells into supercompetitors that rapidly outstrip their normal counterparts. This effect was conferred by a small shift in MYC protein induction kinetics but resulted in aggressive invasive lymphomas, which in humans are linked to dire clinical outcomes. Our findings reveal a delicate evolutionary trade-off between natural selection of B cells to provide immunity and potentially dangerous features that recall the more competitive nature of unicellular organisms.

Most studies of adaptive immunity to SARS-CoV-2 infection focus on peripheral blood, which may not fully reflect immune responses at the site of infection. Using samples from 110 children undergoing tonsillectomy and adenoidectomy during the COVID-19 pandemic, we identified 24 samples with evidence of previous SARS-CoV-2 infection, including neutralizing antibodies in serum and SARS-CoV-2-specific germinal center and memory B cells in the tonsils and adenoids. Single-cell B cell receptor (BCR) sequencing indicated virus-specific BCRs were class-switched and somatically hypermutated, with overlapping clones in the two tissues. Expanded T cell clonotypes were found in tonsils, adenoids and blood post-COVID-19, some with CDR3 sequences identical to previously reported SARS-CoV-2-reactive T cell receptors (TCRs). Pharyngeal tissues from COVID-19-convalescent children showed persistent expansion of germinal center and antiviral lymphocyte populations associated with interferon (IFN)-γ-type responses, particularly in the adenoids, and viral RNA in both tissues. Our results provide evidence for persistent tissue-specific immunity to SARS-CoV-2 in the upper respiratory tract of children after infection., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Myasthenia gravis (MG) is an autoantibody-mediated autoimmune disorder of the neuromuscular junction. A small subset of patients (<10%) with MG, have autoantibodies targeting muscle-specific tyrosine kinase (MuSK). MuSK MG patients respond well to CD20-mediated B cell depletion therapy (BCDT); most achieve complete stable remission. However, relapse often occurs. To further understand the immunomechanisms underlying relapse, we studied autoantibody-producing B cells over the course of BCDT. We developed a fluorescently labeled antigen to enrich for MuSK-specific B cells, which was validated with a novel Nalm6 cell line engineered to express a human MuSK-specific B cell receptor. B cells (≅ 2.6 million) from 12 different samples collected from nine MuSK MG patients were screened for MuSK specificity. We successfully isolated two MuSK-specific IgG4 subclass-expressing plasmablasts from two of these patients, who were experiencing a relapse after a BCDT-induced remission. Human recombinant MuSK mAbs were then generated to validate binding specificity and characterize their molecular properties. Both mAbs were strong MuSK binders, they recognized the Ig1-like domain of MuSK, and showed pathogenic capacity when tested in an acetylcholine receptor (AChR) clustering assay. The presence of persistent clonal relatives of these MuSK-specific B cell clones was investigated through B cell receptor repertoire tracing of 63,977 unique clones derived from longitudinal samples collected from these two patients. Clonal variants were detected at multiple timepoints spanning more than five years and reemerged after BCDT-mediated remission, predating disease relapse by several months. These findings demonstrate that a reservoir of rare pathogenic MuSK autoantibody-expressing B cell clones survive BCDT and reemerge into circulation prior to manifestation of clinical relapse. Overall, this study provides both a mechanistic understanding of MuSK MG relapse and a valuable candidate biomarker for relapse prediction., (© 2022. The Author(s).)

B cells undergo rapid mutation and selection for antibody binding affinity when producing antibodies capable of neutralizing pathogens. This evolutionary process can be intermixed with migration between tissues, differentiation between cellular subsets, and switching between functional isotypes. B cell receptor (BCR) sequence data has the potential to elucidate important information about these processes. However, there is currently no robust, generalizable framework for making such inferences from BCR sequence data. To address this, we develop three parsimony-based summary statistics to characterize migration, differentiation, and isotype switching along B cell phylogenetic trees. We use simulations to demonstrate the effectiveness of this approach. We then use this framework to infer patterns of cellular differentiation and isotype switching from high throughput BCR sequence datasets obtained from patients in a study of HIV infection and a study of food allergy. These methods are implemented in the R package dowser, available at https://dowser.readthedocs.io., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: S.H.K. receives consulting fees from Northrop Grumman and Peraton. K.B.H receives consulting fees from Prellis Biologics.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100A hi /HLA-DR lo classical monocytes and activated LAG-3 hi T cells are hallmarks of progressive disease and highlights the abnormal MHC-II/LAG-3 interaction on myeloid and T cells, respectively. We also find skewed T cell receptor repertories in expanded effector CD8 + clones, unmutated IGHG + B cell clones, and mutated B cell clones with stable somatic hypermutation frequency over time. In conclusion, our in-depth immune profiling reveals dyssynchrony of the innate and adaptive immune interaction in progressive COVID-19., (© 2022. The Author(s).)

Adaptive immune receptor repertoires (AIRRs) are rich with information that can be mined for insights into the workings of the immune system. Gene usage, CDR3 properties, clonal lineage structure, and sequence diversity are all capable of revealing the dynamic immune response to perturbation by disease, vaccination, or other interventions. Here we focus on a conceptual introduction to the many aspects of repertoire analysis and orient the reader toward the uses and advantages of each. Along the way, we note some of the many software tools that have been developed for these investigations and link the ideas discussed to chapters on methods provided elsewhere in this volume., (© 2022. The Author(s).)

The poor efficacy of seasonal influenza virus vaccines is often attributed to pre-existing immunity interfering with the persistence and maturation of vaccine-induced B cell responses. We previously showed that a subset of vaccine-induced B cell lineages are recruited into germinal centers (GCs) following vaccination, suggesting that affinity maturation of these lineages against vaccine antigens can occur. However, it remains to be determined whether seasonal influenza vaccination stimulates additional evolution of vaccine-specific lineages, and previous work has found no significant increase in somatic hypermutation among influenza-binding lineages sampled from the blood following seasonal vaccination in humans. Here, we investigate this issue using a phylogenetic test of measurable immunoglobulin sequence evolution. We first validate this test through simulations and survey measurable evolution across multiple conditions. We find significant heterogeneity in measurable B cell evolution across conditions, with enrichment in primary response conditions such as HIV infection and early childhood development. We then show that measurable evolution following influenza vaccination is highly compartmentalized: while lineages in the blood are rarely measurably evolving following influenza vaccination, lineages containing GC B cells are frequently measurably evolving. Many of these lineages appear to derive from memory B cells. We conclude from these findings that seasonal influenza virus vaccination can stimulate additional evolution of responding B cell lineages, and imply that the poor efficacy of seasonal influenza vaccination is not due to a complete inhibition of vaccine-specific B cell evolution., Competing Interests: KH receives consulting fees from Prellis Biologics, JT is the recipient of a licensing agreement with Abbvie and has received consulting fees from Gerson Lehman Group, FM, RJ, OP No competing interests declared, AE The Ellebedy laboratory received funding under sponsored research agreements from Emergent BioSolutions and AbbVie, SK receives consulting fees from Northrop Grumman and Peraton, (© 2021, Hoehn et al.)

Elevated N-linked glycosylation of IgG V regions (IgG-V N-Glyc ) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-V N-Glyc , we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-V N-Glyc motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-V N-Glyc could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-V N-Glyc , was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes., (Copyright © 2021 by The American Association of Immunologists, Inc.)

In order to better understand how the immune system interacts with environmental triggers to produce organ-specific disease, we here address the hypothesis that B and plasma cells are free to migrate through the mucosal surfaces of the upper and lower respiratory tracts, and that their total antibody repertoire is modified in a common respiratory tract disease, in this case atopic asthma. Using Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) we have catalogued the antibody repertoires of B cell clones retrieved near contemporaneously from multiple sites in the upper and lower respiratory tract mucosa of adult volunteers with atopic asthma and non-atopic controls and traced their migration. We show that the lower and upper respiratory tracts are immunologically connected, with trafficking of B cells directionally biased from the upper to the lower respiratory tract and points of selection when migrating from the nasal mucosa and into the bronchial mucosa. The repertoires are characterized by both IgD-only B cells and others undergoing class switch recombination, with restriction of the antibody repertoire distinct in asthmatics compared with controls. We conclude that B cells and plasma cells migrate freely throughout the respiratory tract and exhibit distinct antibody repertoires in health and disease., Competing Interests: SHK receives consulting fees from Northrop Grumman. KBH receives consulting fees from Prellis Biologics. YJ, CC, TGJ and RH are employed by Bristol Myers Squibb. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor has declared past collaborations with one of the authors SHK at the time of this review., (Copyright © 2021 Ohm-Laursen, Meng, Hoehn, Nouri, Jiang, Clouser, Johnstone, Hause, Sandhar, Upton, Chevretton, Lakhani, Corrigan, Kleinstein and Gould.)

The skin lesion erythema migrans (EM) is an initial sign of the Ixodes tick-transmitted Borreliella spirochetal infection known as Lyme disease. T cells and innate immune cells have previously been shown to predominate the EM lesion and promote the reaction. Despite the established importance of B cells and antibodies in preventing infection, the role of B cells in the skin immune response to Borreliella is unknown. Here, we used single-cell RNA-Seq in conjunction with B cell receptor (BCR) sequencing to immunophenotype EM lesions and their associated B cells and BCR repertoires. We found that B cells were more abundant in EM in comparison with autologous uninvolved skin; many were clonally expanded and had circulating relatives. EM-associated B cells upregulated the expression of MHC class II genes and exhibited preferential IgM isotype usage. A subset also exhibited low levels of somatic hypermutation despite a gene expression profile consistent with memory B cells. Our study demonstrates that single-cell gene expression with paired BCR sequencing can be used to interrogate the sparse B cell populations in human skin and reveals that B cells in the skin infection site in early Lyme disease expressed a phenotype consistent with local antigen presentation and antibody production.

Protective immunity against COVID-19 likely depends on the production of SARS-CoV-2-specific plasma cells and memory B cells postinfection or postvaccination. Previous work has found that germinal center reactions are disrupted in severe COVID-19. This may adversely affect long-term immunity against reinfection. Consistent with an extrafollicular B cell response, patients with severe COVID-19 have elevated frequencies of clonally expanded, class-switched, unmutated plasmablasts. However, it is unclear whether B cell populations in individuals with mild COVID-19 are similarly skewed. In this study, we use single-cell RNA sequencing of B cells to show that in contrast to patients with severe COVID-19, subjects with mildly symptomatic COVID-19 have B cell repertoires enriched for clonally diverse, somatically hypermutated memory B cells ∼30 d after the onset of symptoms. This provides evidence that B cell responses are less disrupted in mild COVID-19 and result in the production of memory B cells., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening post-infectious complication occurring unpredictably weeks after mild or asymptomatic SARS-CoV-2 infection. We profiled MIS-C, adult COVID-19, and healthy pediatric and adult individuals using single-cell RNA sequencing, flow cytometry, antigen receptor repertoire analysis, and unbiased serum proteomics, which collectively identified a signature in MIS-C patients that correlated with disease severity. Despite having no evidence of active infection, MIS-C patients had elevated S100A-family alarmins and decreased antigen presentation signatures, indicative of myeloid dysfunction. MIS-C patients showed elevated expression of cytotoxicity genes in NK and CD8 + T cells and expansion of specific IgG-expressing plasmablasts. Clinically severe MIS-C patients displayed skewed memory T cell TCR repertoires and autoimmunity characterized by endothelium-reactive IgG. The alarmin, cytotoxicity, TCR repertoire, and plasmablast signatures we defined have potential for application in the clinic to better diagnose and potentially predict disease severity early in the course of MIS-C., Competing Interests: Declaration of interests D.A.H. has received research funding from Bristol-Myers Squibb, Novartis, Sanofi, and Genentech. He has been a consultant for Bayer Pharmaceuticals, Bristol Myers Squibb, Compass Therapeutics, EMD Serono, Genentech, Juno Therapeutics, Novartis Pharmaceuticals, Proclara Biosciences, Sage Therapeutics, and Sanofi Genzyme. Further information regarding funding is available on: https://openpaymentsdata.cms.gov/physician/166753/general-payments. N.K. reports personal fees from Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Indalo, Theravance, LifeMax, Three Lake Partners, RohBar in the last 36 months, and Equity in Pliant. N.K. is also a recipient of a grant from Veracyte and non-financial support from Miragen. All outside the submitted work; In addition, N.K. has patents on New Therapies in Pulmonary Fibrosis and ARDS (unlicensed) and Peripheral Blood Gene Expression as biomarkers in IPF (licensed to biotech). S.H.K. receives consulting fees from Northrop Grumman. K.B.H. receives consulting fees from Prellis Biologics. B.S. is a former SomaLogic, Inc. (Boulder, CO, USA) employee and a company shareholder. All other authors declared that they have no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones., Competing Interests: Competing interest statement: K.C.O. has received research support from Ra Pharma and is a consultant and equity shareholder of Cabaletta Bio. K.C.O. is the recipient of a sponsored research subaward from the University of Pennsylvania, the primary financial sponsor of which is Cabaletta Bio. S.H.K. receives consulting fees from Northrop Grumman. R.J.N. has received research support from Alexion Pharmaceuticals, Genentech, Grifols, and Ra Pharma. H.J.K. has served as an advisor to Alnylam Pharmaceuticals, Ra Pharmaceuticals, and UCB Pharmaceuticals, and is Chief Exectutive Officer and Chief Marketing Officer of ARC Biotechnology, LLC, based on US Patent 8,961,98.

Rituximab, a B cell-depleting therapy, is indicated for treating a growing number of autoantibody-mediated autoimmune disorders. However, relapses can occur after treatment, and autoantibody-producing B cell subsets may be found during relapses. It is not understood whether these autoantibody-producing B cell subsets emerge from the failed depletion of preexisting B cells or are generated de novo. To further define the mechanisms that cause postrituximab relapse, we studied patients with autoantibody-mediated muscle-specific kinase (MuSK) myasthenia gravis (MG) who relapsed after treatment. We carried out single-cell transcriptional and B cell receptor profiling on longitudinal B cell samples. We identified clones present before therapy that persisted during relapse. Persistent B cell clones included both antibody-secreting cells and memory B cells characterized by gene expression signatures associated with B cell survival. A subset of persistent antibody-secreting cells and memory B cells were specific for the MuSK autoantigen. These results demonstrate that rituximab is not fully effective at eliminating autoantibody-producing B cells and provide a mechanistic understanding of postrituximab relapse in MuSK MG.

Follicular lymphomas (FLs) are slow-growing, indolent tumors containing extensive follicular dendritic cell (FDC) networks and recurrent EZH2 gain-of-function mutations. Paradoxically, FLs originate from highly proliferative germinal center (GC) B cells with proliferation strictly dependent on interactions with T follicular helper cells. Herein, we show that EZH2 mutations initiate FL by attenuating GC B cell requirement for T cell help and driving slow expansion of GC centrocytes that become enmeshed with and dependent on FDCs. By impairing T cell help, mutant EZH2 prevents induction of proliferative MYC programs. Thus, EZH2 mutation fosters malignant transformation by epigenetically reprograming B cells to form an aberrant immunological niche that reflects characteristic features of human FLs, explaining how indolent tumors arise from GC B cells., Competing Interests: Declaration of Interests A.M.M. is consulting for Epizyme and Constellation Pharmaceuticals, and receives research funding from Janssen Pharmaceuticals; S.H.K. is consulting for Northrop Grumman; C.E.M. is a co-founder and equity stake holder for Onegevity Health and Biotia, Inc.; C.S. has performed consultancy for Seattle Genetics, Curis Inc., Roche, AbbVie, Juno Therapeutics, and Bayer, and has received research funding from Bristol-Myers Squibb and Trillium Therapeutics, Inc. There are no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Nearly all chronic human infections are associated with alterations in the memory B cell (MBC) compartment, including a large expansion of CD19 hi T-bet hi MBC in the peripheral blood of HIV-infected individuals with chronic viremia. Despite their prevalence, it is unclear how these B cells arise and whether they contribute to the inefficiency of antibody-mediated immunity in chronic infectious diseases. We addressed these questions by characterizing T-bet-expressing B cells in lymph nodes (LN) and identifying a strong T-bet signature among HIV-specific MBC associated with poor immunologic outcome. Confocal microscopy and quantitative imaging revealed that T-bet hi B cells in LN of HIV-infected chronically viremic individuals distinctly accumulated outside germinal centers (GC), which are critical for optimal antibody responses. In single-cell analyses, LN T-bet hi B cells of HIV-infected individuals were almost exclusively found among CD19 hi MBC and expressed reduced GC-homing receptors. Furthermore, HIV-specific B cells of infected individuals were enriched among LN CD19 hi T-bet hi MBC and displayed a distinct transcriptome, with features similar to CD19 hi T-bet hi MBC in blood and LN GC B cells (GCBC). LN CD19 hi T-bet hi MBC were also related to GCBC by B cell receptor (BCR)-based phylogenetic linkage but had lower BCR mutation frequencies and reduced HIV-neutralizing capacity, consistent with diminished participation in GC-mediated affinity selection. Thus, in the setting of chronic immune activation associated with HIV viremia, failure of HIV-specific B cells to enter or remain in GC may help explain the rarity of high-affinity protective antibodies., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

In order to produce effective antibodies, B cells undergo rapid somatic hypermutation (SHM) and selection for binding affinity to antigen via a process called affinity maturation. The similarities between this process and evolution by natural selection have led many groups to use phylogenetic methods to characterize the development of immunological memory, vaccination, and other processes that depend on affinity maturation. However, these applications are limited by the fact that most phylogenetic models are designed to be applied to individual lineages comprising genetically diverse sequences, while B cell repertoires often consist of hundreds to thousands of separate low-diversity lineages. Further, several features of affinity maturation violate important assumptions in standard phylogenetic models. Here, we introduce a hierarchical phylogenetic framework that integrates information from all lineages in a repertoire to more precisely estimate model parameters while simultaneously incorporating the unique features of SHM. We demonstrate the power of this repertoire-wide approach by characterizing previously undescribed phenomena in affinity maturation. First, we find evidence consistent with age-related changes in SHM hot-spot targeting. Second, we identify a consistent relationship between increased tree length and signs of increased negative selection, apparent in the repertoires of recently vaccinated subjects and those without any known recent infections or vaccinations. This suggests that B cell lineages shift toward negative selection over time as a general feature of affinity maturation. Our study provides a framework for undertaking repertoire-wide phylogenetic testing of SHM hypotheses and provides a means of characterizing dynamics of mutation and selection during affinity maturation., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

Phylogenetic methods have shown promise in understanding the development of broadly neutralizing antibody lineages (bNAbs). However, the mutational process that generates these lineages, somatic hypermutation, is biased by hotspot motifs which violates important assumptions in most phylogenetic substitution models. Here, we develop a modified GY94-type substitution model that partially accounts for this context dependency while preserving independence of sites during calculation. This model shows a substantially better fit to three well-characterized bNAb lineages than the standard GY94 model. We also demonstrate how our model can be used to test hypotheses concerning the roles of different hotspot and coldspot motifs in the evolution of B-cell lineages. Further, we explore the consequences of the idea that the number of hotspot motifs, and perhaps the mutation rate in general, is expected to decay over time in individual bNAb lineages., (Copyright © 2017 Hoehn et al.)

B-cell receptors (BCRs) are membrane-bound immunoglobulins that recognize and bind foreign proteins (antigens). BCRs are formed through random somatic changes of germline DNA, creating a vast repertoire of unique sequences that enable individuals to recognize a diverse range of antigens. After encountering antigen for the first time, BCRs undergo a process of affinity maturation, whereby cycles of rapid somatic mutation and selection lead to improved antigen binding. This constitutes an accelerated evolutionary process that takes place over days or weeks. Next-generation sequencing of the gene regions that determine BCR binding has begun to reveal the diversity and dynamics of BCR repertoires in unprecedented detail. Although this new type of sequence data has the potential to revolutionize our understanding of infection dynamics, quantitative analysis is complicated by the unique biology and high diversity of BCR sequences. Models and concepts from molecular evolution and phylogenetics that have been applied successfully to rapidly evolving pathogen populations are increasingly being adopted to study BCR diversity and divergence within individuals. However, BCR dynamics may violate key assumptions of many standard evolutionary methods, as they do not descend from a single ancestor, and experience biased mutation. Here, we review the application of evolutionary models to BCR repertoires and discuss the issues we believe need be addressed for this interdisciplinary field to flourish., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Advances in immunoglobulin (Ig) sequencing technology are leading to new perspectives on immune system dynamics. Much research in this nascent field has focused on resolving immune responses to viral infection. However, the dynamics of B-cell diversity in early HIV infection, and in response to anti-retroviral therapy, are still poorly understood. Here, we investigate these dynamics through bulk Ig sequencing of samples collected over 2 years from a group of eight HIV-1 infected patients, five of whom received anti-retroviral therapy during the first half of the study period. We applied previously published methods for visualizing and quantifying B-cell sequence diversity, including the Gini index, and compared their efficacy to alternative measures. While we found significantly greater clonal structure in HIV-infected patients versus healthy controls, within HIV patients, we observed no significant relationships between statistics of B-cell clonal expansion and clinical variables such as viral load and CD4(+) count. Although there are many potential explanations for this, we suggest that important factors include poor sampling resolution and complex B-cell dynamics that are difficult to summarize using simple summary statistics. Importantly, we find a significant association between observed Gini indices and sequencing read depth, and we conclude that more robust analytical methods and a closer integration of experimental and theoretical work is needed to further our understanding of B-cell repertoire diversity during viral infection.

The scuttle fly, Megaselia scalaris, is often cited as a model in which to study early sex chromosome evolution because of its homomorphic sex chromosomes, low but measurable molecular differentiation between sex chromosomes, and occasional transposition of the male-determining element to different chromosomes in laboratory cultures. Counterintuitively, natural isolates consistently show sex linkage to the second chromosome. Frequent natural transposition of the male-determining element should lead to the loss of male specificity of any nontransposed material on the previous sex-linked chromosome pair. Using next-generation sequencing data from a newly obtained natural isolate of M. scalaris, we show that even highly conservative estimates for the size of the male-specific genome are likely too large to be contained within a transposable element. This result strongly suggests that transposition of the male-determining region either is extremely rare or has not persisted recently in natural populations, allowing for differentiation of the sex chromosomes of this species., (Copyright © 2015 Hoehn and Noor.)

Premature termination codon (PTC) mutations can have dramatic effects--both adaptive and deleterious--on gene expression and function. Here, we examine the number and selective effects of PTC mutations within the Drosophila pseudoobscura subclade using 18 resequenced genomes aligned to the reference genome. We located and characterized 1,679 PTC mutations in 605 genes across each of these genomes relative to the D. pseudoobscura reference genome, and use RT-PCR to confirm transcription of a subset of these genes containing PTC mutations. We confirm previous findings that genes containing PTC mutations are less selectively constrained and less broadly expressed than non-PTC-containing genes, suggesting that the most of these mutations are at least mildly deleterious. Further, we find highly significant codon usage bias in regions downstream of the PTC in 38 of these PTC-containing genes, suggesting that some of these PTC mutations--if not alternatively spliced out of the transcript--have neutral effects. Ultimately, these analyzes support the view that the PTC mutations are mostly detrimental, but are nonetheless common enough in genomes that a subset could be effectively neutral.

Chromosomal inversions disrupt recombination in heterozygotes by both reducing crossing-over within inverted regions and increasing it elsewhere in the genome. The reduction of recombination in inverted regions facilitates the maintenance of hybridizing species, as outlined by various models of chromosomal speciation. We present a comprehensive comparison of the effects of inversions on recombination rates and on nucleotide divergence. Within an inversion differentiating Drosophila pseudoobscura and Drosophila persimilis, we detected one double recombinant among 9,739 progeny from F(1) hybrids screened, consistent with published double-crossover frequencies observed within species. Despite similar rates of exchange within and between species, we found no sequence-based evidence of ongoing gene exchange between species within this inversion, but significant exchange was inferred within species. We also observed greater differentiation at regions near inversion breakpoints between species versus within species. Moreover, we observed strong "interchromosomal effect" (higher recombination in inversion heterozygotes between species) with up to 9-fold higher recombination rates along collinear segments of chromosome two in hybrids. Further, we observed that regions most susceptible to changes in recombination rates corresponded to regions with lower recombination rates in homokaryotypes. Finally, we showed that interspecies nucleotide divergence is lower in regions with greater increases in recombination rate, potentially resulting from greater interspecies exchange. Overall, we have identified several similarities and differences between inversions segregating within versus between species in their effects on recombination and divergence. We conclude that these differences are most likely due to lower frequency of heterokaryotypes and to fitness consequences from the accumulation of various incompatibilities between species. Additionally, we have identified possible effects of inversions on interspecies gene exchange that had not been considered previously.