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.
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.
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.
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.
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.
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.
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•scRNA-seq of PBMCs from MIS-C patients reveals immunopathology signatures•MIS-C patients display elevated alarmins and NK/CD8+ T cell cytotoxicity effectors•TRBV11-2-expressing CD4+ and CD8+ memory T cells are expanded in severe MIS-C•Increased plasmablasts and endothelium-reactive IgG are features of severe MIS-C
Multisystem inflammatory syndrome in children (MIS-C) is a life-threatening and unpredictable condition of unknown etiology. Ramaswamy et al. use peripheral blood single-cell transcriptomic profiling along with other techniques to define key innate and adaptive signatures that characterize MIS-C.
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.
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.
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.
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
/HLA-DR
classical monocytes and activated LAG-3
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.
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