High-throughput single-cell profiling of B cell responses following inactivated influenza vaccination in young and older adults.

Seasonal influenza contributes to a substantial disease burden, resulting in approximately 10 million hospital visits and 50 thousand deaths in a typical year in the United States. 70 - 85% of the mortality occurs in people over the age of 65. Influenza vaccination is the best protection against the virus, but it is less effective for the elderly, which may be in part due to differences in the quantity or type of B cells induced by vaccination. To investigate this possibility, we sorted pre- and post-vaccination peripheral blood B cells from three young and three older adults with strong antibody responses to the inactivated influenza vaccine and employed single-cell technology to simultaneously profile the gene expression and the B cell receptor (BCR) of the B cells. Prior to vaccination, we observed a higher somatic hypermutation frequency and a higher abundance of activated B cells in older adults than in young adults. Following vaccination, young adults mounted a more clonal response than older adults. The expanded clones included a mix of plasmablasts, activated B cells, and resting memory B cells in both age groups, with a decreased proportion of plasmablasts in older adults. Differential abundance analysis identified additional vaccine-responsive cells that were not part of expanded clones, especially in older adults. We observed broadly consistent gene expression changes in vaccine-responsive plasmablasts and greater heterogeneity among activated B cells between age groups. These quantitative and qualitative differences in the B cells provide insights into age-related changes in influenza vaccination response.

Intranasal priming induces local lung-resident B cell populations that secrete protective mucosal antiviral IgA.

Antibodies secreted at the mucosal surface play an integral role in immune defense by serving to neutralize the pathogen and promote its elimination at the site of entry. Secretory immunoglobulin A (IgA) is a predominant Ig isotype at mucosal surfaces whose epithelial cells express polymeric Ig receptor capable of transporting dimeric IgA to the lumen. Although the role of IgA in intestinal mucosa has been extensively studied, the cell types responsible for secreting the IgA that protects the host against pathogens in the lower respiratory tract are less clear. Here, using a mouse model of influenza virus infection, we demonstrate that intranasal, but not systemic, immunization induces local IgA secretion in the bronchoalveolar space. Using single-cell RNA sequencing, we found a heterogeneous population of IgA-expressing cells within the respiratory mucosa, including tissue-resident memory B cells, plasmablasts, and plasma cells. IgA-secreting cell establishment within the lung required CXCR3. An intranasally administered protein-based vaccine also led to the establishment of IgA-secreting cells in the lung, but not when given intramuscularly or intraperitoneally. Last, local IgA secretion correlated with superior protection against secondary challenge with homologous and heterologous virus infection than circulating antibodies alone. These results provide key insights into establishment of protective immunity in the lung based on tissue-resident IgA-secreting B cells and inform vaccine strategies designed to elicit highly effective immune protection against respiratory virus infections.

Human B cell lineages associated with germinal centers following influenza vaccination are measurably evolving.

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.

When the immune system encounters a disease-causing pathogen, it releases antibodies that can bind to specific regions of the bacterium or virus and help to clear the infection. These proteins are generated by B cells which, upon detecting the pathogen, can begin to mutate and alter the structure of the antibody they produce: the better the antibody is at binding to the pathogen, the more likely the B cell is to survive. This process of evolution produces B cells that make more effective antibodies. After the infection, some of these cells become 'memory B cells' which can be stimulated in to action when the pathogen invades again. Many vaccines also depend on this process to trigger the production of memory B cells that can fight off a specific disease-causing agent. However, it is unclear to what extent memory B cells that already exist are able to continue to evolve and modify their antibodies. This is particularly important for the flu vaccine, as the virus that causes influenza rapidly mutates. To provide high levels of protection, the memory B cells formed following the vaccine may therefore need to evolve to make different antibodies that recognize mutated forms of the virus. It is thought that the low effectiveness of the flu vaccine is partially because the response it triggers does not stimulate additional evolution of memory B cells. To test this theory, Hoehn et al. developed a computational method that can detect the evolution of B cells over time. The tool was applied to samples collected from the blood and lymph nodes (organ where immune cells reside) of people who recently received the flu vaccine. The results were then compared to B cells taken from people after different infections, vaccinations, and other conditions. Hoehn et al. found the degree to which B cells evolve varies significantly between conditions. For example, B cells produced during chronic HIV infections frequently evolved over time, while such evolution was rarely observed during the autoimmune disease myasthenia gravis. The analysis also showed that memory B cells produced by the flu vaccine were able to evolve if recruited to the lymph nodes, but this was rarely detected in B cells in the blood. These findings suggest the low efficacy of the flu vaccine is not due to a complete lack of B cell evolution, but likely due to other factors. For instance, it is possible the evolutionary process it stimulates is not as robust as in other conditions, or is less likely to produce long-lived B cells that release antibodies. More research is needed to explore these ideas and could lead to the development of more effective flu vaccines.

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

Elevated N-linked glycosylation of IgG V regions (IgG-VN-Glyc) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-VN-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-VN-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-VN-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-VN-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.

Risk factor identification and predictive models for central line requirements for patients on vasopressors.

Vasopressors are ubiquitous in intensive care units. While central venous catheters are the preferred route of infusion, recent evidence suggests peripheral administration may be safe for short, single-agent courses. Here, we identify risk factors and develop a predictive model for patient central venous catheter requirement using the Medical Information Mart for Intensive Care, a single-centre dataset of patients admitted to an intensive care unit between 2008 and 2019. Using prior literature, a composite endpoint of prolonged single-agent courses (>24 hours) or multi-agent courses of any duration was used to identify likely central venous catheter requirement. From a cohort of 69,619 intensive care unit stays, there were 17,053 vasopressor courses involving one or more vasopressors that met study inclusion criteria. In total, 3807 (22.3%) vasopressor courses involved a single vasopressor for less than six hours, 7952 (46.6%) courses for less than 24 hours and 5757 (33.8%) involved multiple vasopressors of any duration. Of these, 3047 (80.0%) less than six-hour and 6423 (80.8%) less than 24-hour single vasopressor courses used a central venous catheter. Logistic regression models identified associations between the composite endpoint and intubation (odds ratio (OR) 2.36, 95% confidence intervals (CI) 2.16 to 2.58), cardiac diagnosis (OR 0.72, CI 0.65 to 0.80), renal impairment (OR 1.61, CI 1.50 to 1.74), older age (OR 1.002, Cl 1.000 to 1.005) and vital signs in the hour before initiation (heart rate, OR 1.006, CI 1.003 to 1.009; oxygen saturation, OR 0.996, CI 0.993 to 0.999). A logistic regression model predicting the composite endpoint had an area under the receiver operating characteristic curve (standard deviation) of 0.747 (0.013) and an accuracy of 0.691 (0.012). This retrospective study reveals a high prevalence of short vasopressor courses in intensive care unit settings, a majority of which were administered using central venous catheters. We identify several important risk factors that may help guide clinicians deciding between peripheral and central venous catheter administration, and present a predictive model that may inform future prospective trials.

Single-cell immunophenotyping of the skin lesion erythema migrans identifies IgM memory B cells.

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.

Divergent and self-reactive immune responses in the CNS of COVID-19 patients with neurological symptoms.

Individuals with coronavirus disease 2019 (COVID-19) frequently develop neurological symptoms, but the biological underpinnings of these phenomena are unknown. Through single-cell RNA sequencing (scRNA-seq) and cytokine analyses of cerebrospinal fluid (CSF) and blood from individuals with COVID-19 with neurological symptoms, we find compartmentalized, CNS-specific T cell activation and B cell responses. All affected individuals had CSF anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies whose target epitopes diverged from serum antibodies. In an animal model, we find that intrathecal SARS-CoV-2 antibodies are present only during brain infection and not elicited by pulmonary infection. We produced CSF-derived monoclonal antibodies from an individual with COVID-19 and found that these monoclonal antibodies (mAbs) target antiviral and antineural antigens, including one mAb that reacted to spike protein and neural tissue. CSF immunoglobulin G (IgG) from 5 of 7 patients showed antineural reactivity. This immune survey reveals evidence of a compartmentalized immune response in the CNS of individuals with COVID-19 and suggests a role of autoimmunity in neurologic sequelae of COVID-19.

Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis.

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.

Exploratory neuroimmune profiling identifies CNS-specific alterations in COVID-19 patients with neurological involvement.

One third of COVID-19 patients develop significant neurological symptoms, yet SARS-CoV-2 is rarely detected in central nervous system (CNS) tissue, suggesting a potential role for parainfectious processes, including neuroimmune responses. We therefore examined immune parameters in cerebrospinal fluid (CSF) and blood samples from a cohort of patients with COVID-19 and significant neurological complications. We found divergent immunological responses in the CNS compartment, including increased levels of IL-12 and IL-12-associated innate and adaptive immune cell activation. Moreover, we found increased proportions of B cells in the CSF relative to the periphery and evidence of clonal expansion of CSF B cells, suggesting a divergent intrathecal humoral response to SARS-CoV-2. Indeed, all COVID-19 cases examined had anti-SARS-CoV-2 IgG antibodies in the CSF whose target epitopes diverged from serum antibodies. We directly examined whether CSF resident antibodies target self-antigens and found a significant burden of CNS autoimmunity, with the CSF from most patients recognizing neural self-antigens. Finally, we produced a panel of monoclonal antibodies from patients' CSF and show that these target both anti-viral and anti-neural antigens-including one mAb specific for the spike protein that also recognizes neural tissue. This exploratory immune survey reveals evidence of a compartmentalized and self-reactive immune response in the CNS meriting a more systematic evaluation of neurologically impaired COVID-19 patients.

Affinity maturation is required for pathogenic monovalent IgG4 autoantibody development in myasthenia gravis.

Pathogenic muscle-specific tyrosine kinase (MuSK)-specific IgG4 autoantibodies in autoimmune myasthenia gravis (MG) are functionally monovalent as a result of Fab-arm exchange. The development of these unique autoantibodies is not well understood. We examined MG patient-derived monoclonal autoantibodies (mAbs), their corresponding germline-encoded unmutated common ancestors (UCAs), and monovalent antigen-binding fragments (Fabs) to investigate how affinity maturation contributes to binding and immunopathology. Mature mAbs, UCA mAbs, and mature monovalent Fabs bound to MuSK and demonstrated pathogenic capacity. However, monovalent UCA Fabs bound to MuSK but did not have measurable pathogenic capacity. Affinity of the UCA Fabs for MuSK was 100-fold lower than the subnanomolar affinity of the mature Fabs. Crystal structures of two Fabs revealed how mutations acquired during affinity maturation may contribute to increased MuSK-binding affinity. These findings indicate that the autoantigen drives autoimmunity in MuSK MG through the accumulation of somatic mutations such that monovalent IgG4 Fab-arm-exchanged autoantibodies reach a high-affinity threshold required for pathogenic capacity.

High-throughput investigation of molecular and cellular biomarkers in NMOSD.

OBJECTIVE: To identify candidate biomarkers associated with neuromyelitis optica spectrum disorder (NMOSD) using high-throughput technologies that broadly assay the concentrations of serum analytes and frequencies of immune cell subsets. METHODS: Sera, peripheral blood mononuclear cells (PBMCs), and matched clinical data from participants with NMOSD and healthy controls (HCs) were obtained from the Collaborative International Research in Clinical and Longitudinal Experience Study NMOSD biorepository. Flow cytometry panels were used to measure the frequencies of 39 T-cell, B-cell, regulatory T-cell, monocyte, natural killer (NK) cell, and dendritic cell subsets in unstimulated PBMCs. In parallel, multiplex proteomics assays were used to measure 46 serum cytokines and chemokines in 2 independent NMOSD and HC cohorts. Multivariable regression models were used to assess molecular and cellular profiles in NMOSD compared with HC. RESULTS: NMOSD samples had a lower frequency of CD16+CD56+ NK cells. Both serum cohorts and multivariable logistic regression revealed increased levels of B-cell activating factor associated with NMOSD. Interleukin 6, CCL22, and CCL3 were also elevated in 1 NMOSD cohort of the 2 analyzed. Multivariable linear regression of serum analyte levels revealed a correlation between CX3CL1 (fractalkine) levels and the number of days since most recent disease relapse. CONCLUSIONS: Integrative analyses of cytokines, chemokines, and immune cells in participants with NMOSD and HCs provide congruence with previously identified biomarkers of NMOSD and highlight CD16+CD56+ NK cells and CX3CL1 as potential novel biomarker candidates.

Autoimmune Pathology in Myasthenia Gravis Disease Subtypes Is Governed by Divergent Mechanisms of Immunopathology.

Myasthenia gravis (MG) is a prototypical autoantibody mediated disease. The autoantibodies in MG target structures within the neuromuscular junction (NMJ), thus affecting neuromuscular transmission. The major disease subtypes of autoimmune MG are defined by their antigenic target. The most common target of pathogenic autoantibodies in MG is the nicotinic acetylcholine receptor (AChR), followed by muscle-specific kinase (MuSK) and lipoprotein receptor-related protein 4 (LRP4). MG patients present with similar symptoms independent of the underlying subtype of disease, while the immunopathology is remarkably distinct. Here we highlight these distinct immune mechanisms that describe both the B cell- and autoantibody-mediated pathogenesis by comparing AChR and MuSK MG subtypes. In our discussion of the AChR subtype, we focus on the role of long-lived plasma cells in the production of pathogenic autoantibodies, the IgG1 subclass mediated pathology, and contributions of complement. The similarities underlying the immunopathology of AChR MG and neuromyelitis optica (NMO) are highlighted. In contrast, MuSK MG is caused by autoantibody production by short-lived plasmablasts. MuSK MG autoantibodies are mainly of the IgG4 subclass which can undergo Fab-arm exchange (FAE), a process unique to this subclass. In FAE IgG4, molecules can dissociate into two halves and recombine with other half IgG4 molecules resulting in bispecific antibodies. Similarities between MuSK MG and other IgG4-mediated autoimmune diseases, including pemphigus vulgaris (PV) and chronic inflammatory demyelinating polyneuropathy (CIDP), are highlighted. Finally, the immunological distinctions are emphasized through presentation of biological therapeutics that provide clinical benefit depending on the MG disease subtype.

Single-cell repertoire tracing identifies rituximab-resistant B cells during myasthenia gravis relapses.

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.

Migrant memory B cells secrete luminal antibody in the vagina.

Antibodies secreted into mucosal barriers serve to protect the host from a variety of pathogens, and are the basis for successful vaccines1. In type I mucosa (such as the intestinal tract), dimeric IgA secreted by local plasma cells is transported through polymeric immunoglobulin receptors2 and mediates robust protection against viruses3,4. However, owing to the paucity of polymeric immunoglobulin receptors and plasma cells, how and whether antibodies are delivered to the type II mucosa represented by the lumen of the lower female reproductive tract remains unclear. Here, using genital herpes infection in mice, we show that primary infection does not establish plasma cells in the lamina propria of the female reproductive tract. Instead, upon secondary challenge with herpes simplex virus 2, circulating memory B cells that enter the female reproductive tract serve as the source of rapid and robust antibody secretion into the lumen of this tract. CD4 tissue-resident memory T cells secrete interferon-γ, which induces expression of chemokines, including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in a CXCR3-dependent manner, and secrete virus-specific IgG2b, IgG2c and IgA into the lumen. These results reveal that circulating memory B cells act as a rapidly inducible source of mucosal antibodies in the female reproductive tract.

Early B cell tolerance defects in neuromyelitis optica favour anti-AQP4 autoantibody production.

Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients.

Control of T helper 2 responses by transcription factor IRF4-dependent dendritic cells.

CD4⁺ T cell differentiation is regulated by specialized antigen-presenting cells. Dendritic cells (DCs) produce cytokines that promote naive CD4⁺ T cell differentiation into T helper 1 (Th1), Th17, and inducible T regulatory (iTreg) cells. However, the initiation of Th2 cell responses remains poorly understood, although it is likely that more than one mechanism might be involved. Here we have defined a specific DC subset that is involved in Th2 cell differentiation in vivo in response to a protease allergen, as well as infection with Nippostrongylus brasiliensis. We have demonstrated that this subset is controlled by the transcription factor interferon regulatory factor 4 (IRF4), which is required for their differentiation and Th2 cell-inducing function. IRF4 is known to control Th2 cell differentiation and Th2 cell-associated suppressing function in Treg cells. Our finding suggests that IRF4 also plays a role in DCs where it controls the initiation of Th2 cell responses.