Trafficking of B cell antigen in lymph nodes.

The clonal selection theory first proposed by Macfarlane Burnet is a cornerstone of immunology (1). At the time, it revolutionized the thinking of immunologists because it provided a simple explanation for lymphocyte specificity, immunological memory, and elimination of self-reactive clones (2). The experimental demonstration by Nossal & Lederberg (3) that B lymphocytes bear receptors for a single antigen raised the central question of where B lymphocytes encounter antigen. This question has remained mostly unanswered until recently. Advances in techniques such as multiphoton intravital microscopy (4, 5) have provided new insights into the trafficking of B cells and their antigen. In this review, we summarize these advances in the context of our current view of B cell circulation and activation.

Clonal Evolution of Autoreactive Germinal Centers.

Germinal centers (GCs) are the primary sites of clonal B cell expansion and affinity maturation, directing the production of high-affinity antibodies. This response is a central driver of pathogenesis in autoimmune diseases, such as systemic lupus erythematosus (SLE), but the natural history of autoreactive GCs remains unclear. Here, we present a novel mouse model where the presence of a single autoreactive B cell clone drives the TLR7-dependent activation, expansion, and differentiation of other autoreactive B cells in spontaneous GCs. Once tolerance was broken for one self-antigen, autoreactive GCs generated B cells targeting other self-antigens. GCs became independent of the initial clone and evolved toward dominance of individual clonal lineages, indicating affinity maturation. This process produced serum autoantibodies to a breadth of self-antigens, leading to antibody deposition in the kidneys. Our data provide insight into the maturation of the self-reactive B cell response, contextualizing the epitope spreading observed in autoimmune disease.

Mechanosensing by Peyer's patch stroma regulates lymphocyte migration and mucosal antibody responses.

Fibroblastic reticular cells (FRCs) and their specialized collagen fibers termed 'conduits' form fundamental structural units supporting lymphoid tissues. In lymph nodes, conduits are known to transport interstitial fluid and small molecules from afferent lymphatics into the nodal parenchyma. However, the immunological contributions of conduit function have remained elusive. Here, we report that intestinal Peyer's patches (PPs) contain a specialized conduit system that directs the flow of water absorbed across the intestinal epithelium. Notably, PP FRCs responded to conduit fluid flow via the mechanosensitive ion channel Piezo1. Disruption of fluid flow or genetic deficiency of Piezo1 on CCL19-expressing stroma led to profound structural alterations in perivascular FRCs and associated high endothelial venules. This in turn impaired lymphocyte entry into PPs and initiation of mucosal antibody responses. These results identify a critical role for conduit-mediated fluid flow in the maintenance of PP homeostasis and mucosal immunity.

The CLEC-2-podoplanin axis controls the contractility of fibroblastic reticular cells and lymph node microarchitecture.

In lymph nodes, fibroblastic reticular cells (FRCs) form a collagen-based reticular network that supports migratory dendritic cells (DCs) and T cells and transports lymph. A hallmark of FRCs is their propensity to contract collagen, yet this function is poorly understood. Here we demonstrate that podoplanin (PDPN) regulates actomyosin contractility in FRCs. Under resting conditions, when FRCs are unlikely to encounter mature DCs expressing the PDPN receptor CLEC-2, PDPN endowed FRCs with contractile function and exerted tension within the reticulum. Upon inflammation, CLEC-2 on mature DCs potently attenuated PDPN-mediated contractility, which resulted in FRC relaxation and reduced tissue stiffness. Disrupting PDPN function altered the homeostasis and spacing of FRCs and T cells, which resulted in an expanded reticular network and enhanced immunity.

B cell homeostasis and follicle confines are governed by fibroblastic reticular cells.

Fibroblastic reticular cells (FRCs) are known to inhabit T cell-rich areas of lymphoid organs, where they function to facilitate interactions between T cells and dendritic cells. However, in vivo manipulation of FRCs has been limited by a dearth of genetic tools that target this lineage. Here, using a mouse model to conditionally ablate FRCs, we demonstrated their indispensable role in antiviral T cell responses. Unexpectedly, loss of FRCs also attenuated humoral immunity due to impaired B cell viability and follicular organization. Follicle-resident FRCs established a favorable niche for B lymphocytes via production of the cytokine BAFF. Thus, our study indicates that adaptive immunity requires an intact FRC network and identifies a subset of FRCs that control B cell homeostasis and follicle identity.

Follicular Dendritic Cell Activation by TLR Ligands Promotes Autoreactive B Cell Responses.

A hallmark of autoimmunity in murine models of lupus is the formation of germinal centers (GCs) in lymphoid tissues where self-reactive B cells expand and differentiate. In the host response to foreign antigens, follicular dendritic cells (FDCs) maintain GCs through the uptake and cycling of complement-opsonized immune complexes. Here, we examined whether FDCs retain self-antigens and the impact of this process in autoantibody secretion in lupus. We found that FDCs took up and retained self-immune complexes composed of ribonucleotide proteins, autoantibody, and complement. This uptake, mediated through CD21, triggered endosomal TLR7 and led to the secretion of interferon (IFN) α via an IRF5-dependent pathway. Blocking of FDC secretion of IFN-α restored B cell tolerance and reduced the amount of GCs and pathogenic autoantibody. Thus, FDCs are a critical source of the IFN-α driving autoimmunity in this lupus model. This pathway is conserved in humans, suggesting that it may be a viable therapeutic target in systemic lupus erythematosus.

Retraction Note: Microglia-dependent synapse loss in type I interferon-mediated lupus.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Transcriptional profiling of stroma from inflamed and resting lymph nodes defines immunological hallmarks.

Lymph node stromal cells (LNSCs) closely regulate immunity and self-tolerance, yet key aspects of their biology remain poorly elucidated. Here, comparative transcriptomic analyses of mouse LNSC subsets demonstrated the expression of important immune mediators, growth factors and previously unknown structural components. Pairwise analyses of ligands and cognate receptors across hematopoietic and stromal subsets suggested a complex web of crosstalk. Fibroblastic reticular cells (FRCs) showed enrichment for higher expression of genes relevant to cytokine signaling, relative to their expression in skin and thymic fibroblasts. LNSCs from inflamed lymph nodes upregulated expression of genes encoding chemokines and molecules involved in the acute-phase response and the antigen-processing and antigen-presentation machinery. Poorly studied podoplanin (gp38)-negative CD31(-) LNSCs showed similarities to FRCs but lacked expression of interleukin 7 (IL-7) and were identified as myofibroblastic pericytes that expressed integrin α(7). Together our data comprehensively describe the transcriptional characteristics of LNSC subsets.

Lupus Susceptibility Loci Predispose Mice to Clonal Lymphocytic Responses and Myeloid Expansion.

Lupus susceptibility results from the combined effects of numerous genetic loci, but the contribution of these loci to disease pathogenesis has been difficult to study due to the large cellular heterogeneity of the autoimmune immune response. We performed single-cell RNA, BCR, and TCR sequencing of splenocytes from mice with multiple polymorphic lupus susceptibility loci. We not only observed lymphocyte and myeloid expansion, but we also characterized changes in subset frequencies and gene expression, such as decreased CD8 and marginal zone B cells and increased Fcrl5- and Cd5l-expressing macrophages. Clonotypic analyses revealed expansion of B and CD4 clones, and TCR repertoires from lupus-prone mice were distinguishable by algorithmic specificity prediction and unsupervised machine learning classification. Myeloid differential gene expression, metabolism, and altered ligand-receptor interaction were associated with decreased Ag presentation. This dataset provides novel mechanistic insight into the pathophysiology of a spontaneous model of lupus, highlighting potential therapeutic targets for autoantibody-mediated disease.

Disparities in Telemedicine Use Among Children Seen in Surgical Clinics During the COVID-19 Pandemic: Experience of One Tertiary Care Freestanding Children's Hospital.

Background: Telemedicine use dramatically increased during the COVID-19 pandemic. However, the effects of telemedicine on pre-existing disparities in pediatric surgical access have not been well described. We describe our center's early experience with telemedicine and disparities in patients' access to outpatient surgical care. Methods: A retrospective study of outpatient visits within all surgical divisions from May to December 2020 was conducted. We assessed the rates of scheduled telemedicine visits during that period, as well as the rate of completing a visit after it has been scheduled. Descriptive and logistic regression analyses were used to test for associations between these rates and patient characteristics. Results: Over the study period, 109,601 visits were scheduled. Telemedicine accounted for 6.1% of all visits with lower cancellation rates than in-person visits (26.9% vs. 34.7%). More scheduled telemedicine encounters were observed for older patients, White, English speakers, those with private insurance, and those living in rural areas. Lower odds of telemedicine visit completion were observed among patients with public insurance (odds ratio [OR] 0.7, 95% confidence interval [CI] 0.64-0.77), Spanish language preference (OR 0.84, 95% CI 0.72-0.97), and those living in rural areas (OR 0.73, 95% CI 0.64-0.84). In contrast, higher odds of telemedicine visit completion were associated with a higher Social Deprivation Index score (OR 1.41, 95% CI 1.27-1.58). Telemedicine visit completion was also associated with increasing community-level income and distance from the hospital. Conclusions: Telemedicine use for outpatient surgical care was generally low during the peak of the pandemic, and certain populations were less likely to utilize it. These findings call for further action to bridge gaps in telemedicine use.

Spatial enrichment of the type 1 interferon signature in the brain of a neuropsychiatric lupus murine model.

Among systemic lupus erythematosus (SLE) patients, neuropsychiatric symptoms are highly prevalent, being observed in up to 80% of adult and 95% of pediatric patients. Type 1 interferons, particularly interferon alpha (IFNα), have been implicated in the pathogenesis of SLE and its associated neuropsychiatric symptoms (NPSLE). However, it remains unclear how type 1 interferon signaling in the central nervous system (CNS) might result in neuropsychiatric sequelae. In this study, we validate an NPSLE mouse model and find an elevated peripheral type 1 interferon signature alongside clinically relevant NPSLE symptoms such as anxiety and fatigue. Unbiased single-nucleus sequencing of the hindbrain and hippocampus revealed that interferon-stimulated genes (ISGs) were among the most highly upregulated genes in both regions and that gene pathways involved in cellular interaction and neuronal development were generally repressed among astrocytes, oligodendrocytes, and neurons. Using image-based spatial transcriptomics, we found that the type 1 interferon signature is enriched as spatially distinct patches within the brain parenchyma of these mice. Our results suggest that type 1 interferon in the CNS may play an important mechanistic role in mediating NPSLE behavioral phenotypes by repressing general cellular communication pathways, and that type 1 interferon signaling modulators are a potential therapeutic option for NPSLE.

Artificial intelligence-based clinical decision support in pediatrics.

Machine learning models may be integrated into clinical decision support (CDS) systems to identify children at risk of specific diagnoses or clinical deterioration to provide evidence-based recommendations. This use of artificial intelligence models in clinical decision support (AI-CDS) may have several advantages over traditional "rule-based" CDS models in pediatric care through increased model accuracy, with fewer false alerts and missed patients. AI-CDS tools must be appropriately developed, provide insight into the rationale behind decisions, be seamlessly integrated into care pathways, be intuitive to use, answer clinically relevant questions, respect the content expertise of the healthcare provider, and be scientifically sound. While numerous machine learning models have been reported in pediatric care, their integration into AI-CDS remains incompletely realized to date. Important challenges in the application of AI models in pediatric care include the relatively lower rates of clinically significant outcomes compared to adults, and the lack of sufficiently large datasets available necessary for the development of machine learning models. In this review article, we summarize key concepts related to AI-CDS, its current application to pediatric care, and its potential benefits and risks. IMPACT: The performance of clinical decision support may be enhanced by the utilization of machine learning-based algorithms to improve the predictive performance of underlying models. Artificial intelligence-based clinical decision support (AI-CDS) uses models that are experientially improved through training and are particularly well suited toward high-dimensional data. The application of AI-CDS toward pediatric care remains limited currently but represents an important area of future research.

Novel approaches to capturing and using continuous cardiorespiratory physiological data in hospitalized children.

Continuous cardiorespiratory physiological monitoring is a cornerstone of care in hospitalized children. The data generated by monitoring devices coupled with machine learning could transform the way we provide care. This scoping review summarizes existing evidence on novel approaches to continuous cardiorespiratory monitoring in hospitalized children. We aimed to identify opportunities for the development of monitoring technology and the use of machine learning to analyze continuous physiological data to improve the outcomes of hospitalized children. We included original research articles published on or after January 1, 2001, involving novel approaches to collect and use continuous cardiorespiratory physiological data in hospitalized children. OVID Medline, PubMed, and Embase databases were searched. We screened 2909 articles and performed full-text extraction of 105 articles. We identified 58 articles describing novel devices or approaches, which were generally small and single-center. In addition, we identified 47 articles that described the use of continuous physiological data in prediction models, but only 7 integrated multidimensional data (e.g., demographics, laboratory results). We identified three areas for development: (1) further validation of promising novel devices; (2) more studies of models integrating multidimensional data with continuous cardiorespiratory data; and (3) further dissemination, implementation, and validation of prediction models using continuous cardiorespiratory data. IMPACT: We performed a comprehensive scoping review of novel approaches to capture and use continuous cardiorespiratory physiological data for monitoring, diagnosis, providing care, and predicting events in hospitalized infants and children, from novel devices to machine learning-based prediction models. We identified three key areas for future development: (1) further validation of promising novel devices; (2) more studies of models integrating multidimensional data with continuous cardiorespiratory data; and (3) further dissemination, implementation, and validation of prediction models using cardiorespiratory data.

Microglia-dependent synapse loss in type I interferon-mediated lupus.

Systemic lupus erythematosus (SLE) is an incurable autoimmune disease characterized by autoantibody deposition in tissues such as kidney, skin and lungs. Notably, up to 75% of patients with SLE experience neuropsychiatric symptoms that range from anxiety, depression and cognitive impairment to seizures and, in rare cases, psychosis-collectively this is referred to as central nervous system (CNS) lupus. In some cases, certain autoantibodies, such as anti-NMDAR or anti-phospholipid antibodies, promote CNS lupus. However, in most patients, the mechanisms that underlie these symptoms are unknown. CNS lupus typically presents at lupus diagnosis or within the first year, suggesting that early factors contributing to peripheral autoimmunity may promote CNS lupus symptoms. Here we report behavioural phenotypes and synapse loss in lupus-prone mice that are prevented by blocking type I interferon (IFN) signalling. Furthermore, we show that type I IFN stimulates microglia to become reactive and engulf neuronal and synaptic material in lupus-prone mice. These findings and our observation of increased type I IFN signalling in post-mortem hippocampal brain sections from patients with SLE may instruct the evaluation of ongoing clinical trials of anifrolumab, a type I IFN-receptor antagonist. Moreover, identification of IFN-driven microglia-dependent synapse loss, along with microglia transcriptome data, connects CNS lupus with other CNS diseases and provides an explanation for the neurological symptoms observed in some patients with SLE.

A narrative review of the mechanisms and consequences of intermittent hypoxia and the role of advanced analytic techniques in pediatric autonomic disorders.

Disorders of autonomic functions are typically characterized by disturbances in multiple organ systems. These disturbances are often comorbidities of common and rare diseases, such as epilepsy, sleep apnea, Rett syndrome, congenital heart disease or mitochondrial diseases. Characteristic of many autonomic disorders is the association with intermittent hypoxia and oxidative stress, which can cause or exaggerate a variety of other autonomic dysfunctions, making the treatment and management of these syndromes very complex. In this review we discuss the cellular mechanisms by which intermittent hypoxia can trigger a cascade of molecular, cellular and network events that result in the dysregulation of multiple organ systems. We also describe the importance of computational approaches, artificial intelligence and the analysis of big data to better characterize and recognize the interconnectedness of the various autonomic and non-autonomic symptoms. These techniques can lead to a better understanding of the progression of autonomic disorders, ultimately resulting in better care and management.

Cerebral Autoregulation during Orthostatic Challenge in Congenital Central Hypoventilation Syndrome.

Rationale: Congenital central hypoventilation syndrome (CCHS) is a rare autonomic disorder with altered regulation of breathing, heart rate (HR), and blood pressure (BP). Aberrant cerebral oxygenation in response to hypercapnia/hypoxia in CCHS raises the concern that altered cerebral autoregulation may contribute to CCHS-related, variably impaired neurodevelopment. Objectives: To evaluate cerebral autoregulation in response to orthostatic challenge in CCHS cases versus controls. Methods: CCHS and age- and sex-matched control subjects were studied with head-up tilt (HUT) testing to induce orthostatic stress. Fifty CCHS and 100 control HUT recordings were included. HR, BP, and cerebral oxygen saturation (regional oxygen saturation) were continuously monitored. The cerebral oximetry index (COx), a real-time measure of cerebral autoregulation based on these measures, was calculated. Measurements and Main Results: HUT resulted in a greater mean BP decrease from baseline in CCHS versus controls (11% vs. 6%; P < 0.05) and a diminished increase in HR in CCHS versus controls (11% vs. 18%; P < 0.01) in the 5 minutes after tilt-up. Despite a similar COx at baseline, orthostatic provocation within 5 minutes of tilt-up caused a 50% greater increase in COx (P < 0.01) and a 29% increase in minutes of impaired autoregulation (P < 0.02) in CCHS versus controls (4.0 vs. 3.1 min). Conclusions: Cerebral autoregulatory mechanisms appear to be intact in CCHS, but the greater hypotension observed in CCHS consequent to orthostatic provocation is associated with greater values of COx/impaired autoregulation when BP is below the lower limits of autoregulation. Effects of repeated orthostatic challenges in everyday living in CCHS necessitate further study to determine their influence on neurodevelopmental disease burden.

Capture of influenza by medullary dendritic cells via SIGN-R1 is essential for humoral immunity in draining lymph nodes.

A major pathway for B cell acquisition of lymph-borne particulate antigens relies on antigen capture by subcapsular sinus macrophages of the lymph node. Here we tested whether this mechanism is also important for humoral immunity to inactivated influenza virus. By multiple approaches, including multiphoton intravital imaging, we found that antigen capture by sinus-lining macrophages was important for limiting the systemic spread of virus but not for the generation of influenza-specific humoral immunity. Instead, we found that dendritic cells residing in the lymph node medulla use the lectin receptor SIGN-R1 to capture lymph-borne influenza virus and promote humoral immunity. Thus, our results have important implications for the generation of durable humoral immunity to viral pathogens through vaccination.

T cell help in the autoreactive germinal center.

The germinal center serves as a site of B cell selection and affinity maturation, critical processes for productive adaptive immunity. In autoimmune disease tolerance is broken in the germinal center reaction, leading to production of autoreactive B cells that may propagate disease. Follicular T cells are crucial regulators of this process, providing signals necessary for B cell survival in the germinal center. Here, we review the emerging roles of follicular T cells in the autoreactive germinal center. Recent advances in immunological techniques have allowed study of the gene expression profiles and repertoire of follicular T cells at unprecedented resolution. These studies provide insight into the potential role follicular T cells play in preventing or facilitating germinal center loss of tolerance. Improved understanding of the mechanisms of T cell help in autoreactive germinal centers provides novel therapeutic targets for diseases of germinal center dysfunction.