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.

Extrafollicular B cell responses correlate with neutralizing antibodies and morbidity in COVID-19.

A wide spectrum of clinical manifestations has become a hallmark of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) COVID-19 pandemic, although the immunological underpinnings of diverse disease outcomes remain to be defined. We performed detailed characterization of B cell responses through high-dimensional flow cytometry to reveal substantial heterogeneity in both effector and immature populations. More notably, critically ill patients displayed hallmarks of extrafollicular B cell activation and shared B cell repertoire features previously described in autoimmune settings. Extrafollicular activation correlated strongly with large antibody-secreting cell expansion and early production of high concentrations of SARS-CoV-2-specific neutralizing antibodies. Yet, these patients had severe disease with elevated inflammatory biomarkers, multiorgan failure and death. Overall, these findings strongly suggest a pathogenic role for immune activation in subsets of patients with COVID-19. Our study provides further evidence that targeted immunomodulatory therapy may be beneficial in specific patient subpopulations and can be informed by careful immune profiling.

Dysregulated naive B cells and de novo autoreactivity in severe COVID-19.

Severe SARS-CoV-2 infection1 has been associated with highly inflammatory immune activation since the earliest days of the COVID-19 pandemic2-5. More recently, these responses have been associated with the emergence of self-reactive antibodies with pathologic potential6-10, although their origins and resolution have remained unclear11. Previously, we and others have identified extrafollicular B cell activation, a pathway associated with the formation of new autoreactive antibodies in chronic autoimmunity12,13, as a dominant feature of severe and critical COVID-19 (refs. 14-18). Here, using single-cell B cell repertoire analysis of patients with mild and severe disease, we identify the expansion of a naive-derived, low-mutation IgG1 population of antibody-secreting cells (ASCs) reflecting features of low selective pressure. These features correlate with progressive, broad, clinically relevant autoreactivity, particularly directed against nuclear antigens and carbamylated proteins, emerging 10-15 days after the onset of symptoms. Detailed analysis of the low-selection compartment shows a high frequency of clonotypes specific for both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against the glomerular basement membrane. We further identify the contraction of this pathway on recovery, re-establishment of tolerance standards and concomitant loss of acute-derived ASCs irrespective of antigen specificity. However, serological autoreactivity persists in a subset of patients with postacute sequelae, raising important questions as to the contribution of emerging autoreactivity to continuing symptomology on recovery. In summary, this study demonstrates the origins, breadth and resolution of autoreactivity in severe COVID-19, with implications for early intervention and the treatment of patients with post-COVID sequelae.

Systems biology of B cells in COVID-19.

The integration of multi-'omic datasets into complex systems-wide assessments has become a mainstay in immunologic investigation. This focus on high-dimensional data collection and analysis was on full display in the investigation of COVID-19, the respiratory illness resulting from infection by the novel coronavirus SARS-CoV-2. Particularly in the area of B cell biology, tremendous efforts in both cellular and serologic investigation have resulted in an increasingly detailed mapping of the coordinated effector, memory, and antibody secreting cell responses that underpin the development of humoral immunity in response to primary viral infection. Further, the rapid development and deployment of effective vaccines has allowed for the assessment of developing memory responses across a wide variety of immune contexts, including in patients with compromised immune function. The result has been a period of rapid gains in the understanding of B cell biology unrestricted to the study of COVID-19. Here, we outline the systems-level technologies that have been routinely implemented in these investigations throughout the pandemic, and discuss how their use has led to clear and applicable gains in pursuance of the amelioration of human infectious disease and beyond.

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.

Distinct Effector B Cells Induced by Unregulated Toll-like Receptor 7 Contribute to Pathogenic Responses in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is characterized by B cells lacking IgD and CD27 (double negative; DN). We show that DN cell expansions reflected a subset of CXCR5- CD11c+ cells (DN2) representing pre-plasma cells (PC). DN2 cells predominated in African-American patients with active disease and nephritis, anti-Smith and anti-RNA autoantibodies. They expressed a T-bet transcriptional network; increased Toll-like receptor-7 (TLR7); lacked the negative TLR regulator TRAF5; and were hyper-responsive to TLR7. DN2 cells shared with activated naive cells (aNAV), phenotypic and functional features, and similar transcriptomes. Their PC differentiation and autoantibody production was driven by TLR7 in an interleukin-21 (IL-21)-mediated fashion. An in vivo developmental link between aNAV, DN2 cells, and PC was demonstrated by clonal sharing. This study defines a distinct differentiation fate of autoreactive naive B cells into PC precursors with hyper-responsiveness to innate stimuli, as well as establishes prominence of extra-follicular B cell activation in SLE, and identifies therapeutic targets.

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.

COVID-19 and plasma cells: Is there long-lived protection?

Infection with SARS-CoV-2, the etiology of the ongoing COVID-19 pandemic, has resulted in over 450 million cases with more than 6 million deaths worldwide, causing global disruptions since early 2020. Memory B cells and durable antibody protection from long-lived plasma cells (LLPC) are the mainstay of most effective vaccines. However, ending the pandemic has been hampered by the lack of long-lived immunity after infection or vaccination. Although immunizations offer protection from severe disease and hospitalization, breakthrough infections still occur, most likely due to new mutant viruses and the overall decline of neutralizing antibodies after 6 months. Here, we review the current knowledge of B cells, from extrafollicular to memory populations, with a focus on distinct plasma cell subsets, such as early-minted blood antibody-secreting cells and the bone marrow LLPC, and how these humoral compartments contribute to protection after SARS-CoV-2 infection and immunization.

Patients taking benralizumab, dupilumab, or mepolizumab have lower postvaccination SARS-CoV-2 immunity.

BACKGROUND: Biologic therapies inhibiting the IL-4 or IL-5 pathways are very effective in the treatment of asthma and other related conditions. However, the cytokines IL-4 and IL-5 also play a role in the generation of adaptive immune responses. Although these biologics do not cause overt immunosuppression, their effect in primary severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunization has not been studied completely. OBJECTIVE: Our aim was to evaluate the antibody and cellular immunity after SARS-CoV-2 mRNA vaccination in patients on biologics (PoBs). METHODS: Patients with severe asthma or atopic dermatitis who were taking benralizumab, dupilumab, or mepolizumab and had received the initial dose of the 2-dose adult SARS-CoV-2 mRNA vaccine were enrolled in a prospective, observational study. As our control group, we used a cohort of immunologically healthy subjects (with no significant immunosuppression) who were not taking biologics (NBs). We used a multiplexed immunoassay to measure antibody levels, neutralization assays to assess antibody function, and flow cytometry to quantitate Spike-specific lymphocytes. RESULTS: We analyzed blood from 57 patients in the PoB group and 46 control subjects from the NB group. The patients in the PoB group had lower levels of SARS-CoV-2 antibodies, pseudovirus neutralization, live virus neutralization, and frequencies of Spike-specific B and CD8 T cells at 6 months after vaccination. In subgroup analyses, patients with asthma who were taking biologics had significantly lower pseudovirus neutralization than did subjects with asthma who were not taking biologics. CONCLUSION: The patients in the PoB group had reduced SARS-CoV-2-specific antibody titers, neutralizing activity, and virus-specific B- and CD8 T-cell counts. These results have implications when considering development of a more individualized immunization strategy in patients who receive biologic medications blocking IL-4 or IL-5 pathways.

Extrafollicular responses in humans and SLE.

Chronic autoimmune diseases, and in particular Systemic Lupus Erythematosus (SLE), are endowed with a long-standing autoreactive B-cell compartment that is presumed to reactivate periodically leading to the generation of new bursts of pathogenic antibody-secreting cells (ASC). Moreover, pathogenic autoantibodies are typically characterized by a high load of somatic hypermutation and in some cases are highly stable even in the context of prolonged B-cell depletion. Long-lived, highly mutated antibodies are typically generated through T-cell-dependent germinal center (GC) reactions. Accordingly, an important role for GC reactions in the generation of pathogenic autoreactivity has been postulated in SLE. Nevertheless, pathogenic autoantibodies and autoimmune disease can be generated through B-cell extrafollicular (EF) reactions in multiple mouse models and human SLE flares are characterized by the expansion of naive-derived activated effector B cells of extrafollicular phenotype. In this review, we will discuss the properties of the EF B-cell pathway, its relationship to other effector B-cell populations, its role in autoimmune diseases, and its contribution to human SLE. Furthermore, we discuss the relationship of EF B cells with Age-Associated B cells (ABCs), a TLR-7-driven B-cell population that mediates murine autoimmune and antiviral responses.

Understanding and measuring human B-cell tolerance and its breakdown in autoimmune disease.

The maintenance of immunological tolerance of B lymphocytes is a complex and critical process that must be implemented as to avoid the detrimental development of autoreactivity and possible autoimmunity. Murine models have been invaluable to elucidate many of the key components in B-cell tolerance; however, translation to human homeostatic and pathogenic immune states can be difficult to assess. Functional autoreactive, flow cytometric, and single-cell cloning assays have proven to be critical in deciphering breaks in B-cell tolerance within autoimmunity; however, newer approaches to assess human B-cell tolerance may prove to be vital in the further exploration of underlying tolerance defects. In this review, we supply a comprehensive overview of human immune tolerance checkpoints with associated mechanisms of enforcement, and highlight current and future methodologies which are likely to benefit future studies into the mechanisms that become defective in human autoimmune conditions.

B cell subset composition segments clinically and serologically distinct groups in chronic cutaneous lupus erythematosus.

OBJECTIVE: While the contribution of B-cells to SLE is well established, its role in chronic cutaneous lupus erythematosus (CCLE) remains unclear. Here, we compare B-cell and serum auto-antibody profiles between patients with systemic lupus erythematosus (SLE), CCLE, and overlap conditions. METHODS: B-cells were compared by flow cytometry amongst healthy controls, CCLE without systemic lupus (CCLE+/SLE-) and SLE patients with (SLE+/CCLE+) or without CCLE (SLE+/CCLE-). Serum was analyed for autoreactive 9G4+, anti-double-stranded DNA, anti-chromatin and anti-RNA antibodies by ELISA and for anti-RNA binding proteins (RBP) by luciferase immunoprecipitation. RESULTS: Patients with CCLE+/SLE- share B-cell abnormalities with SLE including decreased unswitched memory and increased effector B-cells albeit at a lower level than SLE patients. Similarly, both SLE and CCLE+/SLE- patients have elevated 9G4+ IgG autoantibodies despite lower levels of anti-nucleic acid and anti-RBP antibodies in CCLE+/SLE-. CCLE+/SLE- patients could be stratified into those with SLE-like B-cell profiles and a separate group with normal B-cell profiles. The former group was more serologically active and more likely to have disseminated skin lesions. CONCLUSION: CCLE displays perturbations in B-cell homeostasis and partial B-cell tolerance breakdown. Our study demonstrates that this entity is immunologically heterogeneous and includes a disease segment whose B-cell compartment resembles SLE and is clinically associated with enhanced serological activity and more extensive skin disease. This picture suggests that SLE-like B-cell changes in primary CCLE may help identify patients at risk for subsequent development of SLE. B-cell profiling in CCLE might also indentify candidates who would benefit from B-cell targeted therapies.

Podoplanin-rich stromal networks induce dendritic cell motility via activation of the C-type lectin receptor CLEC-2.

To initiate adaptive immunity, dendritic cells (DCs) move from parenchymal tissues to lymphoid organs by migrating along stromal scaffolds that display the glycoprotein podoplanin (PDPN). PDPN is expressed by lymphatic endothelial and fibroblastic reticular cells and promotes blood-lymph separation during development by activating the C-type lectin receptor, CLEC-2, on platelets. Here, we describe a role for CLEC-2 in the morphodynamic behavior and motility of DCs. CLEC-2 deficiency in DCs impaired their entry into lymphatics and trafficking to and within lymph nodes, thereby reducing T cell priming. CLEC-2 engagement of PDPN was necessary for DCs to spread and migrate along stromal surfaces and sufficient to induce membrane protrusions. CLEC-2 activation triggered cell spreading via downregulation of RhoA activity and myosin light-chain phosphorylation and triggered F-actin-rich protrusions via Vav signaling and Rac1 activation. Thus, activation of CLEC-2 by PDPN rearranges the actin cytoskeleton in DCs to promote efficient motility along stromal surfaces.

GLaMST: grow lineages along minimum spanning tree for b cell receptor sequencing data.

BACKGROUND: B cell affinity maturation enables B cells to generate high-affinity antibodies. This process involves somatic hypermutation of B cell immunoglobulin receptor (BCR) genes and selection by their ability to bind antigens. Lineage trees are used to describe this microevolution of B cell immunoglobulin genes. In a lineage tree, each node is one BCR sequence that mutated from the germinal center and each directed edge represents a single base mutation, insertion or deletion. In BCR sequencing data, the observed data only contains a subset of BCR sequences in this microevolution process. Therefore, reconstructing the lineage tree from experimental data requires algorithms to build the tree based on partially observed tree nodes. RESULTS: We developed a new algorithm named Grow Lineages along Minimum Spanning Tree (GLaMST), which efficiently reconstruct the lineage tree given observed BCR sequences that correspond to a subset of the tree nodes. Through comparison using simulated and real data, GLaMST outperforms existing algorithms in simulations with high rates of mutation, insertion and deletion, and generates lineage trees with smaller size and closer to ground truth according to tree features that highly correlated with selection pressure. CONCLUSIONS: GLaMST outperforms state-of-art in reconstruction of the BCR lineage tree in both efficiency and accuracy. Integrating it into existing BCR sequencing analysis frameworks can significant improve lineage tree reconstruction aspect of the analysis.

Early impact of the coronavirus disease (COVID-19) pandemic and physical distancing measures on routine childhood vaccinations in England, January to April 2020.

Using electronic health records, we assessed the early impact of coronavirus disease (COVID-19) on routine childhood vaccination in England by 26 April 2020. Measles-mumps-rubella vaccination counts fell from February 2020, and in the 3 weeks after introduction of physical distancing measures were 19.8% lower (95% confidence interval: -20.7 to -18.9) than the same period in 2019, before improving in mid-April. A gradual decline in hexavalent vaccination counts throughout 2020 was not accentuated by physical distancing.

Concurrent spinal epidural empyema and endocarditis in a dog.

A 9-year-old neutered male Rhodesian ridgeback cross dog was evaluated for progressive non-ambulatory paraparesis, fever, and leukocytosis. The dog was diagnosed with spinal epidural empyema (SEE) and infectious endocarditis (IE) of the mitral valve based on the findings of contrast-enhanced computed tomography (CT), CT myelography, echocardiography, and bacterial culture. The report herein describes the clinical presentation, CT findings, clinical and surgical management of this case, together with the electrocardiography, and echocardiography findings. To the authors' knowledge, this is the first reported case of spinal epidural empyema likely to be caused by infectious endocarditis of the mitral valve in a dog.

Empyème épidural spinal concomitant à une endocardite chez un chien. Un chien mâle castré croisé Rhodesian Ridgeback âgé de 9 ans a été évalué pour une paraparésie progressive non-ambulatoire, de la fièvre et une leucocytose. Un diagnostic d'empyème épidural spinal (SEE) et d'endocardite infectieuse (IE) de la valvule mitrale a été émis basé sur les trouvailles de la tomodensitométrie (CT), d'une myélographie CT, de l'échocardiographie, et de la culture bactérienne. Le présent rapport décrit la présentation clinique, les trouvailles de CT, la gestion clinique et chirurgicale de ce cas, de même que les trouvailles par électrocardiographie et échocardiographie. À la connaissance des auteurs, ceci représente le premier cas rapporté d'empyème épidural spinal à être causé par une endocardite infectieuse de la valvule mitrale chez un chien.(Traduit par Dr Serge Messier).