Increased IgD and CD27 Double Negative (DN) B cell population in pediatric onset autoimmune hepatitis.

Autoimmune hepatitis (AIH) is a chronic, inflammatory liver disease of unknown aetiology which requires lifelong immunosuppression. Most therapeutic and outcome studies of AIH have been conducted predominantly in Caucasian (European Ancestry, EA) cohorts, with the exclusion of African American (AA) patients due to inadequate sample size. It is known that AA patients have a severe phenotype of autoimmune diseases and demonstrate a poor response to conventional medical therapy. Understanding cellular and molecular pathways which determine AIH severity and progression in AA patients is likely to lead to the discovery of novel, personalised and better tolerated therapies. The aim of the study is to determine the distinct effector B cell phenotypes which contribute to disease severity and progression of AIH in AA children as compared to their EA cohorts. PBMCs were isolated from blood samples collected from patients visiting Children's Healthcare of Atlanta (CHOA) and were grouped into AA, (n = 12), EA, (n = 11) and controls (n = 12) and were processed for flow cytometry. Markers of B cell development, maturation and activation were assessed namely CD19, CD21, IgD, CD27, CD38, CD11c, CD24, CD138. AA children with AIH demonstrated an expansion of CD19 + ve, Activated Naïve (aN), (CD19+ IgD-/CD27- Double Negative (DN2) ([CD19+/IgD-/CD27++CD38++) cells. Plasmablasts were significantly higher along with Signalling Lymphocytic activation molecule F7 (SLAMF7). Unswitched memory [CD19+] IgD+CD27+ (USM) B cells were significantly contracted in AA patients with AIH. B cell phenotyping reveals a distinct profile in AA AIH patients with a major skewing towards the expansion of effector pathways which have been previously characterised in severe SLE in AA patients. These results suggest that the quantification and therapeutic target of B cell pathway could contribute substantially to the clinical approach to AIH especially in the AA population.

Affinity maturation generates pathogenic antibodies with dual reactivity to DNase1L3 and dsDNA in systemic lupus erythematosus.

Anti-dsDNA antibodies are pathogenically heterogeneous, implying distinct origins and antigenic properties. Unexpectedly, during the clinical and molecular characterization of autoantibodies to the endonuclease DNase1L3 in patients with systemic lupus erythematosus (SLE), we identified a subset of neutralizing anti-DNase1L3 antibodies previously catalogued as anti-dsDNA. Based on their variable heavy-chain (VH) gene usage, these antibodies can be divided in two groups. One group is encoded by the inherently autoreactive VH4-34 gene segment, derives from anti-DNase1L3 germline-encoded precursors, and gains cross-reactivity to dsDNA - and some additionally to cardiolipin - following somatic hypermutation. The second group, originally defined as nephritogenic anti-dsDNA antibodies, is encoded by diverse VH gene segments. Although affinity maturation results in dual reactivity to DNase1L3 and dsDNA, their binding efficiencies favor DNase1L3 as the primary antigen. Clinical, transcriptional and monoclonal antibody data support that cross-reactive anti-DNase1L3/dsDNA antibodies are more pathogenic than single reactive anti-dsDNA antibodies. These findings point to DNase1L3 as the primary target of a subset of antibodies classified as anti-dsDNA, shedding light on the origin and pathogenic heterogeneity of antibodies reactive to dsDNA in SLE.

Generation of human long-lived plasma cells by developmentally regulated epigenetic imprinting.

Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1, BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1, CASP3, and CASP8 Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

Extrafollicular IgD+ B cells generate IgE antibody secreting cells in the nasal mucosa.

Increased IgE is a typical feature of allergic rhinitis. Local class-switch recombination has been intimated but B cell precursors and mechanisms remain elusive. Here we describe the dynamics underlying the generation of IgE-antibody secreting cells (ASC) in human nasal polyps (NP), mucosal tissues rich in ASC without germinal centers (GC). Using VH next generation sequencing, we identified an extrafollicular (EF) mucosal IgD+ naïve-like intermediate B cell population with high connectivity to the mucosal IgE ASC. Mucosal IgD+ B cells, express germline epsilon transcripts and predominantly co-express IgM. However, a small but significant fraction co-express IgG or IgA instead which also show connectivity to ASC IgE. Phenotypically, NP IgD+ B cells display an activated profile and molecular evidence of BCR engagement. Transcriptionally, mucosal IgD+ B cells reveal an intermediate profile between naïve B cells and ASC. Single cell IgE ASC analysis demonstrates lower mutational frequencies relative to IgG, IgA, and IgD ASC consistent with IgE ASC derivation from mucosal IgD+ B cell with low mutational load. In conclusion, we describe a novel mechanism of GC-independent, extrafollicular IgE ASC formation at the nasal mucosa whereby activated IgD+ naïve B cells locally undergo direct and indirect (through IgG and IgA), IgE class switch.

Partial hepatectomy enhances the growth of CC531 rat colorectal cancer cells both in vitro and in vivo.

Partial hepatectomy (PHx) is the gold standard for the treatment of colorectal cancer liver metastases. However, after removing a substantial amount of hepatic tissue, growth factors are released to induce liver regeneration, which may promote the proliferation of liver micrometastases or circulating tumour cells still present in the patient. The aim of this study is to assess the effect of PHx on the growth of liver metastases induced by intrasplenic cell inoculation as well as on in vitro proliferation of the same cancer cell line. Liver tumours were induced in 18 WAG/RijHsd male rats, by seeding 250,000 syngeneic colorectal cancer cells (CC531) into the spleen. The left lateral lobe of the liver was mobilized and in half of the animals it was removed to achieve a 40% hepatectomy. Twenty-eight days after tumour induction, the animals were sacrificed and the liver was removed and sliced to assess the relative tumour surface area (RTSA%). CC531 cells were cultured in presence of foetal calf serum, non-hepatectomised (NRS) or hepatectomized rat serum (HRS), and their proliferation rate at 24, 48, and 72 h was measured. RTSA% was significantly higher in animals which had undergone PHx than in the controls (non-hepatectomised) (46.98 ± 8.76% vs. 18.73 ± 5.65%; p < 0.05). Analysing each lobe separately, this difference in favour of hepatectomized animals was relevant and statistically significant in the paramedian and caudate lobes. But in the right lobe the difference was scarce and not significant. In vitro, 2.5% HRS achieved stronger proliferative rates than the control cultures (10% FCS) or their equivalent of NRS. In this experimental model, a parallelism has been shown between the effect of PHx on the growth of colorectal cancer cells in the liver and the effect of the serum on those cells in vitro.

Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection.

BACKGROUND: SARS-CoV-2 has caused over 36,000,000 cases and 1,000,000 deaths globally. Comprehensive assessment of the multifaceted anti-viral antibody response is critical for diagnosis, differentiation of severe disease, and characterization of long-term immunity. Initial observations suggest that severe disease is associated with higher antibody levels and greater B cell/plasmablast responses. A multi-antigen immunoassay to define the complex serological landscape and clinical associations is essential. METHODS: We developed a multiplex immunoassay and evaluated serum/plasma from adults with RT-PCR-confirmed SARS-CoV-2 infections during acute illness (N=52) and convalescence (N=69); and pre-pandemic (N=106) and post-pandemic (N=137) healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 Nucleocapsid (N), Spike domain 1 (S1), receptor binding domain (S1-RBD) and S1-N-terminal domain (S1-NTD). RESULTS: To diagnose infection, the combined [IgA+IgG+IgM] or IgG for N, S1, and S1-RBD yielded AUC values -0.90 by ROC curves. From days 6-30 post-symptom onset, the levels of antigen-specific IgG, IgA or [IgA+IgG+IgM] were higher in patients with severe/critical compared to mild/moderate infections. Consistent with excessive concentrations of antibodies, a strong prozone effect was observed in sera from severe/critical patients. Notably, mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared to severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 months. CONCLUSION: This SARS-CoV-2 multiplex immunoassay measures the magnitude, complexity and kinetics of the antibody response against multiple viral antigens. The IgG and combined-isotype SARS-CoV-2 multiplex assay is highly diagnostic of acute and convalescent disease and may prognosticate severity early in illness. ONE SENTENCE SUMMARY: In contrast to patients with moderate infections, those with severe COVID-19 develop prominent, early antibody responses to S1 and N proteins.

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.

DNA methylation changes on immune cells in Systemic Lupus Erythematosus.

DNA methylation as a process that regulates gene expression is crucial in immune cells biology. Global and gene specific methylation changes have been described in autoimmunity, especially in Systemic Lupus Erythematosus. These changes not only contribute to the understanding of the disease, but also some have been proposed as diagnostic or disease activity biomarkers. The present review compiles the most recent discoveries on this field on each type of immune cells, including specific changes in signalling pathways, genes of interest and its possible applications on diagnosis or treatment.

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.

Factors Affecting Early Antibody Secreting Cell Maturation Into Long-Lived Plasma Cells.

Antibody secreting cells (ASCs) are terminally differentiated cells of the humoral immune response and must adapt morphologically, transcriptionally, and metabolically to maintain high-rates of antibody (Ab) secretion. ASCs differentiate from activated B cells in lymph nodes and transiently circulate in the blood. Most of the circulating ASCs undergo apoptosis, but a small fraction of early ASCs migrate to the bone marrow (BM) and eventually mature into long-lived plasma cells (LLPCs). LLPC survival is controlled both intrinsically and extrinsically. Their differentiation and maintenance programs are governed by many intrinsic mechanisms involving anti-apoptosis, autophagy, and metabolism. The extrinsic factors involved in LLPC generation include BM stromal cells, cytokines, and chemokines, such as APRIL, IL-6, and CXCL12. In humans, the BM CD19-CD38hiCD138+ ASC subset is the main repository of LLPCs, and our recent development of an in vitro BM mimic provides essential tools to study environmental cues that support LLPC survival and the critical molecular mechanisms of maturation from early minted blood ASCs to LLPCs. In this review, we summarize the evidence of LLPC generation and maintenance and provide novel paradigms of LLPC maturation.

Epigenetic programming underpins B cell dysfunction in human SLE.

Systemic lupus erythematosus (SLE) is characterized by the expansion of extrafollicular pathogenic B cells derived from newly activated naive cells. Although these cells express distinct markers, their epigenetic architecture and how it contributes to SLE remain poorly understood. To address this, we determined the DNA methylomes, chromatin accessibility profiles and transcriptomes from five human B cell subsets, including a newly defined effector B cell subset, from subjects with SLE and healthy controls. Our data define a differentiation hierarchy for the subsets and elucidate the epigenetic and transcriptional differences between effector and memory B cells. Importantly, an SLE molecular signature was already established in resting naive cells and was dominated by enrichment of accessible chromatin in motifs for AP-1 and EGR transcription factors. Together, these factors acted in synergy with T-BET to shape the epigenome of expanded SLE effector B cell subsets. Thus, our data define the molecular foundation of pathogenic B cell dysfunction in SLE.

Differential transcriptome and development of human peripheral plasma cell subsets.

Human antibody-secreting cells (ASCs) triggered by immunization are globally recognized as CD19loCD38hiCD27hi. Yet, different vaccines give rise to antibody responses of different longevity, suggesting ASC populations are heterogeneous. We define circulating-ASC heterogeneity in vaccine responses using multicolor flow cytometry, morphology, VH repertoire, and RNA transcriptome analysis. We also tested differential survival using a human cell-free system that mimics the bone marrow (BM) microniche. In peripheral blood, we identified 3 CD19+ and 2 CD19- ASC subsets. All subsets contributed to the vaccine-specific responses and were characterized by in vivo proliferation and activation. The VH repertoire demonstrated strong oligoclonality with extensive interconnectivity among the 5 subsets and switched memory B cells. Transcriptome analysis showed separation of CD19+ and CD19- subsets that included pathways such as cell cycle, hypoxia, TNF-α, and unfolded protein response. They also demonstrated similar long-term in vitro survival after 48 days. In summary, vaccine-induced ASCs with different surface markers (CD19 and CD138) are derived from shared proliferative precursors yet express distinctive transcriptomes. Equal survival indicates that all ASC compartments are endowed with long-lived potential. Accordingly, in vivo survival of peripheral long-lived plasma cells may be determined in part by their homing and residence in the BM microniche.

Author Correction: Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion.

The original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: 'A patent has been applied for by Emory University with F.E.L, I.S. and D.C. N. as named inventors. The patent application number is PCT/US2016/036650'. This has now been corrected in both the PDF and HTML versions of the Article.

Factors of the bone marrow microniche that support human plasma cell survival and immunoglobulin secretion.

Human antibody-secreting cells (ASC) in peripheral blood are found after vaccination or infection but rapidly apoptose unless they migrate to the bone marrow (BM). Yet, elements of the BM microenvironment required to sustain long-lived plasma cells (LLPC) remain elusive. Here, we identify BM factors that maintain human ASC > 50 days in vitro. The critical components of the cell-free in vitro BM mimic consist of products from primary BM mesenchymal stromal cells (MSC), a proliferation-inducing ligand (APRIL), and hypoxic conditions. Comparative analysis of protein-protein interactions between BM-MSC proteomics with differential RNA transcriptomics of blood ASC and BM LLPC identify two major survival factors, fibronectin and YWHAZ. The MSC secretome proteins and hypoxic conditions play a role in LLPC survival utilizing mechanisms that downregulate mTORC1 signaling and upregulate hypoxia signatures. In summary, we identify elements of the BM survival niche critical for maturation of blood ASC to BM LLPC.

Understanding B-cell activation and autoantibody repertoire selection in systemic lupus erythematosus: A B-cell immunomics approach.

Understanding antibody repertoires and in particular, the properties and fates of B cells expressing potentially pathogenic antibodies is critical to define the mechanisms underlying multiple immunological diseases including autoimmune and allergic conditions as well as transplant rejection. Moreover, an integrated knowledge of the antibody repertoires expressed by B cells and plasma cells (PC) of different functional properties and longevity is essential to develop new therapeutic strategies, better biomarkers for disease segmentation, and new assays to measure restoration of B-cell tolerance or, at least, of normal B-cell homeostasis. Reaching these goals, however, will require a more precise phenotypic, functional and molecular definition of B-cell and PC populations, and a comprehensive analysis of the antigenic reactivity of the antibodies they express. While traditionally hampered by technical and ethical limitations in human experimentation, new technological advances currently enable investigators to address these questions in a comprehensive fashion. In this review, we shall discuss these concepts as they apply to the study of Systemic Lupus Erythematosus.

BALDR: a computational pipeline for paired heavy and light chain immunoglobulin reconstruction in single-cell RNA-seq data.

B cells play a critical role in the immune response by producing antibodies, which display remarkable diversity. Here we describe a bioinformatic pipeline, BALDR (BCR Assignment of Lineage using De novo Reconstruction) that accurately reconstructs the paired heavy and light chain immunoglobulin gene sequences from Illumina single-cell RNA-seq data. BALDR was accurate for clonotype identification in human and rhesus macaque influenza vaccine and simian immunodeficiency virus vaccine induced vaccine-induced plasmablasts and naïve and antigen-specific memory B cells. BALDR enables matching of clonotype identity with single-cell transcriptional information in B cell lineages and will have broad application in the fields of vaccines, human immunodeficiency virus broadly neutralizing antibody development, and cancer.BALDR is available at https://github.com/BosingerLab/BALDR .

Role of Myocardial Collagen in Severe Aortic Stenosis With Preserved Ejection Fraction and Symptoms of Heart Failure.

INTRODUCTION AND OBJECTIVES: We investigated the anatomical localization, biomechanical properties, and molecular phenotype of myocardial collagen tissue in 40 patients with severe aortic stenosis with preserved ejection fraction and symptoms of heart failure. METHODS: Two transmural biopsies were taken from the left ventricular free wall. Mysial and nonmysial regions of the collagen network were analyzed. Myocardial collagen volume fraction (CVF) was measured by picrosirius red staining. Young's elastic modulus (YEM) was measured by atomic force microscopy in decellularized slices to assess stiffness. Collagen types I and III were measured as CIVF and CIIIVF, respectively, by confocal microscopy in areas with YEM evaluation. RESULTS: Compared with controls, patients exhibited increased mysial and nonmysial CVF and nonmysial:mysial CVF ratio (P < .05). In patients, nonmysial CVF (r = 0.330; P = .046) and the nonmysial:mysial CVF ratio (r = 0.419; P = .012) were directly correlated with the ratio of maximal early transmitral flow velocity in diastole to early mitral annulus velocity in diastole. Both the CIVF:CIIIVF ratio and YEM were increased (P ≤ .001) in nonmysial regions compared with mysial regions in patients, with a direct correlation (r = 0.895; P < .001) between them. CONCLUSIONS: These findings suggest that, in patients with severe aortic stenosis with preserved ejection fraction and symptoms of heart failure, diastolic dysfunction is associated with increased nonmysial deposition of collagen, predominantly type I, resulting in increased extracellular matrix stiffness. Therefore, the characteristics of collagen tissue may contribute to diastolic dysfunction in these patients.

Digestion of Chromatin in Apoptotic Cell Microparticles Prevents Autoimmunity.

Antibodies to DNA and chromatin drive autoimmunity in systemic lupus erythematosus (SLE). Null mutations and hypomorphic variants of the secreted deoxyribonuclease DNASE1L3 are linked to familial and sporadic SLE, respectively. We report that DNASE1L3-deficient mice rapidly develop autoantibodies to DNA and chromatin, followed by an SLE-like disease. Circulating DNASE1L3 is produced by dendritic cells and macrophages, and its levels inversely correlate with anti-DNA antibody response. DNASE1L3 is uniquely capable of digesting chromatin in microparticles released from apoptotic cells. Accordingly, DNASE1L3-deficient mice and human patients have elevated DNA levels in plasma, particularly in circulating microparticles. Murine and human autoantibody clones and serum antibodies from human SLE patients bind to DNASE1L3-sensitive chromatin on the surface of microparticles. Thus, extracellular microparticle-associated chromatin is a potential self-antigen normally digested by circulating DNASE1L3. The loss of this tolerance mechanism can contribute to SLE, and its restoration may represent a therapeutic opportunity in the disease.