Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Positive selection of IgG+ over IgM+ B cells in the germinal center reaction.

Positive selection of high-affinity B cells within germinal centers (GCs) drives affinity maturation of antibody responses. Here, we examined the mechanism underlying the parallel transition from immunoglobulin M (IgM) to IgG. Early GCs contained mostly unswitched IgM+ B cells; IgG+ B cells subsequently increased in frequency, dominating GC responses 14-21 days after antigen challenge. Somatic hypermutation and generation of high-affinity clones occurred with equal efficiency among IgM+ and IgG+ GC B cells, and inactivation of Ig class-switch recombination did not prevent depletion of IgM+ GC B cells. Instead, high-affinity IgG+ GC B cells outcompeted high-affinity IgM+ GC B cells via a selective advantage associated with IgG antigen receptor structure but independent of the extended cytoplasmic tail. Thus, two parallel forms of GC B-cell-positive selection, based on antigen receptor variable and constant regions, respectively, operate in tandem to ensure high-affinity IgG antibodies predominate in mature serum antibody responses.

Transitioning single-cell genomics into the clinic.

The use of genomics is firmly established in clinical practice, resulting in innovations across a wide range of disciplines such as genetic screening, rare disease diagnosis and molecularly guided therapy choice. This new field of genomic medicine has led to improvements in patient outcomes. However, most clinical applications of genomics rely on information generated from bulk approaches, which do not directly capture the genomic variation that underlies cellular heterogeneity. With the advent of single-cell technologies, research is rapidly uncovering how genomic data at cellular resolution can be used to understand disease pathology and mechanisms. Both DNA-based and RNA-based single-cell technologies have the potential to improve existing clinical applications and open new application spaces for genomics in clinical practice, with oncology, immunology and haematology poised for initial adoption. However, challenges in translating cellular genomics from research to a clinical setting must first be overcome.

CCR6 Defines Memory B Cell Precursors in Mouse and Human Germinal Centers, Revealing Light-Zone Location and Predominant Low Antigen Affinity.

Memory B cells (MBCs) and plasma cells (PCs) constitute the two cellular outputs of germinal center (GC) responses that together facilitate long-term humoral immunity. Although expression of the transcription factor BLIMP-1 identifies cells undergoing PC differentiation, no such marker exists for cells committed to the MBC lineage. Here, we report that the chemokine receptor CCR6 uniquely marks MBC precursors in both mouse and human GCs. CCR6+ GC B cells were highly enriched within the GC light zone (LZ), were the most quiescent of all GC B cells, exhibited a cell-surface phenotype and gene expression signature indicative of an MBC transition, and possessed the augmented response characteristics of MBCs. MBC precursors within the GC LZ predominantly possessed a low affinity for antigen but also included cells from within the high-affinity pool. These data indicate a fundamental dichotomy between the processes that drive MBC and PC differentiation during GC responses.

Isolation and characterisation of PR3-specific B cells and their immunoglobulin sequences.

BACKGROUND: PR3 autoantibodies are essential to the diagnosis and monitoring of granulomatosus with polyangiitis, but to date no PR3 autoantibody sequences have been published. OBJECTIVES: To identify and characterise PR3-specific B cells from the peripheral blood of patients with PR3 autoantibodies. METHODS: Peripheral blood mononuclear cells from seven patients with PR3 autoantibodies were stained with PR3. B cells that bound PR3 underwent single cell sorting, transcriptome sequencing, and their immunoglobulin sequences expressed as antibodies and tested for PR3-specificity by ELISA. RESULTS: We identified 19 PR3-specific B cells from only one PR3-seropositive patient at a low frequency (0.0075 % of B cells) in the peripheral blood. These were polyclonal, IgG+ and enriched for IgG4, lambda pairing, IGHJ6 gene usage, CDRH3 length, IGHE and CD71 expression. They demonstrated relatively low levels of somatic hypermutation and variably reduced PR3 binding when reverted to germline. CONCLUSIONS: Identifying PR3-specific B cells in the peripheral blood is possible but challenging and those we did identify exhibited features suggesting that PR3-self reactivity may occur early in B-cell development.

T follicular helper cells have distinct modes of migration and molecular signatures in naive and memory immune responses.

B helper follicular T (Tfh) cells are critical for long-term humoral immunity. However, it remains unclear how these cells are recruited and contribute to secondary immune responses. Here we show that primary Tfh cells segregate into follicular mantle (FM) and germinal center (GC) subpopulations that display distinct gene expression signatures. Restriction of the primary Tfh cell subpopulation in the GC was mediated by downregulation of chemotactic receptor EBI2. Following collapse of the GC, memory T cells persisted in the outer follicle where they scanned CD169(+) subcapsular sinus macrophages. Reactivation and intrafollicular expansion of these follicular memory T cells in the subcapsular region was followed by their extrafollicular dissemination via the lymphatic flow. These data suggest that Tfh cells integrate their antigen-experience history to focus T cell help within the GC during primary responses but act rapidly to provide systemic T cell help after re-exposure to the antigen.

Gastric Cancer Screening in Common Variable Immunodeficiency.

Individuals with common variable immunodeficiency (CVID) have an increased risk of gastric cancer, and gastrointestinal lymphoma, yet screening for premalignant gastric lesions is rarely offered routinely to these patients. Proposed screening protocols are not widely accepted and are based on gastric cancer risk factors that are not applicable to all CVID patients. Fifty-two CVID patients were recruited for screening gastroscopy irrespective of symptoms or blood results and were compared to 40 controls presenting for gastroscopy for other clinical indications. Overall, 34% of CVID patients had intestinal metaplasia (IM), atrophic gastritis or moderate to severe non-atrophic gastritis, which can increase the risk of gastric cancer, compared to 7.5% of controls (p < 0.01). Focal nodular lymphoid hyperplasia, a precursor lesion for gastrointestinal lymphoma, was seen in eight CVID patients (16%), one of whom was diagnosed with gastrointestinal lymphoma on the same endoscopy. High-risk gastric pathology was associated with increased time since diagnosis of CVID, smoking, Helicobacter pylori, a low-serum pepsinogen I concentration, and diarrhea, but not pepsinogen I/II ratio, iron studies, vitamin B12 levels or upper gastrointestinal symptoms. There was a lower rate of detection of IM when fewer biopsies were taken, and IM and gastric atrophy were rarely predicted by the endoscopist macroscopically, highlighting the need for standardized biopsy protocols. The prevalence of premalignant gastric lesions in patients with CVID highlights the need for routine gastric screening. We propose a novel gastric screening protocol to detect early premalignant lesions and reduce the risk of gastric cancer and gastric lymphoma in these patients.

A novel cause of chronic viral meningoencephalitis: Cache Valley virus.

OBJECTIVE: Immunodeficient patients are particularly vulnerable to neuroinvasive infections that can be challenging to diagnose. Metagenomic next generation sequencing can identify unusual or novel microbes and is therefore well suited for investigating the etiology of chronic meningoencephalitis in immunodeficient patients. METHODS: We present the case of a 34-year-old man with X-linked agammaglobulinemia from Australia suffering from 3 years of meningoencephalitis that defied an etiologic diagnosis despite extensive conventional testing, including a brain biopsy. Metagenomic next generation sequencing of his cerebrospinal fluid and brain biopsy tissue was performed to identify a causative pathogen. RESULTS: Sequences aligning to multiple Cache Valley virus genes were identified via metagenomic next generation sequencing. Reverse transcription polymerase chain reaction and immunohistochemistry subsequently confirmed the presence of Cache Valley virus in the brain biopsy tissue. INTERPRETATION: Cache Valley virus, a mosquito-borne orthobunyavirus, has only been identified in 3 immunocompetent North American patients with acute neuroinvasive disease. The reported severity ranges from a self-limiting meningitis to a rapidly fatal meningoencephalitis with multiorgan failure. The virus has never been known to cause a chronic systemic or neurologic infection in humans. Cache Valley virus has also never previously been detected on the Australian continent. Our research subject traveled to North and South Carolina and Michigan in the weeks prior to the onset of his illness. This report demonstrates that metagenomic next generation sequencing allows for unbiased pathogen identification, the early detection of emerging viruses as they spread to new locales, and the discovery of novel disease phenotypes. Ann Neurol 2017;82:105-114.

Isolated anti-Ro52 identifies a severe subset of Sjögren's syndrome patients.

Introduction: Serum autoantibodies targeting the SSA/Ro proteins are a key component of the classification criteria for the diagnosis of Sjögren's syndrome (SS). Most patients' serum reacts with both Ro60 and Ro52 proteins. Here we compare the molecular and clinical characteristics of patients diagnosed with SS with anti-Ro52 in the presence or absence of anti-Ro60/La autoantibodies. Methods: A cross-sectional study was performed. Patients in the SS biobank at Westmead Hospital (Sydney, Australia) that were positive for anti-Ro52 were included and stratified based on the absence (isolated) or presence (combined) of anti-Ro60/La, measured by line immunoassay. We examined clinical associations and the serological and molecular characteristics of anti-Ro52 using ELISA and mass spectrometry in serological groups. Results: A total of 123 SS patients were included for study. SS patients with isolated anti-Ro52 (12%) identified a severe serological subset characterised by higher disease activity, vasculitis, pulmonary involvement, rheumatoid factor (RhF) and cryoglobulinaemia. Serum antibodies reacting with Ro52 in the isolated anti-Ro52 subset displayed less isotype switching, less immunoglobulin variable region subfamily usage and a lower degree of somatic hypermutation than the combined anti-Ro52 subset. Conclusions: In our cohort of SS patients, isolated anti-Ro52 represents a severe subset of SS, and is associated with the presence of cryoglobulinaemia. We therefore provide clinical relevance to the stratification of SS patients by their sero-reactivities. It is possible that the autoantibody patterns may be immunological epiphenomena of the underlying disease process, and further work is required to unearth the mechanisms of the differential clinical phenotypes.

Coronary artery vasospasm in a patient with Churg-Strauss syndrome.

Patients with Churg-Strauss syndrome often suffer from unusual cardiac manifestations and sudden cardiac death. This differs from patients with other autoimmune disorders, who typically present with premature ischaemic heart disease. We report the case of a 56-year-old man with recurrent coronary vasospasm, including an inferoposterior ST-elevation myocardial infarction, complicated by bradycardic arrest. There was only minor coronary artery disease on coronary angiography. An elevated eosinophil count was noted. His medical history included allergic rhinitis with polyposis, adult-onset asthma and biopsy-proven eosinophilic oesophagitis. Review of his sinus biopsies demonstrated blood vessels with marked accumulation of eosinophils in extravascular areas. The patient, therefore, met the American College of Rheumatology criteria for Churg-Strauss syndrome. The patient was commenced on immunosuppression, with the return of the eosinophil count to within normal limits, and remains free of cardiovascular events over 24 months.

Plasma cell and memory B cell differentiation from the germinal center.

Germinal centers (GCs) form in secondary lymphoid tissues in response to antigenic challenge and are the site of somatic hypermutation, generating GC B cells with increasing affinity for the inciting agent that are positively selected over time. However, it is not until GC B cells differentiate into memory B cells and plasma cells and egress from the GC back into the circulation that effective long-lived humoral immunity is conferred upon the host. Here we review what is known about the signals that initiate the transition from a GC B cell into the memory B cell and plasma cell compartments and the downstream transcriptional regulation of these processes.

Managing two decades of visceral leishmaniasis and HIV co-infection: a case report that illustrates the urgent research needs in the field.

Visceral leishmaniasis and HIV co-infection presents diagnostic, monitoring and treatment challenges. This is a report of a co-infected patient who developed multiple complications and treatment side-effects, including renal and liver failure, pancytopenia with recurrent sepsis, along with anal cancer, depression and poor quality-of-life spanning over two decades. Urgent research specific to this cohort is needed.

Differentiation of germinal center B cells into plasma cells is initiated by high-affinity antigen and completed by Tfh cells.

Plasma cells (PCs) derived from germinal centers (GCs) secrete the high-affinity antibodies required for long-term serological immunity. Nevertheless, the process whereby GC B cells differentiate into PCs is uncharacterized, and the mechanism underlying the selective PC differentiation of only high-affinity GC B cells remains unknown. In this study, we show that differentiation into PCs is induced among a discrete subset of high-affinity B cells residing within the light zone of the GC. Initiation of differentiation required signals delivered upon engagement with intact antigen. Signals delivered by T follicular helper cells were not required to initiate differentiation but were essential to complete the differentiation process and drive migration of maturing PCs through the dark zone and out of the GC. This bipartite or two-signal mechanism has likely evolved to both sustain protective immunity and avoid autoantibody production.

Iodide mumps.

Iodide sialadenitis is a rare, delayed idiosyncratic reaction to iodine-containing contrast media. We present non-contrast computed tomography images of this benign but dramatic adverse reaction occurring in the submandibular glands.

Optimizing fluorescence excitation and detection for intravital two-photon microscopy.

Commercial two-photon microscope systems incorporating turnkey ultrafast lasers have made the technology more user-friendly and accessible to nonspecialized biology laboratories. This has been accompanied by the development of an exciting range of new fluorescent proteins and dyes such as near-infra-red fluorescent proteins and optical highlighters. However, the two-photon absorption properties of these fluorescent molecules are not widely available and cannot be reliably predicted from their single photon absorption spectra. Furthermore, the spectral characteristics of fluorescent proteins in vivo can be affected by the local environment and light scattering by deep tissue and can vary greatly from one laboratory to the next. Here, we describe a simple protocol for determining the two-photon excitation peaks of fluorescent reporters that can be tailored to the relevant tissue samples to suit the imaging goals of individual biological laboratories.

Differentiating patterns of oligoclonal banding in the cerebrospinal fluid improves diagnostic utility for multiple sclerosis.

BACKGROUND: Detection of oligoclonal bands (OCBs) in the cerebrospinal fluid (CSF) is an important adjunct to the diagnosis of multiple sclerosis (MS) and clinically isolated syndromes (CIS). OCBs are considered present if two or more extra IgG bands are present in CSF in comparison to a simultaneously collected serum sample. However, using isoelectric focusing and immunofixation with anti-IgG, we observed two distinct banding patterns, one in which the bands were numerous and prominent (which we termed 'delta') and a much more subtle pattern, with fewer, more indistinct bands ('theta'). AIM: To perform a prospective study to determine the diagnostic implications of the two OCB patterns for multiple sclerosis. METHODS: Over a 2-year period, 56 consecutive CSF samples with OCBs were identified. Clinical information and radiological data were collected and correlated with the two banding patterns. RESULTS: : Of the 56 positive CSF samples, 46 (82%) demonstrated a delta pattern, and 10 (18%), a theta pattern. The delta pattern was associated with MS/CIS in 34 of 46 (74%) samples, compared with zero of the 10 theta samples (0%, p < 0.001). Exclusion of the theta pattern samples increased the positive predictive value of OCB testing from 61% to 74% for MS/CIS. CONCLUSION: The diagnostic significance of oligoclonal bands in CSF for MS/CIS can be improved by restricting interpretation to the delta banding pattern alone.