Machine learning analysis of the T cell receptor repertoire identifies sequence features of self-reactivity.

The T cell receptor (TCR) determines specificity and affinity for both foreign and self-peptides presented by the major histocompatibility complex (MHC). Although the strength of TCR interactions with self-pMHC impacts T cell function, it has been challenging to identify TCR sequence features that predict T cell fate. To discern patterns distinguishing TCRs from naive CD4+ T cells with low versus high self-reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that identifies population-level differences between TCRß sequence sets. This approach revealed that weakly self-reactive T cell populations were enriched for longer CDR3ß regions and acidic amino acids. We tested our ML predictions of self-reactivity using retrogenic mice with fixed TCRß sequences. Extrapolating our analyses to independent datasets, we predicted high self-reactivity for regulatory T cells and slightly reduced self-reactivity for T cells responding to chronic infections. Our analyses suggest a potential trade-off between TCR repertoire diversity and self-reactivity. A record of this paper's transparent peer review process is included in the supplemental information.

The landscape of the immunoglobulin repertoire in endemic pemphigus foliaceus.

Introduction: Primarily driven by autoreactive B cells, pemphigus foliaceus (PF) is an uncommon autoimmune blistering skin disease of sporadic occurrence worldwide. However, PF reaches a prevalence of 3% in the endemic areas of Brazil, the highest ever registered for any autoimmune disease, which indicates environmental factors influencing the immune response in susceptible individuals. We aimed to provide insights into the immune repertoire of patients with PF living in the endemic region of the disease, compared to healthy individuals from the endemic region and a non-endemic area. Methods: We characterized the B-cell repertoire in i) nontreated patients (n=5); ii) patients under immunosuppressive treatment (n=5); iii) patients in remission without treatment (n=6); and two control groups iv) from the endemic (n=6) and v) non-endemic areas in Brazil (n=4). We used total RNA extracted from peripheral blood mononuclear cells and performed a comprehensive characterization of the variable region of immunoglobulin heavy chain (IGH) in IgG and IgM using next-generation sequencing. Results: Compared to individuals from a different area, we observed remarkably lower clonotype diversity in the B-cell immune repertoire of patients and controls from the endemic area (p < 0.02), suggesting that the immune repertoire in the endemic area is under geographically specific and intense environmental pressure. Moreover, we observed longer CDR3 sequences in patients, and we identified differential disease-specific usage of IGHV segments, including increased IGHV3-30 and decreased IGHV3-23 in patients with active disease (p < 0.04). Finally, our robust network analysis discovered clusters of CDR3 sequences uniquely observed in patients with PF. Discussion: Our results indicate that environmental factors, in addition to disease state, impact the characteristics of the repertoire. Our findings can be applied to further investigation of the environmental factors that trigger pemphigus and expand the knowledge for identifying new targeted and more effective therapies.

Molecularly Stratified Treatment Options in Primary Refractory DLBCL/HGBL with MYC and BCL2 or BCL6 Rearrangements (HGBL, NOS with MYC/BCL6).

BACKGROUND: There is growing evidence supporting multidisciplinary molecular tumor boards (MTB) in solid tumors whereas hematologic malignancies remain underrepresented in this regard. OBJECTIVE: The present study aimed to assess the clinical relevance of MTBs in primary refractory diffuse large B-cell lymphomas/high-grade B-cell lymphomas with MYC and BCL2 rearrangements (prDLBCL/HGBL-MYC/BCL2) (n = 13) and HGBL, not otherwise specified (NOS), with MYC and BCL6 rearrangements (prHGBL, NOS-MYC/BCL6) (n = 6) based on our previously published whole-exome sequencing (WES) cohort. PATIENTS AND METHODS: For genomic analysis, the institutional MTB WES pipeline (University Cancer Center Schleswig-Holstein: UCCSH), certified for routine clinical diagnostics, was employed and supplemented by a comprehensive immunohistochemical work-up. Consecutive database research and annotation according to established evidence levels for molecularly stratified therapies was performed (NCT-DKTK/ESCAT). RESULTS: Molecularly tailored treatment options with NCT-DKTK evidence level of at least m2A were identified in each case. We classified mutations in accordance with biomarker/treatment baskets and detected a heterogeneous spectrum of targetable alterations affecting immune evasion (IE; n = 30), B-cell targets (BCT; n = 26), DNA damage repair (DDR; n = 20), tyrosine kinases (TK; n = 13), cell cycle (CC; n = 7), PI3K-MTOR-AKT pathway (PAM; n = 2), RAF-MEK-ERK cascade (RME; n = 1), and others (OTH; n = 11). CONCLUSION: Our virtual MTB approach identified potential molecularly targeted treatment options alongside targetable genomic signatures for both prDLBCL/HGBL-MYC/BCL2 and prHGBL, NOS-MYC/BCL6. These results underline the potential of MTB consultations in difficult-to-treat lymphomas early in the treatment sequence.

Case report: Schnitzler-like syndrome without monoclonal gammopathy.

Schnitzler syndrome is a rare autoinflammatory disorder characterized by urticarial rash, joint pain, recurrent fever, leucocytosis, elevated C-reactive protein (CRP) and serum amyloid A (SAA), and monoclonal IgM or IgG gammopathy. According to the Strasbourg criteria, both urticarial rash and gammopathy are mandatorily required for the diagnosis of Schnitzler's syndrome. However, incomplete variants lacking either skin symptoms or monoclonal gammopathy have also been described. Here, we report a case in which the diagnosis of Schnitzler-like syndrome was made despite the absence of gammopathy, based on neutrophilic dermal inflammation, episodic and excessive increase in inflammatory parameters, and prompt response to anakinra, a soluble IL1 receptor antagonist (sIL-1RA). In addition, we detected neutrophil epitheliotropism, which is highly suggestive of autoinflammatory disease. Using whole-exome sequencing, we were unable to find a causative pathogenic mutation but did find several mutations possibly related to the inflammatory processes in this patient. This and other cases highlight that the existing Strasbourg criteria are too strict to capture Schnitzler-like syndromes that may respond well and rapidly to IL1 inhibition. Recurrent episodes of disease with normalization of inflammatory symptoms in the interval, rapid response to anakinra, and neutrophilic epitheliotropism in a lesional skin biopsy may help confirm the diagnosis of Schnitzler-like syndrome.

Primary refractory plasmablastic lymphoma: A precision oncology approach.

Introduction: Hematologic malignancies are currently underrepresented in multidisciplinary molecular-tumor-boards (MTB). This study assesses the potential of precision-oncology in primary-refractory plasmablastic-lymphoma (prPBL), a highly lethal blood cancer. Methods: We evaluated clinicopathological and molecular-genetic data of 14 clinically annotated prPBL-patients from initial diagnosis. For this proof-of-concept study, we employed our certified institutional MTB-pipeline (University-Cancer-Center-Schleswig-Holstein, UCCSH) to annotate a comprehensive dataset within the scope of a virtual MTB-setting, ultimately recommending molecularly stratified therapies. Evidence-levels for MTB-recommendations were defined in accordance with the NCT/DKTK and ESCAT criteria. Results: Median age in the cohort was 76.5 years (range 56-91), 78.6% of patients were male, 50% were HIV-positive and clinical outcome was dismal. Comprehensive genomic/transcriptomic analysis revealed potential recommendations of a molecularly stratified treatment option with evidence-levels according to NCT/DKTK of at least m2B/ESCAT of at least IIIA were detected for all 14 prPBL-cases. In addition, immunohistochemical-assessment (CD19/CD30/CD38/CD79B) revealed targeted treatment-recommendations in all 14 cases. Genetic alterations were classified by treatment-baskets proposed by Horak et al. Hereby, we identified tyrosine-kinases (TK; n=4), PI3K-MTOR-AKT-pathway (PAM; n=3), cell-cycle-alterations (CC; n=2), RAF-MEK-ERK-cascade (RME; n=2), immune-evasion (IE; n=2), B-cell-targets (BCT; n=25) and others (OTH; n=4) for targeted treatment-recommendations. The minimum requirement for consideration of a drug within the scope of the study was FDA-fast-track development. Discussion: The presented proof-of-concept study demonstrates the clinical potential of precision-oncology, even in prPBL-patients. Due to the aggressive course of the disease, there is an urgent medical-need for personalized treatment approaches, and this population should be considered for MTB inclusion at the earliest time.

North and East African mitochondrial genetic variation needs further characterization towards precision medicine.

INTRODUCTION: Mitochondria are maternally inherited cell organelles with their own genome, and perform various functions in eukaryotic cells such as energy production and cellular homeostasis. Due to their inheritance and manifold biological roles in health and disease, mitochondrial genetics serves a dual purpose of tracing the history as well as disease susceptibility of human populations across the globe. This work requires a comprehensive catalogue of commonly observed genetic variations in the mitochondrial DNAs for all regions throughout the world. So far, however, certain regions, such as North and East Africa have been understudied. OBJECTIVES: To address this shortcoming, we have created the most comprehensive quality-controlled North and East African mitochondrial data set to date and use it for characterizing mitochondrial genetic variation in this region. METHODS: We compiled 11 published cohorts with novel data for mitochondrial genomes from 159 Sudanese individuals. We combined these 641 mitochondrial sequences with sequences from the 1000 Genomes (n = 2504) and the Human Genome Diversity Project (n = 828) and used the tool haplocheck for extensive quality control and detection of in-sample contamination, as well as Nanopore long read sequencing for haplogroup validation of 18 samples. RESULTS: Using a subset of high-coverage mitochondrial sequences, we predict 15 potentially novel haplogroups in North and East African subjects and observe likely phylogenetic deviations from the established PhyloTree reference for haplogroups L0a1 and L2a1. CONCLUSION: Our findings demonstrate common hitherto unexplored variants in mitochondrial genomes of North and East Africa that lead to novel phylogenetic relationships between haplogroups present in these regions. These observations call for further in-depth population genetic studies in that region to enable the prospective use of mitochondrial genetic variation for precision medicine.

Analysis of T cell repertoires of CD45RO CD4 T cells in cohorts of patients with bullous pemphigoid: A pilot study.

Autoimmune diseases develop over years - starting from a subclinical phenotype to clinically manifest autoimmune disease. The factors that drive this transition are ill-defined. To predict the turning point towards clinical disease and to intervene in the progress of autoimmune-mediated dysfunction, the establishment of new biomarkers is needed. Especially CD4 T cells are crucially involved in autoimmunity: first, during the initiation phase, because they lose their tolerance towards self-peptides, and second, by the subsequent ongoing presentation of self-peptides during the active autoimmune disease. Accordingly, changes in the degree of diversity of T cell receptor (TCR) repertoires in autoimmunity have been reported. These findings led to the hypothesis that transition from pre-disease to autoimmune disease is associated with an increase of abnormally expanded T cell clones that occupy large portions of the TCR repertoire. In this pilot study, we asked whether the ratio and the diversity of the TCR repertoires of circulating memory (CD45RO) and naïve (CD45RA) CD4 T cells could serve as a predictive factor for the development of autoimmunity. To find out, we analyzed the TCRß repertoires of memory and naïve CD4 T cells in a small cohort of four gender- and age-matched elderly patients having the autoimmune blistering disease bullous pemphigoid or non-melanoma skin cancers. We found that the extent of clonal expansions in the TCRß repertoires from the circulating memory and naïve CD4 populations did not differ between the patient groups. This result shows that the diversity of TCR repertoires from peripheral CD4 T cells does not reflect the manifestation of the skin-associated autoimmune disease BP and does not qualify as a prognostic factor. We propose that longitudinal TCR repertoire analysis of younger patients might be more informative.

B-cell receptor physical properties affect relative IgG1 and IgE responses in mouse egg allergy.

Mutated and unmutated IgE and IgG play different and partly opposing roles in allergy development, but the mechanisms controlling their relative production are incompletely understood. Here, we analyzed the IgE-response in murine food allergy. Deep sequencing of the complementary-determining region (CDR) repertoires indicated that an ongoing unmutated extrafollicular IgE response coexists with a germinal center response, even after long-lasting allergen challenges. Despite overall IgG1-dominance, a significant proportion of clonotypes contained several-fold more IgE than IgG1. Clonotypes with differential bias to either IgE or IgG1 showed distinct hypermutation and clonal expansion. Hypermutation rates were associated with different physiochemical binding properties of individual B-cell receptors (BCR). Increasing BCR signaling strength inhibited class switching from IgG1 to IgE in vitro, preferentially constraining IgE formation. These data indicate that antigen-binding properties of individual BCRs determine differential IgE hypermutation and IgE versus IgG1 production on the level of single B-cell clones.

Longitudinal Characterization of the Fungal Skin Microbiota in Healthy Subjects Over a Period of 1 Year.

Beneficial microorganisms on the skin contribute to the first line of defense against attacking pathogens. However, instability of the skin microbiota is associated with skin diseases. Hence, temporal analyses are crucial because they serve as a baseline to understand the development of dysbiosis in disease. In this study, we aim to improve the understanding of the fungal skin microbiota, the mycobiota, in healthy subjects. Skin swabs were taken monthly for a year from four different skin sites, that is, antecubital crease, dorsal neck, glabella, and vertex, and analyzed by DNA sequencing of the internal transcribed spacer 1 region. The mycobiota on the skin was dominated by the class Malasseziomycetes, and the core community was composed of Malassezia restricta, M. globosa, and M. sympodialis at all skin sites. Over the period of 1 year, the intrapersonal mycobiota remained largely stable, with some fluctuations of low abundant non-Malassezia fungi. We conclude that despite fluctuations of low abundant classes, fungal skin communities form a temporally robust and individual fingerprint in healthy subjects.

The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.

Coronavirus disease 2019 (COVID-19) can damage cerebral small vessels and cause neurological symptoms. Here we describe structural changes in cerebral small vessels of patients with COVID-19 and elucidate potential mechanisms underlying the vascular pathology. In brains of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected individuals and animal models, we found an increased number of empty basement membrane tubes, so-called string vessels representing remnants of lost capillaries. We obtained evidence that brain endothelial cells are infected and that the main protease of SARS-CoV-2 (Mpro) cleaves NEMO, the essential modulator of nuclear factor-κB. By ablating NEMO, Mpro induces the death of human brain endothelial cells and the occurrence of string vessels in mice. Deletion of receptor-interacting protein kinase (RIPK) 3, a mediator of regulated cell death, blocks the vessel rarefaction and disruption of the blood-brain barrier due to NEMO ablation. Importantly, a pharmacological inhibitor of RIPK signaling prevented the Mpro-induced microvascular pathology. Our data suggest RIPK as a potential therapeutic target to treat the neuropathology of COVID-19.

PD-L1 Dependent Immunogenic Landscape in Hot Lung Adenocarcinomas Identified by Transcriptome Analysis.

BACKGROUND: Lung cancer is the most frequent cause of cancer-related deaths worldwide. The clinical development of immune checkpoint blockade has dramatically changed the treatment paradigm for patients with lung cancer. Yet, an improved understanding of PD-1/PD-L1 checkpoint blockade-responsive biology is warranted. METHODS: We aimed to identify the landscape of immune cell infiltration in primary lung adenocarcinoma (LUAD) in the context of tumoral PD-L1 expression and the extent of immune infiltration ("hot" vs. "cold" phenotype). The study comprises LUAD cases (n = 138) with "hot" (≥150 lymphocytes/HPF) and "cold" (<150 lymphocytes/HPF) tumor immune phenotype and positive (>50%) and negative (<1%) tumor PD-L1 expression, respectively. Tumor samples were immunohistochemically analyzed for expression of PD-L1, CD4, and CD8, and further investigated by transcriptome analysis. RESULTS: Gene set enrichment analysis defined complement, IL-JAK-STAT signaling, KRAS signaling, inflammatory response, TNF-alpha signaling, interferon-gamma response, interferon-alpha response, and allograft rejection as significantly upregulated pathways in the PD-L1-positive hot subgroup. Additionally, we demonstrated that STAT1 is upregulated in the PD-L1-positive hot subgroup and KIT in the PD-L1-negative hot subgroup. CONCLUSION: The presented study illustrates novel aspects of PD-L1 regulation, with potential biological relevance, as well as relevance for immunotherapy response stratification.

Receptor repertoires of murine follicular T helper cells reveal a high clonal overlap in separate lymph nodes in autoimmunity.

Follicular T helper cells (Tfh) are a specialized subset of CD4 effector T cells that are crucial for germinal center (GC) reactions and for selecting B cells to undergo affinity maturation. Despite this central role for humoral immunity, only few data exist about their clonal distribution when multiple lymphoid organs are exposed to the same antigen (Ag) as it is the case in autoimmunity. Here, we used an autoantibody-mediated disease model of the skin and injected one auto-Ag into the two footpads of the same mouse and analyzed the T cell receptor (TCR)ß sequences of Tfh located in GCs of both contralateral draining lymph nodes. We found that over 90% of the dominant GC-Tfh clonotypes were shared in both lymph nodes but only transiently. The initially dominant Tfh clonotypes especially declined after establishment of chronic disease while GC reaction and autoimmune disease continued. Our data demonstrates a dynamic behavior of Tfh clonotypes under autoimmune conditions and emphasizes the importance of the time point for distinguishing auto-Ag-specific Tfh clonotypes from potential bystander activated ones.

Increased Fibrosis in a Mouse Model of Anti-Laminin 332 Mucous Membrane Pemphigoid Remains Unaltered by Inhibition of Aldehyde Dehydrogenase.

Mucous membrane pemphigoid (MMP) is an autoimmune blistering disease characterized by autoantibodies against the basal membrane zone of skin and surface-close epithelia and predominant mucosal lesions. The oral cavity and conjunctivae are most frequently affected, albeit clinical manifestations can also occur on the skin. MMP-associated lesions outside the oral cavity typically lead to scarring. Mechanisms underlying scarring are largely unknown in MMP and effective treatment options are limited. Herein, we assessed the collagen architecture in tissue samples of an antibody-transfer mouse model of anti-laminin-332 MMP. In MMP mice, increased collagen fibril density was observed in skin and conjunctival lesions compared to mice injected with normal rabbit IgG. The extracellular matrix of MMP skin samples also showed altered post-translational collagen cross-linking with increased levels of both lysine- and hydroxylysine-derived collagen crosslinks supporting the fibrotic phenotype in experimental MMP compared to control animals. In addition, we evaluated a potential anti-fibrotic therapy in experimental anti-laminin-332 MMP using disulfiram, an inhibitor of the aldehyde dehydrogenase (ALDH), which has been implicated in immune-mediated mucosal scarring. In addition, disulfiram also acts as a copper chelator that was shown to block lysyl oxidase activity, an enzyme involved in formation of collagen crosslinks. Topical use of disulfiram (300 µM in 2% [w/v] methocel) did not improve ocular lesions in experimental MMP over the 12-day treatment period in disulfiram-treated mice compared to vehicle-treated mice (n=8/group). Furthermore, C57BL6/J mice (n=8/group) were treated prophylactically with 200 mg/kg p.o. disulfiram or the solvent once daily over a period of 12 days. Systemic treatment did not show any reduction in the severity of oral and ocular lesions in MMP mice, albeit some improvement in skin lesions was observed in disulfiram- vs. vehicle-treated mice (p=0.052). No reduction in fibrosis was seen, as assessed by immunohistochemistry. Whilst blocking of ALDH failed to significantly ameliorate disease activity, our data provide new insight into fibrotic processes highlighting changes in the collagenous matrix and cross-linking patterns in IgG-mediated MMP.

An integrated personal and population-based Egyptian genome reference.

A small number of de novo assembled human genomes have been reported to date, and few have been complemented with population-based genetic variation, which is particularly important for North Africa, a region underrepresented in current genome-wide references. Here, we combine long- and short-read whole-genome sequencing data with recent assembly approaches into a de novo assembly of an Egyptian genome. The assembly demonstrates well-balanced quality metrics and is complemented with variant phasing via linked reads into haploblocks, which we associate with gene expression changes in blood. To construct an Egyptian genome reference, we identify genome-wide genetic variation within a cohort of 110 Egyptian individuals. We show that differences in allele frequencies and linkage disequilibrium between Egyptians and Europeans may compromise the transferability of European ancestry-based genetic disease risk and polygenic scores, substantiating the need for multi-ethnic genome references. Thus, the Egyptian genome reference will be a valuable resource for precision medicine.

Polymorphisms in the Mitochondrial Genome Are Associated With Bullous Pemphigoid in Germans.

Bullous pemphigoid (BP) is the most prevalent autoimmune skin blistering disease and is characterized by the generation of autoantibodies against the hemidesmosomal proteins BP180 (type XVII collagen) and BP230. Most intriguingly, BP is distinct from other autoimmune diseases because it predominantly affects elderly individuals above the age of 75 years, raising the question why autoantibodies and the clinical lesions of BP emerges mostly in this later stage of life, even in individuals harboring known putative BP-associated germline gene variants. The mitochondrial genome (mtDNA) is a potential candidate to provide additional insights into the BP etiology; however, the mtDNA has not been extensively explored to date. Therefore, we sequenced the whole mtDNA of German BP patients (n = 180) and age- and sex-matched healthy controls (n = 188) using next generation sequencing (NGS) technology, followed by the replication study using Sanger sequencing of an additional independent BP (n = 89) and control cohort (n = 104). While the BP and control groups showed comparable mitochondrial haplogroup distributions, the haplogroup T exhibited a tendency of higher frequency in BP patients suffering from neurodegenerative diseases (ND) compared to BP patients without ND (50%; 3 in 6 BP with haplogroup T). A total of four single nucleotide polymorphisms (SNPs) in the mtDNA, namely, m.16263T>C, m.16051A>G, and m.16162A>G in the D-loop region of the mtDNA, and m.11914G>A in the mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4 gene (MT-ND4), were found to be significantly associated with BP based on the meta-analysis of our NGS data and the Sanger sequencing data (p = 0.0017, p = 0.0129, p = 0.0076, and p = 0.0132, respectively, Peto's test). More specifically, the three SNPs in the D-loop region were negatively, and the SNP in the MT-ND4 gene was positively associated with BP. Our study is the first to interrogate the whole mtDNA in BP patients and controls and to implicate multiple novel mtDNA variants in disease susceptibility. Studies using larger cohorts and more diverse populations are warranted to explore the functional consequences of the mtDNA variants identified in this study on immune and skin cells to understand their contributions to BP pathology.

Genetics and Omics Analysis of Autoimmune Skin Blistering Diseases.

Autoimmune blistering diseases (AIBDs) of the skin are characterized by autoantibodies against different intra-/extracellular structures within the epidermis and at the basement membrane zone (BMZ). Binding of the antibodies to their target antigen leads to inflammation at the respective binding site and degradation of these structures, resulting in the separation of the affected skin layers. Clinically, blistering, erythema and lesions of the skin and/or mucous membranes can be observed. Based on the localization of the autoantigen, AIBDs can be divided into pemphigus (intra-epidermal blistering diseases) and pemphigoid diseases (sub-epidermal blistering diseases), respectively. Although autoantigens have been extensively characterized, the underlying causes that trigger the diseases are still poorly understood. Besides the environment, genetic factors seem to play an important role in a predisposition to AIBDs. Here, we review currently known genetic and immunological mechanisms that contribute to the pathogenesis of AIBDs. Among the most commonly encountered genetic predispositions for AIBDs are the HLA gene region, and deleterious mutations of key genes for the immune system. Particularly, HLA class II genes such as the HLA-DR and HLA-DQ alleles have been shown to be prevalent in patients. This has prompted further epidemiological studies as well as unbiased Omics approaches on the transcriptome, microbiome, and proteome level to elucidate common and individual genetic risk factors as well as the molecular pathways that lead to the pathogenesis of AIBDs.

CD154 Costimulation Shifts the Local T-Cell Receptor Repertoire Not Only During Thymic Selection but Also During Peripheral T-Dependent Humoral Immune Responses.

CD154 is a transmembrane cytokine expressed transiently on activated CD4 T cells upon T-cell receptor (TCR) stimulation that interacts with CD40 on antigen-presenting cells. The signaling via CD154:CD40 is essential for B-cell maturation and germinal center formation and also for the final differentiation of CD4 T cells during T-dependent humoral immune responses. Recent data demonstrate that CD154 is critically involved in the selection of T-cell clones during the negative selection process in the thymus. Whether CD154 signaling influences the TCR repertoire during peripheral T-dependent humoral immune responses has not yet been elucidated. To find out, we used CD154-deficient mice and assessed the global TCRß repertoire in T-cell zones (TCZ) of spleens by high-throughput sequencing after induction of a Th2 response to the multiepitopic antigen sheep red blood cells. Qualitative and quantitative comparison of the splenic TCZ-specific TCRß repertoires revealed that CD154 deficiency shifts the distribution of Vß-Jß genes after antigen exposure. This data led to the conclusion that costimulation via CD154:CD40 during the interaction of T cells with CD40-matured B cells contributes to the recruitment of T-cell clones into the immune response and thereby shapes the peripheral TCR repertoire.

Deep Sequencing Reveals Transient Segregation of T Cell Repertoires in Splenic T Cell Zones during an Immune Response.

Immunological differences between hosts, such as diverse TCR repertoires, are widely credited for reducing the risk of pathogen spread and adaptation in a population. Within-host immunological diversity might likewise be important for robust pathogen control, but to what extent naive TCR repertoires differ across different locations in the same host is unclear. T cell zones (TCZs) in secondary lymphoid organs provide secluded microenvironmental niches. By harboring distinct TCRs, such niches could enhance within-host immunological diversity. In contrast, rapid T cell migration is expected to dilute such diversity. In this study, we combined tissue microdissection and deep sequencing of the TCR ß-chain to examine the extent to which TCR repertoires differ between TCZs in murine spleens. In the absence of Ag, we found little evidence for differences between TCZs of the same spleen. Yet, 3 d after immunization with sheep RBCs, we observed a >10-fold rise in the number of clones that appeared to localize to individual zones. Remarkably, these differences largely disappeared at 4 d after immunization, when hallmarks of an ongoing immune response were still observed. These data suggest that in the absence of Ag, any repertoire differences observed between TCZs of the same host can largely be attributed to random clone distribution. Upon Ag challenge, TCR repertoires in TCZs first segregate and then homogenize within days. Such "transient mosaic" dynamics could be an important barrier for pathogen adaptation and spread during an immune response.

Low-level mitochondrial heteroplasmy modulates DNA replication, glucose metabolism and lifespan in mice.

Mutations in mitochondrial DNA (mtDNA) lead to heteroplasmy, i.e., the intracellular coexistence of wild-type and mutant mtDNA strands, which impact a wide spectrum of diseases but also physiological processes, including endurance exercise performance in athletes. However, the phenotypic consequences of limited levels of naturally arising heteroplasmy have not been experimentally studied to date. We hence generated a conplastic mouse strain carrying the mitochondrial genome of an AKR/J mouse strain (B6-mtAKR) in a C57BL/6 J nuclear genomic background, leading to >20% heteroplasmy in the origin of light-strand DNA replication (OriL). These conplastic mice demonstrate a shorter lifespan as well as dysregulation of multiple metabolic pathways, culminating in impaired glucose metabolism, compared to that of wild-type C57BL/6 J mice carrying lower levels of heteroplasmy. Our results indicate that physiologically relevant differences in mtDNA heteroplasmy levels at a single, functionally important site impair the metabolic health and lifespan in mice.

ClonoCalc and ClonoPlot: immune repertoire analysis from raw files to publication figures with graphical user interface.

BACKGROUND: Next generation sequencing (NGS) technologies enable studies and analyses of the diversity of both T and B cell receptors (TCR and BCR) in human and animal systems to elucidate immune functions in health and disease. Over the last few years, several algorithms and tools have been developed to support respective analyses of raw sequencing data of the immune repertoire. These tools focus on distinct aspects of the data processing and require a strong bioinformatics background. To facilitate the analysis of T and B cell repertoires by less experienced users, software is needed that combines the most common tools for repertoire analysis. RESULTS: We introduce a graphical user interface (GUI) providing a complete analysis pipeline for processing raw NGS data for human and animal TCR and BCR clonotype determination and advanced differential repertoire studies. It provides two applications. ClonoCalc prepares the raw data for downstream analyses. It combines a demultiplexer for barcode splitting and employs MiXCR for paired-end read merging and the extraction of human and animal TCR/BCR sequences. ClonoPlot wraps the R package tcR and further contributes self-developed plots for the descriptive comparative investigation of immune repertoires. CONCLUSION: This workflow reduces the amount of programming required to perform the respective analyses and supports both communication and training between scientists and technicians, and across scientific disciplines. The Open Source development in Java and R is modular and invites advanced users to extend its functionality. Software and documentation are freely available at https://bitbucket.org/ClonoSuite/clonocalc-plot .