Abstract
Myasthenia gravis (MG) is a neurological disease caused by autoantibodies against neuromuscular-associated proteins. While MG frequently develops in thymoma patients, the etiologic factors ...for MG are not well understood. Here, by constructing a comprehensive atlas of thymoma using bulk and single-cell RNA-sequencing, we identify ectopic expression of neuromuscular molecules in MG-type thymoma. These molecules are found within a distinct subpopulation of medullary thymic epithelial cells (mTECs), which we name neuromuscular mTECs (nmTECs). MG-thymoma also exhibits microenvironments dedicated to autoantibody production, including ectopic germinal center formation, T follicular helper cell accumulation, and type 2 conventional dendritic cell migration. Cell–cell interaction analysis also predicts the interaction between nmTECs and T/B cells via
CXCL12
-
CXCR4
. The enrichment of nmTECs presenting neuromuscular molecules within MG-thymoma is further confirmed immunohistochemically and by cellular composition estimation from the MG-thymoma transcriptome. Altogether, this study suggests that nmTECs have a significant function in MG pathogenesis via ectopic expression of neuromuscular molecules.
The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-lineage stability. Here, we present a protocol using bisulfite sequencing to assess ...Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed by bisulfite conversion in unmethylated CpG DNA, bisulfite PCR and cloning, and sequencing to define the TSDR methylation. This protocol uses lymph nodes and spleen tissues and can be adapted to assess the methylation status of Tregs in other tissue types.
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•Bisulfite sequencing of Treg-specific demethylation regions (TSDR)•Cell isolation, antibody staining, and sorting of CD4+ T cells•Detailed procedures for bisulfite conversion of unmodified cytosine into uracil•Methylation status in TSDR indicates the stability of Treg cell-lineage
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
The establishment of regulatory T cells (Treg)-specific demethylation regions (TSDRs) is essential for the Treg-linage stability. Here, we present a protocol using bisulfite sequencing to assess Treg-lineage stability. The protocol describes the isolation of lymphocytes and DNA extraction, followed by bisulfite conversion in unmethylated CpG DNA, bisulfite PCR and cloning, and sequencing to define the TSDR methylation. This protocol uses lymph nodes and spleen tissues and can be adapted to assess the methylation status of Tregs in other tissue types.
The contribution of FOXP3-expressing naturally occurring regulatory T (Treg) cells to common polygenic autoimmune diseases remains ambiguous. Here, we characterized genome-wide epigenetic profiles ...(CpG methylation and histone modifications) of human Treg and conventional T (Tconv) cells in naive and activated states. We found that single-nucleotide polymorphisms (SNPs) associated with common autoimmune diseases were predominantly enriched in CpG demethylated regions (DRs) specifically present in naive Treg cells but much less enriched in activation-induced DRs common in Tconv and Treg cells. Naive Treg cell-specific DRs were largely included in Treg cell-specific super-enhancers and closely associated with transcription and other epigenetic changes in naive and effector Treg cells. Thus, naive Treg cell-specific CpG hypomethylation had a key role in controlling Treg cell-specific gene transcription and epigenetic modification. The results suggest possible contribution of altered function or development of natural Treg cells to the susceptibility to common autoimmune diseases.
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•Autoimmune SNPs are enriched in naive Treg cell-specific CpG hypomethylated regions•Treg-specific CpG hypomethylation is associated with Treg cell super-enhancers•Naive Treg cell-specific epigenetics controls activated Treg cell function
The contribution of natural Treg cells to polygenic autoimmune diseases remains unclear. Ohkura et al. characterized genome-wide epigenetic profiles of human Treg and Tconv cells and found that autoimmune disease SNPs were enriched in CpG-demethylated regions in naive Treg cells that were associated with Treg cell-specific super-enhancers, suggesting that altered Treg cell function contributes to susceptibility to common autoimmune diseases.
The transcription factor Foxp3 plays crucial roles for Treg cell development and function. Conserved non-coding sequences (CNSs) at the Foxp3 locus control Foxp3 transcription, but how they ...developmentally contribute to Treg cell lineage specification remains obscure. Here, we show that among Foxp3 CNSs, the promoter-upstream CNS0 and the intergenic CNS3, which bind distinct transcription factors, were activated at early stages of thymocyte differentiation prior to Foxp3 promoter activation, with sequential genomic looping bridging these regions and the promoter. While deletion of either CNS0 or CNS3 partially compromised thymic Treg cell generation, deletion of both completely abrogated the generation and impaired the stability of Foxp3 expression in residual Treg cells. As a result, CNS0 and CNS3 double-deleted mice succumbed to lethal systemic autoimmunity and inflammation. Thus, hierarchical and coordinated activation of Foxp3 CNS0 and CNS3 initiates and stabilizes Foxp3 gene expression, thereby crucially controlling Treg cell development, maintenance, and consequently immunological self-tolerance.
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•Enhancers activated at Foxp3-CNS0 and -CNS3 are indispensable for thymic Treg generation•CNS0 and CNS3 interact with other enhancers and Foxp3 promoter during Treg development•Deletion of both CNS0 and CNS3 causes lethal autoimmunity•CNS0 is responsible for IL-2-dependent induction and maintenance of Foxp3 expression.
The Foxp3+ Treg generation process in the thymus has not been fully understood, especially in the perspective of genomic enhancer coordination. Kawakami et al. reveal that two independently activated enhancers, Foxp3-CNS0 and Foxp3-CNS3, cooperatively induce and maintain Foxp3 expression for the establishment of self-tolerance.
Foxp3-expressing CD25
CD4
regulatory T cells (Tregs) are abundant in tumor tissues. Here, hypothesizing that tumor Tregs would clonally expand after they are activated by tumor-associated antigens to ...suppress antitumor immune responses, we performed single-cell analysis on tumor Tregs to characterize them by T cell receptor clonotype and gene-expression profiles. We found that multiclonal Tregs present in tumor tissues predominantly expressed the chemokine receptor CCR8. In mice and humans, CCR8
Tregs constituted 30 to 80% of tumor Tregs in various cancers and less than 10% of Tregs in other tissues, whereas most tumor-infiltrating conventional T cells (Tconvs) were CCR8
CCR8
tumor Tregs were highly differentiated and functionally stable. Administration of cell-depleting anti-CCR8 monoclonal antibodies (mAbs) indeed selectively eliminated multiclonal tumor Tregs, leading to cure of established tumors in mice. The treatment resulted in the expansion of CD8
effector Tconvs, including tumor antigen-specific ones, that were more activated and less exhausted than those induced by PD-1 immune checkpoint blockade. Anti-CCR8 mAb treatment also evoked strong secondary immune responses against the same tumor cell line inoculated several months after tumor eradication, indicating that elimination of tumor-reactive multiclonal Tregs was sufficient to induce memory-type tumor-specific effector Tconvs. Despite induction of such potent tumor immunity, anti-CCR8 mAb treatment elicited minimal autoimmunity in mice, contrasting with systemic Treg depletion, which eradicated tumors but induced severe autoimmune disease. Thus, specific removal of clonally expanding Tregs in tumor tissues for a limited period by cell-depleting anti-CCR8 mAb treatment can generate potent tumor immunity with long-lasting memory and without deleterious autoimmunity.
CD4+ T cells are key mediators of various autoimmune diseases; however, their role in disease progression remains unclear due to cellular heterogeneity. Here, we evaluated CD4+ T cell subpopulations ...using decomposition-based transcriptome characterization and canonical clustering strategies. This approach identified 12 independent gene programs governing whole CD4+ T cell heterogeneity, which can explain the ambiguity of canonical clustering. In addition, we performed a meta-analysis using public single-cell datasets of over 1.8 million peripheral CD4+ T cells from 953 individuals by projecting cells onto the reference and cataloging cell frequency and qualitative alterations of the populations in 20 diseases. The analyses revealed that the 12 transcriptional programs were useful in characterizing each autoimmune disease and predicting its clinical status. Moreover, genetic variants associated with autoimmune diseases showed disease-specific enrichment within the 12 gene programs. The results collectively provide a landscape of single-cell transcriptomes of CD4+ T cell subpopulations involved in autoimmune disease.
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•Identified 18 cell types and 12 gene programs from blood CD4+ T cells•Meta-analysis enrolling 1.8M cells outlines CD4+ T alterations in autoimmune diseases•Observed disease-specific heritability enrichment within CD4+ T cell gene programs
Yasumizu, Takeuchi, Morimoto, et al. compile a comprehensive reference of circulating CD4+ T cells, encompassing 12 gene programs and 18 clusters. They reveal disease-specific cellular characteristics through the integration of genome-wide association studies and single-cell meta-analysis. This work provides insights for precision medicine.
DNA methylation is a pivotal epigenetic modification that defines cellular identity. While cell deconvolution utilizing this information is considered useful for clinical practice, current methods ...for deconvolution are limited in their accuracy and resolution. In this study, we collected DNA methylation data from 945 human samples derived from various tissues and tumor-infiltrating immune cells and trained a neural network model with them. The model, termed MEnet, predicted abundance of cell population together with the detailed immune cell status from bulk DNA methylation data, and showed consistency to those of flow cytometry and histochemistry. MEnet was superior to the existing methods in the accuracy, speed, and detectable cell diversity, and could be applicable for peripheral blood, tumors, cell-free DNA, and formalin-fixed paraffin-embedded sections. Furthermore, by applying MEnet to 72 intrahepatic cholangiocarcinoma samples, we identified immune cell profiles associated with cancer prognosis. We believe that cell deconvolution by MEnet has the potential for use in clinical settings.
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