Single-cell Multi-omics Defines Tolerogenic Extrathymic Aire-Expressing Populations with Unique Homology to Thymic Epithelium
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ABSTRACT: Adaptive immunity requires multiple complementary mechanisms to maintain self-tolerance. Dedicated tolerogenic lymphoid populations and their key transcriptional regulators have been well characterized, but while recent evidence suggests some myeloid populations, particularly migratory dendritic cells (migDCs), may have similar functions, the identity of these populations and the transcriptional circuits regulating them remain obscure. The Autoimmune Regulator (Aire), well defined in medullary thymic epithelial cells (mTECs), is also expressed in extrathymic Aire-expressing cells (eTACs) in the secondary lymphoid organs. eTACs have been shown to have tolerogenic capabilities (ref), and recently work has shown them to be required for normal immune homeostasis in pregnancy (ref). But the precise identity and function of these cells remain unclear. Here using high-dimensional single-cell multiomics (scRNAseq/ASAPseq), we define the identity and biology of the principal extrathymic Aire-expressing populations at the transcriptional, genomic, and proteomic level, resulting in two related populations: CCR7+ Aire-regulated migratory dendritic cells (ArmDCs) and a novel population co-expressing RAR-related orphan receptor gamma-t (ROR-γt), with significant transcriptional and genomic homology to both migDCs and mTECs, which we termed Janus cells (JCs). We demonstrate these eTAC populations share a distinct transcriptional and genomic homology to medullary epithelium and, like mTECs, depend on RANK-RANK-ligand interactions to drive Aire expression. Lineage-tracing experiments suggest JCs are not a precursor population to the majority of ArmDCs. Further, self-antigen display by eTACs is sufficient to mediate negative selection of T cells that escape thymic selection. This remarkable central and peripheral homology suggests that a core Aire-associated transcriptional program may drive immune tolerance in both the thymus and periphery, and suggests a remarkable convergence of transcriptional programs in two disparate cell lineages in the thymus and periphery.
Project description:We had previously found that ablating autoimmune regulator (Aire)-expressing cells during pregnancy lead to fetal loss, intruterine growth restriction, and the specific expansion of Tfh populations. To investigate transcriptional changes in Aire+ cells, specifically in medullary thymic epithelial cells (mTECs) and extrathymic Aire-expressing cells (eTACs), we used the Aire-Driven Igrp-GFP (Adig) reporter mouse. We sorted mTECs and eTACs from virgin Adig females and E9.5 pregnant Adig dams for bulk RNA sequencing. While we found no differences in the frequency or absolute number of mTECs or eTACs, or of Aire expression in mTECs or eTACs during pregnancy, we did find a significant number of differentially expressed (DE) transcripts during pregnancy in splenic eTACs, and almost no changes in mTECs. Pathway analysis of splenic DE genes revealed enrichment for erythrocyte development and included a number of putative PAAs such as embryonic hemoglobin theta. While these transcriptional changes may represent eTAC-intrinsic expression of PAAs, pregnancy also causes increased maternal hematopoiesis as well as passage of fetal nucleated RBCs into the maternal circulation and the immunologic relevance of this phenomenon requires further study.
Project description:Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. We used sequence conservation analysis and ChIP-seq against the enhancer-associated histone mark H3K27ac to identify a candidate Aire cis-regulatory element. There is enrichment of H3K27ac near this element, ACNS1, in mTECs and the element also has characteristics of being NF-κB-responsive. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance. Two experimental groups (GFP neg mTECs and GFP pos mTECs), each with three samples, and one control sample (D10 Th2 cells).
Project description:Thymic central tolerance is essential to preventing autoimmunity. In medullary thymic epithelial cells (mTECs), the Autoimmune regulator (Aire) gene plays an essential role in this process by driving the expression of a diverse set of tissue-specific antigens (TSAs), which are presented and help tolerize self-reactive thymocytes. Interestingly, Aire has a highly tissue-restricted pattern of expression, with only mTECs and peripheral extrathymic Aire-expressing cells (eTACs) known to express detectable levels in adults. Despite this high level of tissue specificity, the cis-regulatory elements that control Aire expression have remained obscure. We used sequence conservation analysis and ChIP-seq against the enhancer-associated histone mark H3K27ac to identify a candidate Aire cis-regulatory element. There is enrichment of H3K27ac near this element, ACNS1, in mTECs and the element also has characteristics of being NF-κB-responsive. Finally, we find that this element is essential for Aire expression in vivo and necessary to prevent spontaneous autoimmunity, reflecting the importance of this regulatory DNA element in promoting immune tolerance.
Project description:The prevention of autoimmunity requires elimination of self-reactive T cells during their development and maturation. Expression of diverse self-antigens by stromal cells in the thymus is essential to this process, and depends, in part, on the activity of the Autoimmune Regulator (Aire) gene. Here we report the identification of extrathymic Aire-expressing cells (eTACs) resident within the secondary lymphoid organs. These stromally-derived eTACs express a diverse array of unique self-antigens and are capable of interacting with and deleting naive autoreactive T cells. Using two-photon microscopy we observe stable, antigen-specific interactions between eTACs and autoreactive T cells. We propose that such a secondary network of self-antigen-expressing stromal cells may help reinforce immune tolerance by preventing the maturation of autoreactive T cells that escape thymic negative selection. A genetic modification design type is where an organism(s) has had genetic material removed, rearranged, mutagenized or added, such as knock out. Keywords: genetic_modification_design
Project description:Aire is a transcriptional regulator that induces promiscuous expression of thousands of tissue-restricted antigen (TRA) genes in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs regulating its own expression remain elusive. We used Affymetrix microarrays to analyze the gene expression patterns of Aire expressing cells (mature mTECs and Thymic B cells) and compared them to control counterparts, namely immature mTECs, cortical Thymic epithelial cells and splenic B cells of tissue-restricted antigen (TRA) genes in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs regulating its own expression remain elusive. We’ve used Assay for transposase-accessible chromatin using sequencing (ATAC-Seq) on the different thymic epithelial cell populations to assess chromatin accessibility around the Aire locus in these cells. Moreover, we’ve used the indexing-first chromatin immunoprecipitation (iChIP) technique to assess the occupancy of the Irf8 transcription factor in the Aire locus
Project description:Aire is a transcriptional regulator that induces promiscuous expression of thousands of tissue-restricted antigen (TRA) genes in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs regulating its own expression remain elusive. We used Affymetrix microarrays to analyze the gene expression patterns of Aire expressing cells (mature mTECs and Thymic B cells) and compared them to control counterparts, namely immature mTECs, cortical Thymic epithelial cells and splenic B cells of tissue-restricted antigen (TRA) genes in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs regulating its own expression remain elusive. We’ve used Assay for transposase-accessible chromatin using sequencing (ATAC-Seq) on the different thymic epithelial cell populations to assess chromatin accessibility around the Aire locus in these cells. Moreover, we’ve used the indexing-first chromatin immunoprecipitation (iChIP) technique to assess the occupancy of the Irf8 transcription factor in the Aire locus
Project description:Tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing AutoImmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. A second wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens within the first few weeks of life, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new lineage of tolerogenic RORγt+ antigen-presenting cells (APC) with a hybrid dendritic cell (DC)-mTEC phenotype, dubbed Thetis cells (TCs), comprising 4 major sub-groups (TC I-IV), We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While Aire+ TC I and III bore remarkable homology with Aire+ mTECs, including expression of tissue restricted self-antigens, TC IV lacked Aire expression and were enriched for molecules required for pTreg generation, including the TGF-β activating integrin αvβ8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical DCs was neither sufficient nor required for pTreg generation, further implicating TCs as the critical tolerogenic RORγt+ APC. Our studies reveal parallel pathways for establishment of tolerance to self and foreign antigen within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs.
Project description:Tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing AutoImmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. A second wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens within the first few weeks of life, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new lineage of tolerogenic RORγt+ antigen-presenting cells (APC) with a hybrid dendritic cell (DC)-mTEC phenotype, dubbed Thetis cells (TCs), comprising 4 major sub-groups (TC I-IV), We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While Aire+ TC I and III bore remarkable homology with Aire+ mTECs, including expression of tissue restricted self-antigens, TC IV lacked Aire expression and were enriched for molecules required for pTreg generation, including the TGF-β activating integrin αvβ8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical DCs was neither sufficient nor required for pTreg generation, further implicating TCs as the critical tolerogenic RORγt+ APC. Our studies reveal parallel pathways for establishment of tolerance to self and foreign antigen within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs.
Project description:Tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing AutoImmune Regulator, Aire, play a critical role in self-tolerance through deletion of autoreactive T cells and promotion of thymic regulatory T (Treg) cell development. A second wave of Treg cell differentiation occurs in the periphery, upon exposure to dietary and commensal microbiota derived antigens within the first few weeks of life, yet the cell types responsible for the generation of peripheral Treg (pTreg) cells are not known. Here we identified a new lineage of tolerogenic RORγt+ antigen-presenting cells (APC) with a hybrid dendritic cell (DC)-mTEC phenotype, dubbed Thetis cells (TCs), comprising 4 major sub-groups (TC I-IV), We uncovered a developmental wave of TCs within intestinal lymph nodes during a critical early life window, coincident with the wave of pTreg cell differentiation. While Aire+ TC I and III bore remarkable homology with Aire+ mTECs, including expression of tissue restricted self-antigens, TC IV lacked Aire expression and were enriched for molecules required for pTreg generation, including the TGF-β activating integrin αvβ8. Loss of either MHCII or Itgb8 expression by TCs led to a profound impairment in intestinal pTreg differentiation, with onset of intestinal inflammation. In contrast, MHCII expression by RORγt+ group 3 innate lymphoid cells (ILC3) and classical DCs was neither sufficient nor required for pTreg generation, further implicating TCs as the critical tolerogenic RORγt+ APC. Our studies reveal parallel pathways for establishment of tolerance to self and foreign antigen within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs.