GSE176282
Single-cell Multi-omics Defines Tolerogenic Extrathymic Aire-Expressing Populations with Unique Homology to Thymic Epithelium
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-?t), 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. Overall design: A total of 3 WT and 6 Adig B6 mice were sacrificed. Lymph nodes were dissected and digested and pooled between the mice with the same genotype. Cells were then enriched via 1) magnetic depletion and 2) sorted as indicated for the GFP positive sample. For each pool of cells from the mice, both ASAP-seq and scRNA-seq were performed using the 10x Genomics chromium machine.