A novel lineage of ROR?t+ antigen presenting cells instructs microbiota-dependent regulatory T cell differentiation and tolerance during early life [multiome scRNA-seq]

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 ROR?t+ 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-ß activating integrin avß8. 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 ROR?t+ group 3 innate lymphoid cells (ILC3) and classical DCs was neither sufficient nor required for pTreg generation, further implicating TCs as the critical tolerogenic ROR?t+ 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. Overall design: Single cell multiome (ATAC + Gene Expression) sequencing of Lin–RORgt+MHCII+ cells from the mLN of 2-week-old Rorc_Venus-creERT2 pooled from 16 biological replicates.