A novel lineage of RORγt+Aire+ antigen presenting cells promotes peripheral generation of intestinal regulatory T cells and tolerance during early life

Tolerance to self- or innocuous foreign antigens is vital for preservation of organismal health. Within the thymus, medullary thymic epithelial cells (mTECs) expressing A uto i mmune Re gulator, 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+ Aire+ 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 TC I bore remarkable homology with Aire+ mTECs, TC IV were specifically enriched for critical 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 dispensable for pTreg generation, further implicating TCs as the critical tolerogenic RORγt+ APC. Our studies reveal parallel pathways for establishment of tolerance within the thymus and periphery, marked by involvement of shared cellular and transcriptional programs. ### Competing Interest Statement M.vdB. has received research support and stock options from Seres Therapeutics and stock options from Notch Therapeutics and Pluto Therapeutics; he has received royalties from Wolters Kluwer; has consulted, received honorarium from or participated in advisory boards for Seres Therapeutics, WindMIL Therapeutics, Rheos Medicines, Merck & Co, Inc., Magenta Therapeutics, Frazier Healthcare Partners, Nektar Therapeutics, Notch Therapeutics, Forty Seven Inc., Priothera, Ceramedix, Lygenesis, Pluto Therapeutics, GlaskoSmithKline, Da Volterra, Vor BioPharma, Novartis (Spouse), Synthekine (Spouse), and Beigene (Spouse); he has IP Licensing with Seres Therapeutics and Juno Therapeutics; and holds a fiduciary role on the Foundation Board of DKMS (a nonprofit organization). A.Y.R. is a member of SAB and has equity in Surface Oncology, RAPT Therapeutics, and holds an IP licensed to Takeda, which is not related to the content of this study.