Ontogeny and transcriptional regulation of Thetis cells [Smart-seq3]

Thetis cells (TCs), a recently identified family of RORgt+ antigen-presenting cells (APCs), comprise four subsets, including the TC IV subset that instructs tolerance to gut microbiota and food antigens. A developmental wave of TCs creates a window of opportunity for establishing intestinal tolerance during early life. However, the ontogeny of TCs and the cues that determine their abundance and heterogeneity are not known, limiting our ability to interrogate their functional roles and exploit their therapeutic potential. Here, we identify a RORgt+ TC and lymphoid tissue inducer (LTi) cell progenitor (TLP) that differentiates into the immediate RORgt+ TC progenitor (TCP) and the LTi progenitor (LTiP), with the transcription factor PU.1 determining TC fate. Despite their similarity to myeloid-derived classical dendritic cells (cDCs), we found that TCs were descended from the common lymphoid progenitor (CLP) with deletion of the plasmacytoid DC (pDC) lineage-determining transcription factor TCF4 leading to expansion of TLP and TCs, suggesting a shared developmental precursor with pDCs. TLP is enriched in mouse fetal liver, but in contrast to LTi cells, TCs emerge postnatally, pointing to a role for developmentally-timed environmental cues in promoting TCP differentiation. We identify one such cue–provision of RANKL by lymphoid tissue organizer cells–which was essential for TC I differentiation. Together, these findings identify the ontogeny of TCs and define the transcription factors that promote TC differentiation and heterogeneity. These insights will facilitate future investigations of these enigmatic cells, which have potential therapeutic applications in promoting tolerance. Overall design: SMART3 sequencing of mesenteric lymph nodes (mLN), peripheral lymph nodes (pLN) and large intestine (LI) of 2-week-old Rorc-Venus mice. Cells were enriched using the strategies outlined below.