Single-cell transcriptomics reveals heterogenous thymic dendritic cell subsets with distinct functions and requirements for thymocyte-regulated crosstalk

Dendritic cells are essential for establishing thymic central tolerance; however, mechanisms supporting their homeostasis and activation remain unresolved. Through single-cell transcriptomics and functional assays, we identify seven thymic conventional dendritic cell (cDC) subsets and discriminate their abilities to present self-antigens and induce regulatory T cells. Mice blocked at different stages of T-cell development reveal that CD4+ single-positive (SP) and CD8SP thymocytes differentially support homeostasis and activation of cDC1s versus cDC2s/plasmacytoid DCs (pDCs), respectively. CD8SPs indirectly support pDC survival and cDC2 thymic immigration, and they induce interferon signaling in cDCs, partly by promoting Type III interferon expression by medullary thymic epithelial cells (mTECs). In contrast, CD4SPs undergo cognate interactions with cDCs, inducing CD40 signaling required for activation of cDC1s. Activated cDC1s make non-redundant contributions to central tolerance. Altogether, this study comprehensively identifies distinct thymic DC subsets and elucidates requirements for crosstalk with thymocyte subsets that support their homeostasis, activation, and function.