Sequential DC-intrinsic Lymphotoxin Beta Receptor and Retinoic Acid Receptor signals regulate cDC2 fates

Type II conventional dendritic cells (cDC2) are functionally and phenotypically heterogenous. Previous work identified two major cDC2 subsets (cDC2A and cDC2B) and a transcriptionally related monocytic DC3 subset; however, the underlying microenvironmental cues governing their distinct differentiation pathways remain unclear. Here we delineate cDC2 lineage relationships, and the sequential signals required for cDC2A specification and maintenance. We show that T-bet+ cDC2A, arising from a Clec9a+ DC progenitor, express the retinol receptor RBPR2, require cell-intrinsic retinoic acid (RA) signaling for their maintenance in the spleen and mesenteric lymph nodes, and depend on intrinsic RA receptor signaling to sustain downstream Notch signals. Prior to their expression of T-bet, splenic cDC require cell-intrinsic Lymphotoxin beta receptor (LTbR) which is needed to reduce F-actin content and prevents the escape of cDC2 to the blood. We propose a novel mechanism whereby splenic cDC2-intrinsic LTbR signaling anchors cDC2 within the spleen so that they may access macromolecules such as retinol to promote cDC2A development. Overall design: scRNA-seq (gene expression and HTOs) of YFP+ and YFP– CD45+Lin–MHCII+ cells from spleen and mLN of Tbx21RFP-creERT2_Clec9a-cre/cre_R26-YFP (Clec9a fate-mapping) mice, pooled from 4 biological replicates.