Subsets of mononuclear phagocytes, including macrophages and classical dendritic cells (cDC), are highly heterogeneous in peripheral tissues such as the intestine, with each subset playing distinct roles in immune responses. Understanding this complexity at the cellular level has proven difficult due to the expression of overlapping phenotypic markers and the inability to isolate leukocytes of the mucosal lamina propria (LP) effector site, without contamination by the isolated lymphoid follicles (ILFs), which are embedded in the mucosa and which are responsible for the induction of immunity. Here we exploit our novel method for separating lamina propria from isolated lymphoid follicles to carry out single-cell RNA-seq, CITE-seq and flow cytometry analysis of MNPs in the human small intestinal and colonic LP, without contamination by lymphoid follicles. As well as classical monocytes, non-classical monocytes, mature macrophages, cDC1 and CD103+ cDC2, we find that a CD1c+ CD103- cDC subset, which shares features of both cDC2 and monocytes, is similar to the cDC3 that have recently been described in human peripheral blood. As well as differing between the steady-state small intestine and colon, the proportions of the different MNP subsets change during different stages of inflammatory bowel disease (IBD) inflammation. Putative cDC precursors (pre-cDC) were also present in the intestine, and trajectory analysis revealed clear developmental relationships between these and subsets of mature cDC, as well as between tissue monocytes and macrophages. By providing novel insights into the heterogeneity and development of intestinal MNP, our findings should help develop targeted approaches for modulating intestinal immune responses.

NCT02324023.