The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. To comprehensively map cell lineages in the healthy developing, pediatric and adult human gut from ten distinct anatomical regions, as well as draining lymph nodes, we used single-cell RNA-seq and VDJ analysis of roughly one third of a million cells. This reveals the presence of BEST4+ absorptive cells throughout the human intestinal tract, demonstrating the existence of this cell type beyond the colon for the first time. Furthermore, we implicate IgG sensing as a novel function of intestinal tuft cells, and link these cells to the pathogenesis of inflammatory bowel disease. We define novel glial and neuronal cell populations in the developing enteric nervous system, and predict cell-type specific expression of Hirschsprung's disease-associated genes. Finally, using a systems approach, we identify key cell players across multiple cell lineages driving secondary lymphoid tissue formation in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. These data provide an unprecedented catalogue of intestinal cells, and new insights into cellular programs in development, homeostasis and disease.

42Gastrointestinal microbiota and immune cells interact closely and display regional 43 specificity, but little is known about how these communities differ with location. Here, 44we simultaneously assess microbiota and single immune cells across the healthy, 45 adult human colon, with paired characterisation of immune cells in the mesenteric 46 lymph nodes, to delineate colonic immune niches at steady-state. We describe 47 distinct T helper cell activation and migration profiles along the colon and 48 characterise the transcriptional adaptation trajectory of T regulatory cells between 49 lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal 50 expansion and mutational frequency from caecum to sigmoid colon, and link this to 51 the increasing number of reactive bacterial species. 52 53

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