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. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung’s disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue 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. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Differentiation of naïve CD4⁺ T cells into functionally distinct T helper (T_H) subsets is crucial for the orchestration of immune responses. Because of extensive heterogeneity and multiple overlapping transcriptional programs in differentiating T cell populations, this process has remained a challenge for systematic dissection in vivo. By using single-cell transcriptomics and computational analysis with a temporal mixtures of Gaussian processes model, termed GPfates, we reconstructed the developmental trajectories of T_H1 and T_FH (T follicular helper) cells during blood-stage Plasmodium infection in mice. By tracking clonality using endogenous T cell receptor sequences, we first demonstrated that T_H1/T_FH bifurcation had occurred at both population and single-clone levels. Next, we identified genes whose expression was associated with T_H1 or T_FH fates and demonstrated a T cell–intrinsic role for Galectin-1 in supporting T_H1 differentiation. We also revealed the close molecular relationship between T_H1 and interleukin-10–producing Tr1 cells in this infection. T_H1 and T_FH fates emerged from a highly proliferative precursor that up-regulated aerobic glycolysis and accelerated cell cycling as cytokine expression began. Dynamic gene expression of chemokine receptors around bifurcation predicted roles for cell-cell interaction in driving T_H1/T_FH fates. In particular, we found that precursor T_H cells were coached toward a T_H1 but not a T_FH fate by inflammatory monocytes. Thus, by integrating genomic and computational approaches, our study has provided two unique resources: a database, www.PlasmoTH.org, which facilitates discovery of novel factors controlling T_H1/T_FH fate commitment, and, more generally, GPfates, a modeling framework for characterizing cell differentiation toward multiple fates.

The gastrointestinal tract is a multi-organ system crucial for efficient nutrient uptake and barrier immunity. Advances in genomics and a surge in gastrointestinal diseases^1,2 has fuelled efforts to catalogue cells constituting gastrointestinal tissues in health and disease³. Here we present systematic integration of 25 single-cell RNA sequencing datasets spanning the entire healthy gastrointestinal tract in development and in adulthood. We uniformly processed 385 samples from 189 healthy controls using a newly developed automated quality control approach (scAutoQC), leading to a healthy reference atlas with approximately 1.1 million cells and 136 fine-grained cell states. We anchor 12 gastrointestinal disease datasets spanning gastrointestinal cancers, coeliac disease, ulcerative colitis and Crohn’s disease to this reference. Utilizing this 1.6 million cell resource (gutcellatlas.org), we discover epithelial cell metaplasia originating from stem cells in intestinal inflammatory diseases with transcriptional similarity to cells found in pyloric and Brunner’s glands. Although previously linked to mucosal healing⁴, we now implicate pyloric gland metaplastic cells in inflammation through recruitment of immune cells including T cells and neutrophils. Overall, we describe inflammation-induced changes in stem cells that alter mucosal tissue architecture and promote further inflammation, a concept applicable to other tissues and diseases.

Immune cells are characterized by diversity, specificity, plasticity, and adaptability—properties that enable them to contribute to homeostasis and respond specifically and dynamically to the many threats encountered by the body. Single-cell technologies, including the assessment of transcriptomics, genomics, and proteomics at the level of individual cells, are ideally suited to studying these properties of immune cells. In this review we discuss the benefits of adopting single-cell approaches in studying underappreciated qualities of immune cells and highlight examples where these technologies have been critical to advancing our understanding of the immune system in health and disease.