Redefining intestinal immunity with single-cell transcriptomics

Abstract

The intestinal immune system represents the largest collection of immune cells in the body and is continually exposed to antigens from food and the microbiota. Here we discuss the contribution of single-cell transcriptomics in shaping our understanding of this complex system. We consider the impact on resolving early intestine development, engagement with the neighbouring microbiota, diversity of intestinal immune cells, compartmentalisation within the intestines and interactions with non-immune cells. Finally, we offer a perspective on open questions about gut immunity that evolving single-cell technologies are well placed to address.

Fig. 1. ScRNAseq approaches and analysis tools for studying intestinal immunity.

Left: ScRNAseq, multi-omics and spatial platforms; Right: mirrored analysis tools for analysing resulting data. sm-FISH: single molecule fluorescence in situ hybridization; SNPs: single nucleotide polymorphisms.

Fig. 2. ScRNAseq advances in understanding mucosal immunity throughout life and during disease.

(1) Building from observations of lymphocyte maturation in the prenatal intestines, single-cell studies have promoted the concept that T and B cell activation up to second-trimester development is in support of intestinal development rather than in response to microbial seeding^,–. (2) Key players in gut-associated lymphoid tissue (GALT) formation previously identified in developing mouse gut have been identified in scRNAseq studies of human fetal gut, with additional cell heterogeneity identified^,. (3) TNF-α-producing CD4+ T cells resolved at single-cell level are enriched in the preterm intestines, likely contributing to epithelial damage observed in necrotising enterocolitis (NEC). (4) Intestinal stromal heterogeneity has been determined, and expanded subtypes in inflammatory bowel disease linked with pathology^–. (5) Inlaid box depicts the classical idea of discrete immune cell subsets versus a spectrum of phenotypes determined through scRNAseq analyses. LTi lymphoid tissue inducer, LTo lymphoid tissue organiser.

Fig. 3. Key scRNAseq finding of zonation of intestinal immunity.

(1) ScRNAseq of distinct anatomical regions of the human intestines has revealed increasing activation of plasma cells linked to recognition of a richer microbiota from proximal to distal colon and (2) small intestine-specific expression of CFTR by BEST4+ epithelial cells^,. (3) At the microanatomical level, scRNAseq has shown distinctions in gene expression of the epithelium, with cells at the villus tip expressing immune-modulatory genes and follicle-associated cells prioritising efficient sampling of microbial antigens. (4) Unbiased clustering of single intestinal macrophages has revealed their unique signatures within the layers of the intestinal wall indicative of specific functions and the influence of interactions with tissue-resident cells. (5) ScRNAseq of physically separated intraepithelial and lamina propria γδ T has implicated the latter in supporting intestinal epithelium remodelling in mice due to change in diet. (6) In the context of colitis, scRNAseq has revealed decreased abundance of CD8- and γδ-ENTPD1 cells in the intraepithelial space and implicated defective cAMP signalling in disease pathogenesis.