T cells and innate lymphoid cells in the pig intestine

Abstract

The intestinal tract is an interface between body and environment, resulting in an immune landscape composed of a spectrum of immune cell states unique from other anatomical locations. T cells and innate lymphoid cells (ILCs) in the intestinal tract contribute to intestinal health versus dysfunction based on balance between immune response versus regulation, making T cells and ILCs promising targets for immunomodulatory intervention strategies that can influence intestinal health. Pigs (Sus scrofa) are a major global food source and important biomedical model, yet T cells and ILCs in the porcine intestinal tract are poorly understood. Most knowledge of porcine T cells and ILCs is derived from the study of non-intestinal cells (primarily from circulation), leaving question as to whether current knowledge of porcine T cells and ILCs is cross-applicable to respective intestinal cell populations in pigs. Moreover, study of porcine T cells and ILCs is limited by minimal protein immunoreagent availability, and studies of gene expression lack the ability to define specific cell types at high resolution. Single-cell RNA sequencing was performed to obtain high-resolution transcriptomic profiles of T cells and ILCs from porcine blood and ileum, revealing previously undescribed levels of cellular heterogeneity and undocumented cell types. Intestinal group 1 and group 3 ILCs were identified and were transcriptionally dissimilar to natural killer (NK) cells, the only previously described ILC subset in pigs. Ileal T cells and ILCs were transcriptionally distinct from circulating cells, and gene expression supported roles in tissue residency, cellular activation, and modified cellular metabolism within the intestinal tract. Locational context of ileal T cells and ILCs further revealed cells with transcriptional profiles associated with follicular responses were enriched in Peyer’s patches, supporting Peyer’s patches as sites of immune induction, while activated, effector T cells and ILCs were located in the epithelium, suggesting roles in epithelial surveillance and defense. Porcine intraepithelial T lymphocytes (T-IELs) were further investigated across small and large intestinal locations from pigs at multiple ages in the post-weaning period using available protein immunoreagents. As pigs aged, T-IEL compositions diverged by intestinal location, resulting in greatest differences in T-IEL compositions between proximal and distal intestinal locations in older animals. In distal compared to proximal intestine, larger proportions of T-IELs had phenotypes indicative of T cell activation, and a larger proportion of distally-located intestinal T-IELs had phenotypes suggestive of non-conventional, innate-like T cells. Collectively, results emphasize the heterogeneous nature of T cells and ILCs in the intestinal tract of pigs, both in the context of the various compartments of the ileum (e.g. Peyer’s patches, epithelium) at a single snapshot in time and across multiple intestinal locations (e.g. proximal versus distal intestine) and ages (e.g. early versus late post-weaning period). Delineating identities and functions of T cells and ILCs in the porcine intestine is a critical first step for understanding their importance in intestinal health. Knowledge gained herein can be utilized to develop targeted immunomodulatory intervention strategies to harness functional roles of T cells and ILCs for promotion of intestinal health in pigs, while taking into consideration the added complexity of variables including intestinal compartment, intestinal location, and pig age.