Identification of porcine ileal innate lymphoid cells using single-cell RNA sequencing

Abstract Innate lymphoid cells (ILCs) are integral in maintaining intestinal defense and homeostasis, but intestinal ILCs have not been identified in pigs. Pigs are an important global food source and human biomedical model, thus identifying and characterizing porcine intestinal ILCs can have broad implications. Single-cell RNA sequencing of porcine ileal lymphocytes yielded 31,983 cells distributed across 54 clusters. Five clusters were closely related to T cell clusters yet lacked expression of the pan-T cell marker, CD3E. One cluster had high expression of genes related to type 3 immunity, including IL22, RORC, and CXCL8, indicating identification of putative ILC3s. The other four clusters expressed genes encoding for NK receptors (KLRK1, KLRC1, KLRD1) and effector molecules (CCL5, PRF1), yet they lacked expression of genes traditionally expressed by porcine NK cells (CD8A, KLRB1, HCST). Thus, we putatively identified the four clusters as ILC1s. Profiles of putative porcine ILC clusters were further compared to transcriptomic signatures of murine intestinal ILC subsets. Results indicated a relative enrichment of murine ILC3 gene signatures in the porcine ILC3 cluster, while relative enrichment of murine ILC1 gene signatures was observed for the other four clusters of porcine ILC1s. We further utilized single-cell gene expression profiles to develop a gating strategy to identify porcine intestinal ILCs as CD45+CD2+CD3ɛ−CD79α− lymphocytes at the protein level by flow cytometry. Altogether, the data allowed us to create the first transcriptomic descriptions of porcine intestinal ILCs and develop approaches to identify ILCs at the protein level, which will be applicable for future research.