Exploring Ly49+ CD8+ T cell ontogeny and their connection to Intraepithelial Lymphocytes 2309007

Introduction KIR+CD8+ T cells are present in diverse tissues and disease states, including autoimmunity, infection, and cancer. Interest in these cells has recently increased due to their detection by single-cell transcriptomics in these contexts. However, their development and function remain poorly understood. They show gene expression and T cell receptor (TCR) features associated with autoreactivity and agonist selection, a developmental pathway shared by intraepithelial lymphocytes (IELs), another unconventional T cell population thought to contribute to peripheral tolerance in the small intestine. This study investigates the ontogeny of Ly49+CD8+ T cells, the murine equivalent of human KIR+CD8+ T cells, through single cell transcriptomic analysis of IEL precursors, with the aim of identifying Klra6+ cells, a specific marker for Ly49+CD8+ T cells. Methods Mature IEL precursors were isolated from wild-type C57BL/6 mouse thymi using a defined antibody panel (CD1d-Tet- CD25- CD4- CD8- CD5+ TCRβ+ H-2kb+ CD122+). Hashtag antibodies identified the mouse of origin and CITE-seq antibodies distinguished type-A (PD1+ NK1.1- CD44- CXCR3-) from type-B (PD1- NK1.1+ CD44+ CXCR3+) precursors. Single-cell gene expression, CITE-seq, and TCR library data were generated with the 10x Genomics platform and analyzed using Seurat in R. Results IEL precursors separated into two major groups, forming 10 clusters. Previous findings have shown that IEL precursors can be separated into two groups named type A and type B. To determine if this distinction drove clustering, module scores based on PD1, NK1.1, CD44, and CXCR3 expression were used to assign clusters as A-like or B-like. This analysis confirmed that A/B identity explained the major separation. Conclusion Klra6+ cells were distributed across both A-like and B-like clusters, rather than clustering together, suggesting the existence of two distinct Ly49+CD8+ T cell populations: one potentially functioning within the thymus and another contributing to peripheral tolerance. Funding Source St.