Single-cell transcriptomic profiling reveals innate-like cytotoxic intraepithelial lymphocyte expansion during Salmonella Enteritidis infection in chickens

On day 2 post infection (2 dpi) and 6 dpi, eight birds per group were euthanized by CO2 asphyxiation to collect liver and ceca for bacteriology and ileum for IEL acquisition. Body weights were recorded at 0, 2, and 6 dpi. Bacteriological findings confirmed the challenge: the SE group harbored S. Enteritidis at both time points, while body weight gain was only lower at 2 dpi in the SE group, indicating a transient dip in performance due to Salmonella infection.

Flow cytometry results showed that Salmonella challenge increased the proportion of TCRγδ+CD8αβ+ cytotoxic IELs at 2 dpi, as well as the overall IEL proportion at 2 and 6 dpi. Notably, scRNA-seq identified clusters of progenitor T cells and innate-like cytotoxic T cells that significantly expanded in SE-challenged birds at 2 dpi, indicating rapid mobilization of an innate-like cytotoxic response. Integration of flow cytometry and scRNA-seq data provided evidence that cytotoxic T cells expressing CD8αβ acquire innate-like transcriptional signatures within the intestinal epithelial compartment, suggesting functional reprogramming that enables rapid antigen responses.

Trajectory analysis identified a robust differentiation pathway from progenitor T cells through activated CD8⁺ T cells to innate-like CTL as the terminal fate, with quiescent stem-like resident memory T cells serving as an upstream reservoir. These findings reveal a previously uncharacterized innate-like cytotoxic IEL response as a critical early defense mechanism against Salmonella in poultry and identify self-renewing stem-like Trm cells as a reservoir for rapid IEL effector differentiation.