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

ABSTRACT: Salmonella is a major foodborne pathogen that causes approximately 1.35 million infections annually in the US and remains a leading cause of poultry-associated foodborne illness. To improve chickens' resistance to this pathogen, it is important to understand the mucosal immune mechanisms that govern intestinal defense. Intraepithelial lymphocytes (IELs) positioned between intestinal epithelial cells provide frontline immune surveillance against enteric pathogens. However, a comprehensive characterization of IEL subtype responses to Salmonella infection remains incomplete. Therefore, we conducted this study to examine IEL subtypes and their mechanisms in response to a Salmonella enterica serovar Enteritidis (S. Enteritidis) challenge using a combination of spectral flow cytometry and single-cell RNA sequencing (scRNA-seq). Fifty specific-pathogen-free (SPF) day-old chicks were reared to 21 days of age and then assigned to S. Enteritidis-challenged (SE; 1.62 × 10⁸ CFU/bird, oral gavage) or control (CN; PBS) groups (n = 25/group). 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.

ORGANISM(S): Gallus gallus

PROVIDER: GSE326338 | GEO | 2026/06/24

REPOSITORIES: GEO