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

<i>Salmonella</i> 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 <i>Salmonella</i> infection remains incomplete. Therefore, we conducted this study to examine IEL subtypes and their mechanisms in response to a <i>Salmonella</i> enterica serovar Enteritidis (<i>S.</i> Enteritidis) challenge using a combination of spectral flow cytometry and single-cell RNA sequencing (scRNA-seq). Fifty specific-pathogen free (SPF) chicks were reared to 21 days of age and then assigned to <i>S.</i> 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 sampled to collect liver and ceca for bacteriology and ileum for IEL acquisition. Bacteriological findings confirmed the challenge: the SE group harbored <i>S.</i> Enteritidis at both time points. Flow cytometry results showed that <i>Salmonella</i> 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 that significantly expanded and innate-like cytotoxic T cells, which emerged 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 transcriptionally inferred trajectory from progenitor T cells through activated CD8⁺ T cells to innate-like CTL as the predicted terminal cluster, with quiescent stem-like resident memory T cells transcriptionally positioned as a reservoir. These findings reveal a previously uncharacterized innate-like cytotoxic IEL response as a critical early defense mechanism against <i>Salmonella</i> in poultry and identify self-renewing stem-like Trm cells as a reservoir for rapid IEL effector differentiation.