Using time-resolved single-cell RNA sequencing on murine model of S. Aureus, we define immune cell dynamics in the skin during infection, resolution, and memory

Staphylococcus aureus is a leading cause of skin and soft tissue infections (SSTIs), for which effective vaccines remain unavailable, in part due to poorly defined correlates of protective immunity. While CD4? T cells are known to contribute to host defense against S. aureus, their tissue-resident counterparts have not been comprehensively examined in the context of SSTIs. Here, using a self-resolving murine model of S. aureus SSTI in C57BL/6 mice, longitudinal single-cell RNA sequencing was combined with FACS-analysis to define immune cell dynamics in the skin during infection, resolution, and memory. Single-cell profiling of CD45?Ly6G? cells across acute infection, adaptive responses, and post-resolution timepoints revealed the accumulation and long-term persistence of an effector memory CD4? T cell population within the skin. These cells adopted a transcriptional and phenotypic profile consistent with tissue-resident memory T cells (Trms), characterized by expression of CD69±CD103, and genes associated with tissue retention and residency. Upon secondary S. aureus challenge, skin-derived CD4? Trms rapidly produced IL-17A and IFN-?, and were associated with enhanced neutrophil recruitment, reduced bacterial burdens and weight loss. Importantly, pharmacological blockade of lymphocyte egress from secondary lymphoid organs did not impair protective immunity during reinfection, supporting a role for resident, rather than circulating, T cells. Together, these findings identify infection-induced, skin-resident CD4? memory T cells as a durable and functionally responsive population that contributes to protective immunity during recurrent S. aureus skin infection, highlighting CD4? Trms as a potential target for future vaccine strategies.