Barrier immune memory is programmed by intestinal epithelial cell presentation of cytosol-delivered bacterial antigens.

Antigen compartmentalization is known to influence CD4 and CD8 T cell recognition but its impact on immune responses to different antigens expressed by the same pathogen is poorly understood. By tracking the clonal CD4 T cell response to a single epitope shuttled between different virulence proteins of Citrobacter rodentium, we found that response magnitude and quality depended on antigen localization. Antigens retained within bacteria elicited limited responses, whereas antigens injected into colonocyte cytosol through a type III secretion system generated robust mucosal CD4 T cell responses and promoted formation of epithelial-resident memory T cells. This process required direct presentation by infected epithelial cells. Single-cell transcriptomic analyses revealed that sustained, bidirectional communication between epithelial cells and T cells was essential to elicit both barrier-protective functions and transcriptional programs favoring tissue residency over central memory differentiation. These findings identify nonprofessional antigen-presenting cells as key regulators of CD4 T cell memory fate.