Subcellular antigen localization in commensal E. coli is critical for T cell activation and induction of specific tolerance.

Oral tolerance to soluble antigens is critically important for the maintenance of immunological homeostasis in the gut. The mechanisms of tolerance induction to antigens of the gut microbiota are still less well understood. Here, we investigate whether the subcellular localization of antigens within non-pathogenic E. coli has a role for its ability to induce antigen-specific tolerance. E. coli that express an ovalbumin (OVA) peptide in the cytoplasm, at the outer membrane or as secreted protein were generated. Intestinal colonization of mice with non-pathogenic E. coli expressing OVA at the membrane induced the expansion of antigen-specific Foxp3+ Tregs and mediated systemic immune tolerance. In contrast, cytoplasmic OVA was ignored by antigen-specific CD4+ T cells and failed to induce tolerance. In vitro experiments revealed that surface-displayed OVA of viable E. coli was about two times of magnitude more efficient to activate antigen-specific CD4+ T cells than soluble antigens, surface-displayed antigens of heat-killed E. coli or cytoplasmic antigen of viable or heat-killed E. coli. This effect was independent of the antigen uptake efficiency in dendritic cells. In summary, our results show that subcellular antigen localization in viable E. coli strongly influences antigen-specific CD4+ cell expansion and tolerance induction upon intestinal colonization.