Investigating Polyreactivity of CD4 ⁺ T Cells to the Intestinal Microbiota

Antigen‐specific recognition of microbiota by T cells enforces tolerance at homeostasis. Conversely, dysbiosis leads to imbalanced T‐cell responses, triggering inflammatory and autoimmune diseases. Despite their significance, the identities of immunogenic microbial antigens remain largely enigmatic. Here, we leveraged a sensitive, unbiased, genome‐wide screening platform to identify peptides from Akkermansia muciniphila (AKK) and Bacteroides thetaiotaomicron (BT) recognized by CD4 ⁺ T cells. The platform is based on screening peptide libraries using an NFAT‐fluorescence reporter cell line transduced with a retrovirus encoding an MHC‐TCR (MCR) hybrid molecule. We discovered several novel epitopes from AKK and BT. T‐cell hybridomas reactive to AKK and BT bacteria demonstrated polyreactivity to microbiota‐derived peptides in co‐cultures with MCR reporter cells. Steady‐state T cells recognized these epitopes in an MHC‐restricted fashion. Intriguingly, most of the identified epitopes are broadly conserved within the given phylum and originate from membrane and intracellular proteins. Ex vivo stimulation of CD4 ⁺ T cells from mice vaccinated with the identified peptides revealed mono‐specific IFN‐γ and IL‐17 responses. Our work showcases the potential of the MCR system for identifying immunogenic microbial epitopes, providing a valuable resource. Our study facilitates decoding antigen specificity in immune system‐bacterial interactions, with applications in understanding microbiome and pathogenic bacterial immunity.