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.