Investigating Polyreactivity of CD4+ T Cells to the Intestinal Microbiota

The symbiotic relationship between host and microbiota plays a pivotal role in training and development of the host’s innate and adaptive immune systems. Antigen-specific recognition of microbiota by T cells enforces tolerance at homeostasis. Conversely, dysbiosis—characterized by alterations in microbiota diversity and abundance—leads to imbalanced T cell responses and triggering of inflammatory and autoimmune diseases. Despite their significance, the identities of immunogenic microbial antigens are still largely enigmatic. Here, we leveraged an in-house developed antigen screening platform, the MCR system ¹ , to delineate CD4+ T cell reactivity against Akkermansia muciniphila (AKK) and Bacteroides thetaiotaomicron (BT), —two prominent members of the gut microbiota. T-cell hybridomas reactive to AKK and BT bacteria showed polyreactivity to select microbiota-derived peptides in MCR co-cultures. We discovered 13 novel antigenic epitopes from AKK and 14 from BT. Steady-state T cells recognized these epitopes in an MHC-restricted fashion. Ex vivo stimulation of peptide-specific T cells revealed induction of type 1 and type 17 immune responses, albeit with non-overlapping specificities, contrary to MCR system results. Our findings further demonstrated that most identified epitopes are broadly conserved within the given phylum and originate from both membrane and intracellular proteins. Our work showcases the potential of the MCR system for identifying immunogenic microbial epitopes, providing a valuable resource. Additionally, it indicates the existence of mucosal T cells with a tropism toward broadly conserved bacterial epitopes. Overall, our study forms the basis for decoding antigen specificity in immune system-bacterial interactions, with applications in understanding both microbiome and pathogenic bacterial immunity.