Microbial-antigen reactive T cells are the primary effectors of inflammatory damage in animal models of Crohn's disease, causing T cell-mediated colitis only in the presence of gut microbiota. By focusing on a proposed similar key role for T cells in Crohn's inflammation, we hypothesize that anti-flagellin antibodies that are highly shared by Crohn's patients, and characterize an aberrant B cell response, are potential markers for the presence T cell clonotypes that are reactive to the same gut commensal antigens (but different epitopes). Of note, the presence of anti-CBir1 flagellin antibodies is the strongest predictor of severe, complicated Crohn's disease. Our preliminary data demonstrate two features critical to the success of this long-term project. First, there is a disease-specific repertoire of expanded T cells (defined by TCR? CDR3 sequences) that is very highly shared among Crohn's patients, making the study of these T cells' antigen reactivity relevant for the majority of patients. Second, this highly shared T cell repertoire is associated with patterns of shared serum anti-flagellin antibodies that are themselves restricted to Crohn's disease patients, suggesting a potential link between aberrant B cell and T cell responses. To accomplish this study, we will enroll active Crohn's patients as well as remission Crohn's patients, active ulcerative colitis patients and healthy controls to test for significant associations between profiles of serum antibody reactivity to a panel of gut microbial antigens and the TCR? CDR3 repertoire. The goal is to identify high value CDR3 sequences that couple with cognate flagellin antigens. We plan to define the distribution of these TCR? CDR3 sequences among peripheral blood and gut tissue T cells subsets (effector, regulatory and memory) with the goal of identifying sequences that segregate to a particular compartment or phenotype and to see if the memory T cell subset is a reservoir of highly shared TCR? CDR3 clonotypes. Lastly we will measure which TCR? CDR3 clonotypes are expanded in vitro following specific antigen exposure to identify candidate antigen-specific sequences and test for their significant association with antigen seropositivity and cell phenotype. These data will then be used to isolate single T cells from different individuals who share identical expanded TCR? CDR3 clonotypes in order to compare the similarity of structure (?/? pairing), MHC specificity, and recognition of antigen. The focus will be on targeting highly shared TCR? CDR3 sequences that have been robustly linked with antigen-specific responses (cytokine production, proliferation) and seropositivity across multiple Crohn's patients. The long-term goals of this project build on these data and aim to define the hierarchy of mechanisms (antigen peptide binding motifs, MHC promiscuity, e.g.) that contribute to shared antigen reactivity among Crohn's patients, to develop strategies that disrupt the antigen-specific T cell activation, and to assess the feasibility and value of targeting microbial antigen-reactive T cells as a treatment approach in Crohn's disease.
This proposal aims to identify the specific T cells that drive the inflammation in Crohn's disease. We believe that these cells are a primary cause of the inflammatory damage to the gut due to an abnormal inflammatory response to normal resident bacteria of the GI tract. Our preliminary data show that Crohn's patients actually share identical T cell receptor antigen-binding regions and that this sharing can be linked with similarly shared serum antibodies (only seen in Crohn's disease) that are known to recognize specific bacterial proteins. Based on this link we plan to show that these highly shared T cell receptor regions actually react against these same bacterial proteins providing a major mechanism of Crohn's disease. We plan to isolate single T cells so they can be studied more intensively as to how they react to the bacteria and how best to develop T cell-based strategies that block the activity of these inflammatory T cells.
