Impaired Th17 immunity in recurrent C. difficile infection is ameliorated by fecal microbial transplantation

Background & Aims Clostridioides difficile is a leading cause of infectious diarrhea and an urgent antimicrobial resistant threat. Symptoms are caused by its toxins, TcdA and TcdB, with many patients developing recurrent C. difficile infection (CDI), requiring fecal microbiota transplant (FMT). Antibody levels have not been useful in predicting patient outcomes, which is an unmet need. We aimed to characterize T cell-mediated immunity to C. difficile toxins and assess how these responses were affected by FMT. Methods We obtained blood samples from patients with newly acquired CDI, recurrent CDI (with a subset receiving FMT), inflammatory bowel disease with no history of CDI, and healthy individuals (controls). Toxin-specific CD4+ T cell responses were analysed using a whole blood flow cytometry antigen-induced marker assay. Serum antibodies were measured by ELISA. Tetramer guided mapping was used to identify HLA-II-restricted TcdB epitopes and DNA was extracted from TcdB-specific CD4+ T cells for TCR repertoire analysis by Sanger sequencing. Results CD4+ T cell responses to C. difficile toxins were functionally diverse. Compared to controls, individuals with CDI, or inflammatory bowel disease had significantly higher frequencies of TcdB-specific CD4+ T cells. Subjects with recurrent CDI had reduced proportions of TcdB-specific CD4+ Th17 cells, FMT reversed this deficit and increased toxin-specific antibody production. Conclusions These data suggest that effective T cell immunity to C. difficile requires the development of Th17 cells. In addition, they show that an unknown aspect of the therapeutic effect of FMT may be enhanced T and B cell-mediated immunity to TcdB. ![Figure][1] GRAPHICAL ABSTRACT ### Competing Interest Statement M.K.L received research funding from Bristol Myers Squibb, Takeda, CRISPR therapeutics and Sangamo Inc for work not related to this study. T.S.S received research funding from Rebiotix, Seres, NuBiyota, Actelion, Sanofi Pasteur, and Pfizer for studies in CDI not related to this study. L.C received a Young Investigator Award from Adaptive Biotechnologies that funded TCR sequencing work for this study. ### Funding Statement Canadian Institutes of Health Research Project Grant (20R76508) and an Antimicrobial Resistance Point of Care Diagnostics in Human Health grant (20R75988) The Broad Medical Research Program at the Crohns & Colitis Foundation of America (IBD-0326) MISP grant from Merck Canada ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Study protocols were approved by Clinical Research Ethics Boards of the University of British Columbia (H15-01682, H09-01238 and H18-02553) and the Vancouver Coastal Health Authority (V15-01682). FMTs were administered under approved University of British Columbia ethics protocols (H13-01221 and H15-00763). All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes There are no large datasets in this paper. [1]: pending:yes