CD4+ T Cells Specific for C. difficile Toxins are a Marker of Patients with Active Relapsing Disease

Abstract Background The bacterial pathogen Clostridium difficile is the leading cause of nosocomial infectious diarrhea. Although C. difficile infection (CDI) can be treated with antibiotics, approximately 25% of patients relapse after treatment. The pathogenicity of CDI requires the activities of its toxins, TcdA and TcdB, but T cell-mediated responses to these toxins remain uncharacterized. Methods We enrolled two cohorts of patients, one with newly acquired CDI (n = 14) and the other with relapsing CDI (n = 25); and healthy volunteers with no history of CDI (n = 12). We measured peripheral blood CD4+ T cell responses to the toxins using a whole blood flow cytometry assay that identifies antigen-specific CD4+ T cells by co-expression of CD25 and OX40 following 44h incubation with antigen (Fig 1). Results We found that in patients with recurring CDI, T cell responses to TcdB were significantly higher than in healthy controls (median 1.04% vs. 0.18%; P = 0.003, Fig 2). In contrast, TcdA T cell responses and anti-TcdA/TcdB IgG titres were not different between recurring patients and controls. TcdB, but not TcdA, T cell responses were significantly higher in recurring CDI compared with newly acquired CDI (median 1.04% vs. 0.44%; P = 0.032). In both patient cohorts TcdB-specific CD4+ T cells were functionally heterogeneous, on average: 25% expressed the gut homing marker integrin β7; there was a 1:1 ratio of Tregs to T effectors; and T effectors contained Th1, Th2 and Th17 cells at a 1.5:1:3 ratio. The proportion of Th1 and Th17 cells within TcdB-specific CD4+ T cells was also significantly reduced in recurring, compared with newly acquired, CDI (Fig 3). Analysis of sorted TcdB-specific CD25+OX40+ cells confirmed specificity for TcdB and polarization towards Th17 cells, which are important for intestinal anti-pathogen immunity. Conclusion This is the first investigation of T cell immunity to C. difficile toxins. Our data show that anti-TcdB CD4+ T cell responses are a more specific marker of disease than IgG titres. Tracking how toxin-specific CD4+ T cell responses change following treatment and/or vaccination not only has the potential to predict relapse, but also to deliver insight into how CD4+ T cell memory develops in response to this prevalent pathogen. Disclosures All authors: No reported disclosures.