Generation and characterization of a novel MHC-II tetramer for tracking and characterization of toxin B-specific CD4 + T cell responses

Abstract The gastrointestinal pathogen Clostridioides difficile , is a major burden for health systems due to high rates of recurrence. C. difficile pathogenesis is mediated by two virulence factors, toxin A (TcdA) and Toxin B (TcdB). Antibodies specific for TcdA and TcdB are correlated with protection from symptomatic recurrence, however, the role for CD4 + T cells is poorly understood in part due to the lack of tools to study the toxin-specific CD4 + T cell response. Our group recently demonstrated the antibody and CD4 + T cell response to C. difficile toxins is impaired via the glucosyltransferase activity of the toxins; however, tools do not exist to study the protective capacity and the phenotype of toxin-specific CD4 + T cells. Therefore, we developed an MHC-II tetramer to identify TcdB-specific CD4 + T cells via flow cytometry. Herein, we identified an immunodominant epitope (TcdB 1961-1975 ) in the CROPs region of TcdB and optimized an MHC-II tetramer for use in tracking and phenotyping TcdB-specific CD4 + T cell responses following multiple different immunization strategies in mice. Utilizing the tetramer, TcdB-specific T follicular helper (Tfh) cells were detected following TcdB-CROPs mRNA-LNP vaccination validating the advantage of the tetramer. Furthermore, using a modular mRNA vector expressing the TcdB 1961 peptide covalently bound to the beta chain of MHC-II (MHC-IIβ) we were able to generate a robust population of TcdB-specific CD4 + T cells. These data outline the generation of new tools for the C. difficile field and lay the groundwork for future studies of toxin-specific CD4 + T cell responses.