Develop and validate DNA-barcode labelled MHCII multimers for detection of antigen-specific CD4 T cells displayed towards large libraries of epitopes 4185

Description Here we demonstrate the method for broad-scale detection of antigen-specific CD4 T cells. We have established a protocol for MHCII production and peptide exchange (pMHCII complexes), which are multimerized on DNA barcode- and fluorophore-labelled dextrane backbone to detect CD4 T cells. We demonstrate that antigen-specific T cell populations can be identified by amplifying the co-attached barcode by sorting the MHCII-multimer binding CD4 T cell population and that such signals correlate with the CD4 T cell population identified based on fluorescence-labeled MHC II multimers. We demonstrate that individual T cell populations can be identified, with a peptide-MHC II panel consisting of 151 peptides derived from Flu, EBV, HCMV, and HCV loaded on DRB1*01:01 and DRB1*01:04. We could detect T cell populations at frequencies down to 0.001% of all CD4 T cells. Low-frequency responses were validated using peptide-driven expansion of antigen-specific CD4 T cells. This methodology was applied to identify MHC-II epitopes in HCV, recognized by CD4 T cells. Using PBMC samples from a patient cohort previously tested for CD4 functional reactivity to HCV-derived 20mer peptides, we identified 15 minimal epitopes within such sequences, presented in DRB1*01:01 and DRB1*04:01, being recognized by CD4 T cells in these donors. This method will enable detailed analysis of CD4 T cell-specific immune interactions, revealing antigen-driven links between CD4 and CD8 responses in health and disease. Funding Sources Janssen Collaboration Topic Categories Technological Innovations in Immunology (TECH)