Empty-loadable MHC class I tetramers for efficient detection of antigen-specific T cells

Abstract

Abstract The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use for peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. We demonstrate here the generation, analysis, and use of empty-loadable (peptide-free) MHC class I tetramers made from disulfide-stabilized MHC molecules. A disulfide bond links the α1 and α2 helices of MHC class I molecule at the extreme end of the F pocket. It allows in vitro folding of recombinant A2 with a dipeptide and subsequent removal of the dipeptide to yield stable peptide-receptive MHC monomers. Empty-loadable tetramers prepared using disulfide-stabilized MHC monomers can be loaded with peptides within minutes. These tetramers efficiently detects antigen-specific T cells. Furthermore, peptide-MHC tetramers prepared using disulfide-stabilized MHC molecules provide a better staining index for antigen-specific T cell detection compared to multimers prepared with wild-type MHC class I molecules. We demonstrate the value of empty-loadable tetramers that are converted to antigen-specific tetramers by a single-step peptide addition, for the identification of T cells specific to several neo- and cancer-associated antigens in melanoma. Disulfide stabilization has been achieved with several MHC class I allotypes-HLA-A* 02:01, HLA-A*01:01, HLA-A*03:01, HLA-A*11:01, HLA-A*24:02, HLA-B*07:02, and H-2Kb.