Saini et al. developed disulfide-stabilized variants of the human MHC-I molecules HLA-A*02:01, HLA-A*24:02 and the murine MHC molecule H2-Kb that could be efficiently purified while remaining functionally empty and instantly peptide receptive, even to low affinity peptides, and even after long-term storage. Peptide-MHC-I multimers prepared from the empty-loadable MHC-I variants readily identified T cells specific for viral antigens, cancer-associated antigens, and neoantigens, providing a better staining index than those prepared using wild-type MHC-I. Stable, off-the-shelf, empty MHC-I multimers could be easily prepared.
The peptide-dependent stability of MHC class I molecules poses a substantial challenge for their use in peptide-MHC multimer-based approaches to comprehensively analyze T cell immunity. To overcome this challenge, we demonstrate the use of functionally empty MHC class I molecules stabilized by a disulfide bond to link the alpha1 and alpha2 helices close to the F pocket. Peptide-loaded disulfide-stabilized HLA-A*02:01 shows complete structural overlap with wild-type HLA-A*02:01. Peptide-MHC multimers prepared using disulfide-stabilized HLA-A*02:01, HLA-A*24:02, and H-2K(b) can be used to identify antigen-specific T cells, and they provide a better staining index for antigen-specific T cell detection compared with multimers prepared with wild-type MHC class I molecules. Disulfide-stabilized MHC class I molecules can be loaded with peptide in the multimerized form without affecting their capacity to stain T cells. We demonstrate the value of empty-loadable tetramers that are converted to antigen-specific tetramers by a single-step peptide addition through their use to identify T cells specific for mutation-derived neoantigens and other cancer-associated antigens in human melanoma.