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.
Author Info: (1) Department of Health Technology, Technical University of Denmark (DTU), Denmark. (2) Department of Health Technology, Technical University of Denmark (DTU), Denmark. (3) Depart
Author Info: (1) Department of Health Technology, Technical University of Denmark (DTU), Denmark. (2) Department of Health Technology, Technical University of Denmark (DTU), Denmark. (3) Department of Life Sciences and Chemistry, Jacobs University, Bremen, Germany. (4) Department of Life Sciences and Chemistry, Jacobs University, Bremen, Germany. (5) Department of Health Technology, Technical University of Denmark (DTU), Denmark. (6) National Center for Cancer Immune Therapy, Copenhagen University Hospital, Herlev, Denmark. (7) National Center for Cancer Immune Therapy, Copenhagen University Hospital, Herlev, Denmark. (8) Department of Health Technology, Technical University of Denmark (DTU), Denmark. (9) European Molecular Biology Laboratory (EMBL), Hamburg, Germany. (10) Physik-Department, T38, Technical University of Munich, Germany. (11) European Molecular Biology Laboratory (EMBL), Hamburg, Germany. (12) Department of Life Sciences and Chemistry, Jacobs University, Bremen, Germany. (13) Department of Health Technology, Technical University of Denmark (DTU), Denmark. sirha@dtu.dk.
