Abstract B049: Empty MHC class I molecules for improved detection of antigen-specific T-cells

Abstract

Major histocompatibility complex (MHC) class I multimers have been widely used to identify antigen specific T-cells for immune monitoring, epitope discovery, and T-cell isolation. A bottleneck to many peptide-MHC driven applications for T-cell interrogation is the peptide ligand dependent stability of the MHC class I proteins, which thus compels high-affinity peptide dependent in-vitro folding of each MHC protein and the use of a peptide-exchange technology to investigate antigens of interest. To overcome this challenge, we demonstrate the use of empty peptide-receptive MHC class I molecule for detection of antigen specific T-cells. This strategy is based on an HLA-A*02:01 variant which is stabilized by a disulfide bond to link the alpha-1 and alpha-2 helices close to the F pocket. Determined by the crystal structure, peptide-loaded disulfide-stabilized HLA-A*02:01 show complete structural overlap to wild-type HLA-A*02:01. Following peptide loading, we used such disulfide-stabilized HLA-A*02:01 molecules to form fluorescence labeled tetramers and applied them for detections of T-cell responses against common viruses in healthy donor peripheral blood mononuclear cells. In all tested samples, disulfide-stabilized HLA-A*02:01 tetramers detected T-cell with same specificity as wild-type MHC tetramers and they consistently provide a better staining index for antigen-specific T-cell detection. Importantly, disulfide-stabilized MHC class I molecules can be loaded with peptide in the multimerized form without impacting the T-cell staining capacity. We demonstrate the value of empty loadable tetramers, converted to antigen-specific tetramers by a single-step peptide addition, for identification of T-cells specific to several neo- and cancer-associated antigens among tumor-infiltrating lymphocytes in melanoma.To evaluate if the disulfide linkage has an impact on TCR recognition of peptide-MHC complexes, we determined and compared TCR fingerprints of T-cell clones specific to a given peptide-MHC complex using both the wild-type and the disulfide-stabilized HLA-A*02:01 multimers. No differences were observed in the TCR interaction profile between the disulfide optimized and the wild-type MHC class I. In conclusion, disulfide-stabilized empty HLA class I proteins are a potentially powerful tool for interrogating T-cell recognition—offering a fast and flexible transformation from an empty peptide receptive state to a set of personalized reagents generated to match individual tumor characteristics for T-cell monitoring or selection. Citation Format: Tripti Tamhane, Sunil Kumar Saini, Raghavendra Anjanappa, Ankur Saikia, Sofie Ramskov, Marco Donia, Inge Marie Stenfoft Svane, Soren Nyboe Jakobsen, Maria Garcia-Alai, Martin Zacharias, Rob Meijers, Sebastian Springer, Sine Reker Hadrup. Empty MHC class I molecules for improved detection of antigen-specific T-cells [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B049.