Protocol

Epitope Mapping Protocols

Volume 524 of the series Methods in Molecular Biology™ pp 383-405

Date:

High-Throughput T-Cell Epitope Discovery Through MHC Peptide Exchange

  • Sine Reker HadrupAffiliated withDivision of Immunology, The Netherlands Cancer Institute
  • , Mireille ToebesAffiliated withDivision of Immunology, The Netherlands Cancer Institute
  • , Boris RodenkoAffiliated withDivision of Molecular Carcinogenesis, The Netherlands Cancer Institute
  • , Arnold H. BakkerAffiliated withDivision of Immunology, The Netherlands Cancer Institute
  • , David A. EganAffiliated withDivision of Immunology, The Netherlands Cancer Institute
  • , Huib OvaaAffiliated withDivision of Cellular Biochemistry, The Netherlands Cancer Institute
  • , Ton N.M. SchumacherAffiliated withDivision of Immunology, The Netherlands Cancer Institute Email author 

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Summary

Recombinant major histocompatibility complex (MHC) class I molecules complexed with pathogen-specific or other disease-associated antigens have become essential reagents for the analysis of adaptive T-cell responses. However, conventional techniques for the production of recombinant peptide-MHC (pMHC) complexes are highly involved and thereby limit the use of pMHC complexes in terms of antigen diversity. To make pMHC-based techniques suitable for high-throughput analyses we developed an MHC peptide exchange technology based on the use of conditional MHC ligands. This technology enables the parallel production of thousands of different pMHC complexes within hours, allowing the development of high-throughput MHC-based assay systems to identify MHC ligands and cytotoxic T-cell responses. These high-throughput assays should prove valuable for the screening of entire disease-associated proteomes, including pathogen-encoded proteomes, tumor-associated antigens, and autoimmune antigens.

Key words

Conditional ligands MHC peptide exchange High-throughput screening T-cell epitopes CD8+ T cells