Generation of peptide–MHC class I complexes through UV-mediated ligand exchange

Abstract

Major histocompatibility complex (MHC) class I molecules present peptide ligands on the cell surface for recognition by appropriate cytotoxic T cells. MHC-bound peptides are critical for the stability of the MHC complex, and standard strategies for the production of recombinant MHC complexes are based on in vitro refolding reactions with specific peptides. This strategy is not amenable to high-throughput production of vast collections of MHC molecules. We have developed conditional MHC ligands that form stable complexes with MHC molecules but can be cleaved upon UV irradiation. The resulting empty, peptide-receptive MHC molecules can be charged with epitopes of choice under native conditions. Here we describe in-depth procedures for the high-throughput production of peptide-MHC (pMHC) complexes by MHC exchange, the analysis of peptide exchange efficiency by ELISA and the parallel production of MHC tetramers for T-cell detection. The production of the conditional pMHC complex by an in vitro refolding reaction can be achieved within 2 weeks, and the actual high-throughput MHC peptide exchange and subsequent MHC tetramer formation require less than a day.

Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA-A1, -A3, -A11, and -B7

“…All peptides were synthesized by standard Fmoc synthesis. (ϩ/Ϫ)-3-amino-3-(2-nitro)phenyl-propionic acid was generated as described (12). Fluorescent labeling of peptides was performed as described in SI Text and confirmed by LC-MS.…”

“…MHC Class I monomer refolding reactions with E. coliderived ␤2M were performed as described (22) and purified by gel-filtration HPLC in PBS (pH 7.4). Biotinylation and MHC tetramer formation were performed as described (12). pMHC complexes were stored at Ϫ20°C in PBS/16% glycerol, MHC tetramers were stored at Ϫ20°C in PBS/16% glycerol/0.5% BSA.…”

“…Peptide elution and subsequent reverse-phase chromatography was performed as described in SI Text. Sandwich ELISAs were performed as described (12).…”

UV-Induced Ligand Exchange in MHC Class I Protein Crystals

“…To allow efficient release of peptide fragments generated upon UV exposure of MHC crystals, a new conditional ligand, KILGJ 1 VFJ 2 V, was designed for the human MHC class I protein HLA-A2 (where J 1 is (2-nitro)phenylglycine as described 18 and J 2 is 3-amino-3-(2-nitro)phenyl-propionic acid. 29 Because of the presence of two UV-sensitive groups, UV-induced cleavage of this ligand results in formation of three short peptide fragments that each have a low affinity for MHC class I, and this is expected to facilitate fragment release from the protein within crystals ( Figure 1A).…”

“…[15][16][17] Here we present a method that is also based on light-induced chemical reactions, but with the aim to generate structures of novel complexes within the existing crystal. This method is based on the use of peptide tools we have recently described (Figure 1), [18][19][20] and makes pMHC complexes amenable to HTP X-ray crystallographic analysis.…”