High Throughput pMHC-I Multimer Library Production Using Chaperone-Mediated Peptide Exchange

Abstract

Abstract The development of pMHC-multimer technologies has been indispensable for the characterisation of T cell responses to individual viral and tumor peptide antigens. Given the large variety of T cell receptors expressed in polyclonal repertoires, peptide exchange technologies are becoming increasingly important in providing a high throughput means of generating pMHC-multimer libraries, containing a wide range of antigens to probe such repertoires. Here we present a simple method for the isolation of empty MHC-I molecules which can be readily used for high throughput multimer library preparation. We utilise the chaperone TAPBPR, which binds MHC-I molecules and stabilises them in a peptide-receptive conformation, to isolate empty MHC-I molecules through the combination of placeholder peptides and small molecule competitors. We demonstrate that empty H-2Dd:TAPBPR and HLA-A*02:01:TAPBPR complexes can be stored long term and subsequently loaded with peptides of choice in a high throughput fashion to afford pMHC-multimer libraries. The resulting pMHC-multimers show equivalent flow cytometric properties to multimers made from MHC-I molecules refolded in vitro with synthetic peptides and are significantly improved relative to multimers prepared using peptide exchange of conditional ligands. Using this simple system to generate a library of oligonucleotide-labeled pMHC-multimers, combined with our recently described multi-modal cellular indexing technology (ECCITE-seq), we can identify distinct T cell receptor sequences present in polyclonal repertoires, together with their antigen specificities.