High throughput pMHC-I tetramer library production using chaperone-mediated peptide exchange

Sarah A Overall,Nicolas Gonzalez,Sara M O’rourke Sara M O’Rourke,Giora I Morozov,Stephanie Hao,Jugmohit S Toor,Michael R Betts,Alberto Sada Japp,Son Nguyen,Nikolaos G Sgourakis,Danai Moschidi,John M Maris,Mark Yarmarkovich,Peter Smibert

doi:10.1038/s41467-020-15710-1

Abstract

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Here, using the molecular chaperone TAPBPR, we develop a robust method for the capture of stable, empty MHC-I molecules comprising murine H2 and human HLA alleles, which can be readily tetramerized and loaded with peptides of choice in a high-throughput manner. Alternatively, catalytic amounts of TAPBPR can be used to exchange placeholder peptides with high affinity peptides of interest. Using the same system, we describe high throughput assays to validate binding of multiple candidate peptides on empty MHC-I/TAPBPR complexes. Combined with tetramer-barcoding via a multi-modal cellular indexing technology, ECCITE-seq, our approach allows a combined analysis of TCR repertoires and other T cell transcription profiles together with their cognate antigen specificities in a single experiment. The new approach allows TCR/pMHC interactions to be interrogated easily at large scale.

Highlights

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal T cell receptor (TCR) repertoires in various settings
To circumvent the need for photo-cleavable ligands, previously used to demonstrate high-affinity TAPasin Binding Protein Related (TAPBPR) binding to empty MHC-I molecules[15], we explored the use of destabilizing placeholder peptides
We recently described a destabilizing N-terminally truncated mutant of the P18-I10 peptide_GPGRAFVTI16. gP18-I10 showed a high affinity for a free H-2Dd groove during in vitro refolding, but dissociated in the presence of TAPBPR to generate stable, empty H-2Dd/TAPBPR complexes (Supplementary Fig. 1a and d)

Summary

Introduction

Peptide exchange technologies are essential for the generation of pMHC-multimer libraries used to probe diverse, polyclonal TCR repertoires in various settings. Upon cleavage and in the presence of a peptide of interest, a net exchange occurs where the cleaved conditional ligand dissociates and the peptide of interest associates with the MHC-I, thereby forming the desired pMHC-I complex. This work revealed an approximately 5% minor conformation with a widened peptide-binding groove and altered dynamics at the α3/ β2m interface exhibited by some MHC alleles, which enables binding to TAPBPR, consistently with reports that polymorphisms within the F-pocket of the groove relate to recognition by Tapasin[20] and TAPBPR21. We leverage these mechanistic insights to design conditional ligands for the production of peptide deficient MHC-I/

Methods

Results

Conclusion