A novel and efficient approach to high-throughput production of HLA-E/peptide monomer for T-cell epitope screening

Juliette Vaurs,Agnès Fortun,Klara Echasserieau,Karine Bernardeau,Frédéric Pecorari,Romain Oger,Nicolas Jouand,Nadine Gervois,Leslie Hesnard,Mikaël Croyal,Gaël Douchin

doi:10.1038/s41598-021-96560-9

Abstract

Over the past two decades, there has been a great interest in the study of HLA-E-restricted αβ T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket. To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. We characterized the UV exchange by ELISA and improved the peptide exchange readout using size exclusion chromatography. This novel approach for complex quantification is indeed very important to perform tetramerization of MHC/peptide complexes with the high quality required for detection of specific T cells. Our approach allows the rapid screening of peptides capable of binding to the non-classical human HLA-E allele, paving the way for the development of new therapeutic approaches based on the detection of HLA-E-restricted T cells.

Highlights

Over the past two decades, there has been a great interest in the study of Human leukocyte antigen E (HLA-E)-restricted αβ T cells during bacterial and viral infections, including recently SARS-CoV-2 infection
We selected 13 peptides derived from the leader sequence of different HLA class I molecules and/or human cytomegalovirus (HCMV) UL40 (Table 1)[14,36,37]
Using the mutated P8, D2 (VMAPRTVJL) or G2 (VMTPRTLJL) peptides, we obtained a high yield of stable HLA-E/peptide complexes almost fully cleaved by UV exposure

Summary

Introduction

Over the past two decades, there has been a great interest in the study of HLA-E-restricted αβ T cells during bacterial and viral infections, including recently SARS-CoV-2 infection. Phenotyping of these specific HLA-E-restricted T cells requires new tools such as tetramers for rapid cell staining or sorting, as well as for the identification of new peptides capable to bind to the HLA-E pocket To this aim, we have developed an optimal photosensitive peptide to generate stable HLA-E/pUV complexes allowing high-throughput production of new HLA-E/peptide complexes by peptide exchange. Using the conventional batch production protocol, approximately 10 days are required to obtain a validated pMHC tetramer for one peptide of interest[23,24] This production method is not suitable for a screening approach aimed at either designing novel epitopes for a specific MHC allele or producing tetramers for visualization of the antigen-specific T-cell population. For functional validation of the generated MHC/peptide complexes, we compared the labeling of HCMV-specific HLA-E-restricted CD8 T cells, which we g enerated[14] using either conventional batch productions or peptide exchange productions

Methods

Results

Conclusion