# Shared HLA‐E and Mamu‐E Peptide Repertoires With Subtle Peptide Binding Differences Revealed by Combined nDSF‐ and Fluorescence Polarisation‐Based Methods

## TL;DR

This study compares how human and rhesus macaque MHC-E proteins bind peptides, revealing shared repertoires and subtle differences in binding strength using two new methods.

## Contribution

The paper introduces and combines two novel high-throughput methods to analyze MHC-E peptide binding differences between human and rhesus macaque subtypes.

## Key findings

- HLA-E*01:03 and Mamu-E*02:04 share peptide repertoires but show subtype-specific binding hierarchies.
- nDSF and FP methods correlate thermal stability with binding strength, showing small Tm changes lead to large IC50 differences.
- The combined methods provide scalable, detailed insights into MHC-E peptide binding for both human and rhesus macaque allotypes.

## Abstract

The primary function of MHC‐E—human leukocyte antigen (HLA)‐E in humans and Mamu‐E in rhesus macaques—relates to immune surveillance via CD94/NKG2x receptors expressed on NK cells. However, a secondary role where MHC‐E presents immunogenic peptides to CD8+ T cells that provide protective immunity in specific settings has also been described. Given the high sequence homologies between HLA‐E and Mamu‐E molecules, peptide binding similarities are assumed but not systematically explored, with most studies prioritising HLA‐E. Here, we have optimised and developed two complementary techniques to explore the peptide repertoires of specific HLA‐E and Mamu‐E subtypes. We established a label‐free, high‐throughput nano‐differential scanning fluorimetry (nDSF)‐based method, where peptide binding strength is measured through thermal stability (Tm). This method revealed shared repertoires with occasional subtype‐specific peptide binding hierarchies for the MHC‐E types studied here, HLA‐E*01:03 and Mamu‐E*02:04. When combined with a fluorescence polarisation (FP) peptide competition assay, we show that half maximal inhibitory concentrations (IC50) correlate exponentially with nDSF‐acquired Tm data, revealing that modest Tm increments equate to marked IC50 differences, and hence substantial differences in relative peptide binding strengths. Collectively, these methodologies offer high‐throughput, scalable approaches to provide detailed peptide binding information for rhesus and human MHC‐E types.

The MHC‐E peptide binding methods described here—nano‐differential scanning fluorimetry and fluorescence polarisation—demonstrate shared peptide binding repertoires with subtle binding hierarchy differences for the tested HLA‐E and Mamu‐E allotypes. When combined, these methods illustrate how minor MHC‐E‐peptide thermal stability changes equate to significant binding strength differences.