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

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 (T m ). 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 (IC 50 ) correlate exponentially with nDSF‐acquired T m data, revealing that modest T m increments equate to marked IC 50 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.