Pathogen-derived HLA-E bound epitopes reveal broad primary anchor pocket tolerability and conformationally malleable peptide binding

Lucy Walters ,Geraldine M Gillespie,Vitul Jain,Shaheed Abdulhaqq,Justin M Greene,Klaus Früh,Scott G Hansen,K Harlos ,Simon Brackenridge,Emily E Marshall ,Persephone Borrow,T S Walter ,Daniel Rozbeský ,Andrew J Mcmichael,E Y Jones ,Jordan R Barrett,Jonah B Sacha ,C A O’callaghan ,Mireille Toebes,Louis J Picker

doi:10.1038/s41467-018-05459-z

Abstract

Through major histocompatibility complex class Ia leader sequence-derived (VL9) peptide binding and CD94/NKG2 receptor engagement, human leucocyte antigen E (HLA-E) reports cellular health to NK cells. Previous studies demonstrated a strong bias for VL9 binding by HLA-E, a preference subsequently supported by structural analyses. However, Mycobacteria tuberculosis (Mtb) infection and Rhesus cytomegalovirus-vectored SIV vaccinations revealed contexts where HLA-E and the rhesus homologue, Mamu-E, presented diverse pathogen-derived peptides to CD8+ T cells, respectively. Here we present crystal structures of HLA-E in complex with HIV and Mtb-derived peptides. We show that despite the presence of preferred primary anchor residues, HLA-E-bound peptides can adopt alternative conformations within the peptide binding groove. Furthermore, combined structural and mutagenesis analyses illustrate a greater tolerance for hydrophobic and polar residues in the primary pockets than previously appreciated. Finally, biochemical studies reveal HLA-E peptide binding and exchange characteristics with potential relevance to its alternative antigen presenting function in vivo.

Highlights

Since RL9HIV ranked as a strong binder relative to previously reported human leucocyte antigen E (HLA-E) restricted microbial peptides in the micro-refolding ELISA25,28 (Fig. 1a) and elicited Mamu-E restricted CD8+ T cell responses in RM vaccinated with an HIVGag-insert RhCMV68-1 vector (Fig. 1b), it was selected for crystallographic analysis
As Mycobacteria tuberculosis (Mtb)[44] elicited Mamu-E-restricted CD8+ T cell responses in Bacillus Calmette–Guérin (BCG) vaccinated RM (Fig. 1d), it was pursued in crystallographic studies
Mamu-E restricted CD8+ T cell responses have been implicated as immune correlates of protection in RhCMV68-1-vectored SIV vaccination trials, triggering new interest in HLA-E as a potential driver of protective immunity against HIV-113,30–32

Summary

Results

Mtb44*P2-Phe forms three additional H-bonds securing P1 and 2 in the groove Such features align with single-chain trimer-based transfectant data demonstrating that Mtb[44] P2-Phe drives the highest relative levels of HLA-E surface expression. These minor readjustments in peptide positioning due to P2 substitution do not disrupt immune recognition: CD8+ T cells isolated from the spleens of BCG-vaccinated RM mounted responses of similar magnitude when stimulated with Mtb44*P2-Phe, Mtb44*P2-Gln or the index Mtb[44] epitope, emphasising the similarity in positioning of solvent exposed side chains and their antigenicity in vivo (Fig. 3f). Despite the apparent homogenous nature of HLA-E refolded in the presence of RL9HIV, as indicated by size exclusion and ionexchange chromatography, it was not possible to obtain a reproducible thermal melt pattern for this complex in contrast to

Å P7-Ser C

Discussion

Methods