The murine MHC-E molecule Qa-1b is surface displayed in a peptide-free conformation in homeostasis

Gaby Schaap¹Soroush Ghaffari²Jim Middelburg¹Marjolein Sluijter¹Lisa Griffioen¹Tom A.W. Schoufour¹Ruud H.M. Wijdeven¹Jacques Neefjes¹Ramon Arens¹Jon Weidanz¹Thorbald Van Hall^1*

• ¹Leids Universitair Medisch Centrum, Leiden, Netherlands
• ²The University of Texas at Arlington, Arlington, United States

Qa-1b, the murine ortholog of the nonclassical MHC-E family, contains minimal polymorphism and exhibits reduced surface stability compared with classical MHC class I molecules. To investigate Qa-1b conformations and their immunological relevance, we employed two antibodies: EXX-1, which selectively recognizes Qa-1b bound to the canonical leader peptide Qdm, and 6A8.6F10, a broadly used Qa-1b-reactive antibody. Genome-wide CRISPR screens revealed that Qdm presentation was induced by interferon-γ and required the components of the peptide-loading complex (PLC) and endoplasmic reticulum quality control. EXX-1 binding thus reflected broad cellular integrity and mirrored CD94/NKG2x receptor engagement. In contrast, 6A8.6F10 staining occurred independently of PLC components such as ERAP1 and tapasin, and intriguingly increased in their absence. Accordingly, exogenous pulsing with Qa-1b-binding peptides markedly reduced 6A8.6F10 antibody binding and resonance shift assays revealed that 6A8.6F10 selectively recognizes peptide-deficient Qa-1b complexes. These findings suggest an additional layer of regulation beyond the immune checkpoint NKG2x/CD94, involving peptide-free MHC-E.