Peptide occupancy governs HLA-E checkpoint interactions enabling VISTA recognition of destabilized HLA-E conformers

Human leukocyte antigen E (HLA-E) plays a central role in immune regulation across homeostasis, infection, cancer, and transplantation. Canonically, HLA-E presents leader sequence-derived VL9 peptides to engage CD94/NKG2 receptors, while alternative peptides disrupt this interaction. HLA-E also interacts with LILRB1/2 and the checkpoint molecule VISTA, but the role of peptide occupancy in these interactions remains unclear. Here, we demonstrate that HLA-E engages VISTA when peptide occupancy is absent or suboptimal, whereas interaction with LILRB1/2 is peptide independent. Both receptors recognize peptide-deficient or weakly stabilized HLA-E conformers. In contrast, stable peptide loading prevents VISTA binding, indicating that peptide occupancy governs receptor engagement. These findings support a model in which HLA-E exists along a spectrum of conformational states defined by peptide stability, enabling selective interaction with distinct immune checkpoints. This work identifies VISTA as a receptor for destabilized HLA-E conformers and reveals a previously unrecognized layer of immune regulation. VISTA recognizes peptide-deficient HLA-E, revealing peptide occupancy as a conformational switch for checkpoint receptor selectivity distinct from VL9 loaded HLA-E recognition by NKG2A and peptide independent binding by LILRB1/II.