Using peptide-exchange systems to interrogate peptide-specific KIR binding to HLA Class I

The killer-cell immunoglobulin-like receptors (KIR) are a family of activating and inhibitory Class I human leukocyte antigen (HLA-I) binding receptors expressed on natural killer (NK) cells and subsets of T cells. The KIR detect HLA-I molecules in a peptide-dependent manner, with some KIR displaying exquisite peptide specificity. Studying peptide recognition by KIR often uses TAP-deficient cell lines expressing single HLA-I alleles, which are heterogenous and time consuming to generate. Here, we established an alternative approach using peptide-exchange technologies hitherto developed for studying T cell recognition of HLA-I.

We tested two methods; dipeptide-mediated peptide exchange and "open-HLA-I", HLA-I molecules consisting of heavy chain-β₂m disulphide bonded dimers. We combined peptide-exchange technologies with SpyTag-SpyCatcher chemistry to allow rapid detection of KIR binding via HLA-I displayed on plates or cells.

We demonstrated the fidelity of this system with peptides of known KIR specificity bound to HLA-C*05:01. We then screened a peptide library to identify novel strong KIR2DS4 binding peptides presented by HLA-C*04:01. Peptide-exchanged HLA-C was functionally competent, promoting activation of KIR2DS4+ NK cells and inhibiting activation of KIR2DL1+ NK cells.

Together, we show that peptide-exchangeable HLA-I molecules are ligands for KIR, presenting a flexible, efficient system for examining the peptide sequence dependent recognition of HLA-I by KIR.