OR1 The bound peptides are the determinants for the potential of HLA-G mediated immune regulation

Abstract

Aim The essential role of HLA-G in immune tolerance and its expression frequency in immune privileged tissues is well known; however the specific importance of allelic subtypes in immune responses remains unclear. 14 allelic variants exist of which HLA-G*01:01, *01:03 and *01:04 are the most prevalent with these variants differing at residues 31 and 110, respectively. Systematic analysis of these subtypes was the aim of this study. Methods In cytotoxicity assays using artificial K562 cells expressing the HLA-G variants and NK cells as effector cells the differential protective potential of HLA-G variants was analyzed. The inhibitory receptors ILT-2 and KIR2DL4 are known HLA-G ligands. To determine binding of HLA-G to these molecules, recombinant sHLA-G molecules were used to block inhibitory NK surface receptors. These experiments were repeated reciprocally by blocking or silencing the respective receptor and analyze the sHLA-G blocking sites. HLA-G*01:01, G*01:03 and G*01:04 restricted peptide profiles were determined utilizing soluble HLA technology and mass spectrometry. Results An increased protective potential of HLA-G*01:04 could be observed, G*01:04 demonstrated a stronger affinity to the inhibitory receptors on NK cells than G*01:01 or G*01:03 which we attribute to their altered peptide motifs. All variants exhibit a unique repertoire with marginal overlap, while G*01:04 differs in its peptide anchor profile substantially and appears more invariant than G*01:01 or G*01:03. Using the identified peptides bound to the G*01:04 binding groove, structural analysis explains the impact of the L110I mismatch on the HLA-G conformation and its differential interaction with NK cell receptors. Conclusions For the first time a contribution of structural alterations within the HLA-G heavy chain for peptide specificity and NK cell engagement could be observed. These results will be a step towards understanding immune tolerance and guide towards immune therapeutic strategies.