Viral infections can affect the glycosylation pattern of
glycoproteins involved in
antiviral immunity. Given the importance of protein glycosylation for immune function, we investigated the effect that modulation of the highly conserved HLA class I N-
glycan has on KIR:HLA interactions and NK cell function. We focused on
HLA-B*57:01 and its interaction with KIR3DL1, which has been shown to play a critical role in determining the progression of a number of human diseases, including human immunodeficiency virus-1
infection. 721.221 cells stably expressing
HLA-B*57:01 were treated with a panel of glycosylation
enzyme inhibitors, and HLA class I expression and KIR3DL1 binding was quantified. In addition, the functional outcomes of
HLA-B*57:01 N-
glycan disruption/modulation on KIR3DL1ζ+ Jurkat reporter cells and primary human KIR3DL1+ NK cells was assessed. Different glycosylation
enzyme inhibitors had varying effects on
HLA-B*57:01 expression and KIR3DL1-Fc binding. The most remarkable effect was that of
tunicamycin, an inhibitor of the first step of N-glycosylation, which resulted in significantly reduced KIR3DL1-Fc binding despite sustained expression of
HLA-B*57:01 on 721.221 cells. This effect was paralleled by decreased activation of KIR3DL1ζ+ Jurkat reporter cells, as well as increased degranulation of primary human KIR3DL1+ NK cell clones when encountering
HLA-B*57:01-expressing 721.221 cells that were pre-treated with
tunicamycin. Overall, these results demonstrate that N-glycosylation of HLA class I is important for KIR:HLA binding and has an impact on NK cell function.