Introduction: Epstein–Barr virus (EBV) infects more than 90% of adults worldwide and is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Although EBV infection is typically asymptomatic in immune-competent individuals, transplant recipients, especially children, are highly susceptible to EBV-associated PTLD due to the immunosuppression treatment required to prevent graft rejection. Innate immune responses, including Natural Killer (NK) cells, are considered vital early in the infection process. Our group has recently demonstrated that a specific NK cell subset can recognize and respond to autologous B cells latently infected with EBV. This NK subset expresses NKG2A/CD94, an inhibitory receptor that recognizes HLA-E. Since EBV-infected cells induced NK cell activation despite the expression of HLA-E, we hypothesize that EBV encoded peptides play a pivotal role in the recognition and response of NKG2A+ NK cells towards latently infected EBV+ cells.
Methods and Results: Using in silico analysis (NetMHCpan 4.0) a peptide library derived from EBV latent cycle proteins (LMPs and EBNAs) was generated. Initial assessment resulted in a library of 61 peptides which could potentially bind to HLA-E. Then, using target cells, we performed a peptide stabilization assay in vitro to test the ability of individual peptides to be presented by HLA-E.  We identified 37 peptides, expressed during the latency III stage of EBV infection that bound and induced HLA-E expression at the surface of the target cells.
Functional assays, using CD107a as a readout for NK cell activity, demonstrated HLA-E bound EBV-derived peptides were able to inhibit NK cell activation (15 peptides), or prevent NK cell inhibition via the NKG2A receptor, resulting in NK cell activation (12 peptides). We determined that, in general, peptides encoded by LMPs tended to favor NK cell degranulation (NK cell killing) while peptides encoded by EBNAs were mostly inhibitory.
Conclusions: Our results demonstrate that EBV latent cycle proteins can encode for peptides that bind to HLA-E which have the ability to promote NK cell degranulation and therefore may be involved in the elimination of EBV latently infected B cells.  As NK cells are less susceptible to immunosuppression, understanding NK cell interactions with EBV-infected cells will lead to improvements in therapeutic strategies to control EBV diseases post-transplant.