Maria Giovanna Desimio ¹ Daniela Angela Covino ¹ Caterina Cancrini ^1,2 Margherita Doria ^1*

• ¹ Bambino Gesù Children's Hospital (IRCCS), Rome, Italy
• ² University of Rome Tor Vergata, Roma, Lazio, Italy

Epstein-Barr virus (EBV) is usually acquired during infancy as an asymptomatic infection and persists throughout life in a latent state under the control of the host immune system. However, EBV is associated with various malignant diseases that preferentially develop in immunodeficient individuals. Accumulating evidence suggests an important role for NK cells, though the mechanisms by which EBV evades or triggers NK cell responses are poorly understood. Here, we generated EBVimmortalized lymphoblastoid cell lines stably expressing an inducible form of the BZLF1 early lytic viral protein (LCL-Z) to challenge primary NK cells with EBV + targets in either latent or lytic phase of infection. We show that entry into the lytic phase results in drastic down-regulation of HLA-E but not HLA-A,B,C molecules and in increased expression of ligands for the activating NKG2D receptor, with MICB being up-regulated at the cell-membrane and released in a soluble form while ULBP2 and ULBP4 accumulate intracellularly. Furthermore, LCL-Z cells are killed by NK cells in an NKG2D-dependent manner and to a much higher extent during the lytic phase, but HLA-class I molecules constrain killing throughout the viral life cycle; unexpectedly, antibody-mediated block of the inhibitory NKG2A receptor results in reduced lysis of lytic LCL-Z cells that are nearly devoid of the cognate HLA-E ligand. Accordingly, we show that NKG2A + NK cell subsets, specifically CD56 bright and NKG2A + KIR + CD56 dim cells, are those that preferentially respond against cells with lytic EBV replication. Overall, these results shed light on NK/EBV + cell interactions providing new information for improving NK cell-based immunotherapies to treat EBV-induced diseases.