Abstract Text: Epstein–Barr virus (EBV) is a ubiquitous gamma-herpesvirus that persists as a chronic infection in over 90% of the adult human population. EBV infections are mainly asymptomatic and subside with the virus transitioning to latency in a subset of memory B cells. Failure to control latent EBV infection can result in a variety of malignancies, including lymphoproliferative diseases in immunocompromised people. Studies in both experimental models and humans suggest that NK cells are critical in the host defense against EBV. In previous work, we demonstrated NK cells expressing the inhibitory receptor NKG2A were specifically able to recognize and kill latently infected autologous B cell lines. We also showed that HLA-E-presented peptides derived from EBV latent cycle proteins can impair NKG2A recognition and its downstream inhibitory signaling, potentially leading to NK cell activation. To better characterize the phenotype of NK cells that respond to EBV-infected B cells, we generated a panel of EBV-lymphoblastoid cell lines (EBV-LCL) and performed co-culture experiments with primary NK cells and autologous EBV-LCL. We developed a novel mass cytometry panel of NK functional and phenotypic markers and determined that in addition to NKG2A, EBV-LCL-responsive NK cells express increased NKp30 and CD32 and downregulate CD16 which may contribute to NK cell activation and killing of EBV-LCL. Further, we generated HLA-E knockout EBV-LCL using CRISPR and determined that autologous NK cell cytotoxicity increased, suggesting that disruption of the NKG2A:HLA-E immune checkpoint axis enhances killing of EBV-infected B cells and would be an attractive therapeutic option for EBV-associated malignancies.
