Epstein Barr virus (EBV) infection elicits a strong host immune response that is considered to be essential for control of the virus and for preventing EBV-associated lymphomas in transplant recipients, including post-transplant lymphoproliferative disorder (PTLD). Natural killer (NK) cells are lymphocytes capable of responding to viral infection by secretion of cytokines including IFN-γ and by direct killing of target cells via release of cytotoxic granules. Direct evidence for NK cell control of EBV infection in transplant recipients has been lacking, particularly regarding the NK cell response to latently-infected B cells, as seen in PTLD. To assess the ability of NK cells to recognize and respond to latently-infected B cells, we first generated EBV+ lymphoblastoid cell lines (LCL), a model of EBV+ B cell lymphomas, from a cohort of healthy donors. Next, primary human NK cells isolated by negative selection were primed for two days with or without IL-2 prior to co-culture with allogeneic and autologous LCLs. After co-culture, NK cells were assayed by flow cytometry for the degranulation protein CD107a, the NK cell marker CD56, the NK cell maturation marker CD57, the NK cell receptors NKG2A, NKG2C, NKG2D, 2B4, and CD16, and intracellular IFN-γ. IL-2 primed NK cells exhibited cytotoxicity against LCLs, with more robust cytotoxicity against allogeneic LCLs. Similarly, IL-2 primed NK cells produced IFN-γ in response to both autologous and allogeneic LCLs. NK cells did not exhibit any functional responses against primary autologous B cells, suggesting this response was specific to latent EBV infection. Initial analysis revealed that both degranulating (CD107+) and IFNγ-producing (IFNγ+) NK cells displayed greater numbers of NKG2A+ cells and decreased numbers of NKG2C+ and CD16+ cells compared to their non-responding NK populations (CD107-, IFNγ-). Examination of HLA-E expression on primary B cells and autologous LCLs revealed that latent EBV infection increases expression of the NKG2A/NKG2C ligand HLA-E. Thus, we demonstrate that NK cells recognize and respond to EBV-infected B cells in an EBV-specific, IL-2 dependent manner. Further, these findings suggest that immunosuppressive agents that spare IL-2 would support the NK cell response to EBV in vivo. This response involves the NKG2A/NKG2C axis. Regulation of the NKG2A/NKG2C ligand HLA-E may represent a mechanism of NK immune evasion by latent EBV infection.

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