Acute myeloid leukemia (AML) is a common hematologic malignancy with an estimated prevalence of 3.8 cases per 100,000. Patients with relapsed/refractory (rel/ref) AML have poor prognosis and new, well tolerated therapeutic options are needed. Recently, paradigm changing studies of murine natural killer (NK) cells have identified that these innate immune cells have memory-like properties. Our lab has confirmed cytokine-induced memory-like (CIML) NK cells in humans. Further, pre-clinical studies have demonstrated that human CIML NK cells have superior anti-leukemia activity in vitro and in xenograft models. We will utilize samples from the first- in-human study of CIML NK cells in AML patients to perform correlative studies evaluating CIML NK cell expansion, survival, activation, and functions in patients. Samples collected from patients at various time points before and after allogeneic donor NK cell infusion and the indicated parameters evaluated by flow cytometry. We expect that human CIML NK cells will exhibit enhance expansion in vivo as well as enhance effector function against primary tumors ex vivo. Our proposed work will concurrently examine human CIML NK cells in the context of adoptive immunotherapy for the treatment of AML, as well as the underlying mechanisms that contribute to the differentiation, maintenance, and enhanced effector function upon secondary activation. Understanding these mechanisms will expand the clinical potential of CIML NK cells. The T-box transcription factor Eomes has been shown to be involved in NK cell effector function and preliminary data from our lab suggest it is upregulated in CIML NK cells compared to controls. Here we will utilize and loss- and gain-of-function analyses to determine if Eomes is necessary and sufficient, respectively, to promote CIML NK cell differentiation and effector function. This will be achieved by lentivirally transducing human CIML NK cells, resulting in the expression of Eomes (gain-of function) or Eomes shRNA (knock-down). We expect that if Eomes is important for CIML NK cell biology, knock-down of its expression will abrogate CIML NK cell differentiation or function. Likewise, if Eomes is sufficient for CIML induction, its forced expression will lead to the enhanced function of NK cells regardless of cytokine pre-activation. Since CIML NK cell function is passed on to daughter cells, we hypothesize that epigenetic mechanisms contribute to their enhanced functional program. We will examine the histone modifications and DNA methylation status of candidate genes, comparing naive, pre-activated, and CIML NK cells. We will focus on candidate genes that are functionally important and differentially expressed in CIML versus control NK cells. We expect that the enhanced responsiveness observed by CIML NK cells will correlate with candidate genes that display active histone marks and decreased DNA promoter methylation in CIML NK cells compared to controls.