Understanding the fundamental processes that determine an optimal immune memory response has been the bedrock of vaccine therapy and cell-based immunotherapy. Historically, therapeutic strategies have focused on strengthening the adaptive immune arm, the primary agents that effectuate immunological memory. More recently, research has expanded to include an innate immune arm that can retain a durable enhanced capability after initial activation, a phenomenon more commonly known as trained immunity. Natural killer (NK) cells have been pioneers in traversing the borders of innate and adaptive immunity, as best demonstrated in studies on mouse and human cytomegalovirus infection, whereby NK cells can acquire long-lived memory in an antigen-specific manner. At the same time, NK cells can acquire features of trained immunity, whereby activation by proinflammatory cytokines can nonspecifically enhance longevity and functional potential that is maintained weeks after primary exposure. Together, this malleability makes NK cells a promising and versatile tool for cell-based immunotherapy. One of the fundamental mechanisms by which these NK cells and all immune memory cells retain potential is via epigenetic reprogramming. This R01 proposal thus focuses on understanding and dissecting out epigenetic features that establish an immune memory program, and how these programs fundamentally differ between antigen-specific and trained immune memory. We study two related but non-mutually exclusive readouts of epigenetic regulation: chromatin accessibility and DNA methylation. Based on our previous work comparing the in vivo open chromatin landscapes of antigen-specific NK cells and CD8+ T cells, we have identified AP-1 factor JunB as playing a role at least during early antigen-specific responses, and have additional evidence that suggests it may also play a role in trained immunity. Based on performing similar analyses that instead focus on the DNA methylome, we further explore the role of genome-wide active DNA hypomethylation in establishing antigen-specific and trained NK cell memory responses. As a result, Aim 1 focuses on perturbing the open chromatin landscape via JunB-depletion, while Aim 2 disrupts the DNA methylome via Tet2-depletion. By doing so, we will identify key candidate regulatory elements and genes that link epigenetic activity to cellular and functional phenotypes, thereby providing a launchpad for therapeutic strategies that co-opt NK cell memory traits to improve NK-cell based therapies.