This is distinct from HCMV-induced adaptive NK cells, which have a number of intracellular adapter alterations that impact FcRIIIa signaling.30 These results also show for the first time that cytokine-induced memory-like NK cells have enhanced IFN- responses to FcRIIIa triggering, as well as enhanced antibody-dependent cellular cytotoxicity in vitro, suggesting that memory-like NK cells will be effective responders to antibody-opsonized targets. of licensing status. These results demonstrate that both licensed and unlicensed memory-like NK cell populations have enhanced functionality, which may be translated to improve leukemia immunotherapy. strong class=”kwd-title” Keywords: NK cell, adoptive Biotin-PEG3-amine immunotherapy, cytokines, NK cell education, NK cell memory Graphical Abstract Introduction Natural killer (NK) cells are innate lymphoid cells that mediate anti-tumor immune responses, especially against hematologic malignancies.1,2 Distinct from adaptive T and B lymphocytes, NK cells express a Biotin-PEG3-amine variety of germline DNA-encoded, stochastically-expressed activating and inhibitory receptors FA3 that govern functional responses to target cells.3 NK cell activating receptors (e.g. FcRIIIa, NKG2D, natural cytotoxicity receptors) generally recognize cell stress ligands expressed on virally-infected or malignantly-transformed cells, or antibody-opsonized targets. In contrast, inhibitory receptors [e.g. CD94/NKG2A and killer immunoglobulin-like receptors (KIR)] recognize major histocompatibility complex (MHC) class I-type molecules. In addition to modulating functional responsiveness, NK cell inhibitory receptors are critical for promoting NK cell tolerance. In order to become functionally competent, maturing NK cells require a signaling event through an inhibitory receptor engaging its cognate MHC class I ligand, a dynamic process termed licensing or education.4 Unlicensed NK cells that express neither KIR nor CD94/NKG2A, or that only express KIR without a cognate ligand present in that individual, are anergic to activating stimuli,4C8 ensuring NK cell tolerance under normal homeostatic conditions. While licensing can occur through either CD94/NKG2A or KIR, significantly higher effector capacity is achieved when licensing is KIR-mediated.9 Studies have shown that the activation state of murine NK cells can alter unlicensed NK cell responses. Specifically, under inflammatory conditions, traditional licensing rules may not apply.4,8,10 In humans, there have been conflicting reports about the impact of short-term cytokine exposure on licensed or unlicensed NK cell responsiveness.5,6,11,12 Thus, it remains uncertain how Biotin-PEG3-amine specific cytokine receptor signals, such as those induced by interleukin (IL)-12/15/18, may modulate functional response in the context of licensing. Recent reports have demonstrated that NK cell function is also shaped by prior experience.13 Exposure to specific haptens, viral infection, or combined cytokine pre-activation elicits innate memory or memory-like responses from murine NK cells.14C16 Paralleling findings in model organisms, brief combined pre-activation with IL-12, IL-15, and IL-18 results in the differentiation of human memory-like NK cells.17,18 While the initial pre-activating cytokine stimulus results in potent NK cell activation, within days the NK cells return to their baseline activation state, and memory-like differentiation is detectable after 1 week in vitro with survival supported by low-dose IL-15.17 These memory-like NK cells show enhanced responsiveness upon re-stimulation with diverse stimuli, including cytokines or tumor targets, for weeks to months following the initial pre-activation event. Human memory-like NK cell recognition of, and functional response against, leukemia targets is also enhanced both in vitro and in vivo. 19 As a result, cytokine-induced memory has been translated to the clinic in an ongoing memory-like NK cell adoptive immunotherapy trial in patients with relapsed or refractory acute myeloid leukemia (AML, “type”:”clinical-trial”,”attrs”:”text”:”NCT01898793″,”term_id”:”NCT01898793″NCT01898793).19,20 Preliminary results from this trial have demonstrated significant donor memory-like NK cell engraftment of recipient bone marrow, and retention of enhanced memory-like functionality upon ex vivo re-stimulation with tumor targets. In addition, clinical responses were observed in 5 of 9 patients treated with 0.5C10x106 NK cells/kg, demonstrating the immunotherapeutic potential of memory-like NK cells in the setting of AML.19 Based upon results in the hematopoietic cell transplantation setting,21C23 MHC-haploidentical donors are chosen for NK cell-based immunotherapy to facilitate inhibitory KIR to KIR-ligand mismatch, thereby reducing NK cell inhibitory signaling and enhancing anti-tumor activity. However, the impact of donor NK Biotin-PEG3-amine cell licensing status on NK cell adoptive immunotherapy outcomes has not been widely explored. Here, we investigated how the enhanced function of.