Background Allogeneic NK cell adoptive immunotherapy is usually a growing therapeutic option for patients. or IL-15 activation enhanced in vitro cytotoxicity compared to pre-activated cells. There was no difference in final product composition or cytotoxicity between cytokine cohorts. Conclusion Clinical-scale/cGMP production of NK cells using CD3/CD19 cell-depletion effectively minimized T cell and B cell contamination in a single manipulation without compromise to NK cell recovery. Cytokine-activation increased in vitro cytotoxicity compared to column-depleted, pre-activated NK cells. INTRODUCTION Human natural killer (NK) cells are lymphocytes of the innate immune system with effector and regulatory functions that have been exploited for their antitumor and antiviral potential. Approximately 10 to 20 percent of circulating lymphocytes are NK cells that surveil for virally-infected and transformed cells and exert potent cytotoxic activity in the absence of prior antigenic sensitization.1 NK cells are a phenotypically and functionally heterogeneous population distinguished by surface expression of CD56 and lack of T-cell antigens such as the CD3 pan-T-cell marker or the T-cell receptor (CD56+CD3?). CD56 is an isoform of the neural cell adhesion molecule with unknown function, but CD56 density marks the maturation status of NK cells such that CD56bright NK cells differentiate into CD56dim NK cells supporting a linear model of development between these subsets.2 These subsets are functionally divided into a CD56bright immunoregulatory populace that Npy predominate in secondary lymphoid tissue and produce cytokines (e.g., IFN-, TNF-, IL-10, IL-13, and GM-CSF), and a CD56dim cytotoxic populace primarily in circulation.3,4 Subpopulations of the CD56dim cells expressing the low-affinity Fc receptor, CD16, participate in antibody-dependent cell-mediated cytotoxicity.5,6 NK cells lyse target cells in the absence of major histocompatibility complex class I (MHC-I) expression, which is unique from cytotoxic T-lymphocytes that require antigen presentation by MHC-I to become activated for killing.7 However, NK cells do not function independently of MHC-I. In a process known as licensing, na?ve NK cells that engage MHC-I are educated to recognize self AZD6642 and remain quiescent while simultaneously becoming activated to lyse targets that lack or down-regulate this expression.8 Virally-infected and transformed cells commonly down-regulate MHC as a mechanism of immune evasion from cytotoxic T-lymphocytes, and this loss of self renders them sensitive to killing by NK cells.9 NK cell function is tightly regulated by a combination of signals received via inhibitory and stimulatory receptors that recognize MHC-I and other surface ligands.8,10,11 It is the net effect of complex signaling that determines whether NK cells become cytotoxic or remain quiescent. The killer immunoglobulin-like receptor (KIR), lectin NKG2 receptors, and natural cytotoxicity receptors AZD6642 (NCRs) are the main families of known NK AZD6642 cell receptors described.12 KIRs interact with classic HLA ligands (HLA-A, -B, -C) whereas NKG2 receptors interact with non-classic HLA ligands (HLA-E). KIR and NKG2 each have subsets of stimulatory and inhibitory receptors; under normal physiologic conditions the inhibitory signals prevail. It is not enough to remove the inhibitory signals to activate NK cell killing but rather, activating receptors must also interact with their cognate ligand, which are generally only upregulated under conditions that stress the target cell.1,8,12 With each NK cell expressing a different pattern of receptors, they can sense and react to subtle changes in the microenvironment. NK cells have gained significant traction in adoptive immunotherapy clinical trials due to their graft-versus-tumor effect without apparent harm of graft-versus-host disease (GVHD).13,14 NK cells are cytotoxic against a variety of solid organ and hematologic malignancies in vitro and IL-2 or IL-15 exposure has been shown to augment cytotoxicity against NK-resistant AZD6642 cell lines.15 Early trials of autologous NK cells lacked clinically efficacy, 16 presumably due to inhibition by self-MHC, and current immunotherapy is focused on allogeneic sources. Mismatch of KIR-HLA ligands, whereby the recipient is usually missing the cognate ligand to an inhibitory KIR around the donor cells, has been shown to enhance alloreactivity and graft-versus-leukemia in vitro and in vivo.17,18 The greatest clinical benefit for allogeneic NK.