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Purpose: Alternative strategies to EGFR blockage by mAbs is necessary to improve the efficacy of therapy in patients with locally advanced or metastatic pancreatic cancer. One such strategy includes the use of NK cells to clear cetuximab-coated tumor cells, as need for novel therapeutic approaches to enhance the efficacy of cetuximab is evident. We show that IL-21 enhances NK cell-mediated effector functions against cetuximab-coated pancreatic tumor cells irrespective of KRAS mutation status., Experimental Design: NK cells from normal donors or donors with pancreatic cancer were used to assess ADCC, IFN-γ release, and T-cell chemotaxis toward human pancreatic cancer cell lines. The in vivo efficacy of IL-21 in combination with cetuximab was evaluated in a subcutaneous and intraperitoneal model of pancreatic cancer., Results: NK cell lysis of cetuximab-coated wild-type and mutant kras pancreatic cancer cell lines were significantly higher following NK cell IL-21 treatment. In response to cetuximab-coated pancreatic tumor cells, IL-21-treated NK cells secreted significantly higher levels of IFN-γ and chemokines, increased chemotaxis of T cells, and enhanced NK cell signal transduction via activation of ERK and STAT1. Treatment of mice bearing subcutaneous or intraperitoneal EGFR-positive pancreatic tumor xenografts with mIL-21 and cetuximab led to significant inhibition of tumor growth, a result further enhanced by the addition of gemcitabine., Conclusions: These results suggest that cetuximab treatment in combination with IL-21 adjuvant therapy in patients with EGFR-positive pancreatic cancer results in significant NK cell activation, irrespective of KRAS mutation status, and may be a potential therapeutic strategy. Clin Cancer Res; 23(2); 489-502. ©2016 AACR., Competing Interests: The authors have no conflicts of interest to disclose., (©2016 American Association for Cancer Research.)

Background: Traditionally, the CD56(dim)CD16(+) subset of Natural Killer (NK) cells has been thought to mediate cellular cytotoxicity with modest cytokine secretion capacity. However, studies have suggested that this subset may exert a more diverse array of immunological functions. There exists a lack of well-developed functional models to describe the behavior of activated NK cells, and the interactions between signaling pathways that facilitate effector functions are not well understood. In the present study, a combination of genome-wide microarray analyses and systems-level bioinformatics approaches were utilized to elucidate the transcriptional landscape of NK cells activated via interactions with antibody-coated targets in the presence of interleukin-12 (IL-12)., Methods: We conducted differential gene expression analysis of CD56(dim)CD16(+) NK cells following FcR stimulation in the presence or absence of IL-12. Next, we functionally characterized gene sets according to patterns of gene expression and validated representative genes using RT-PCR. IPA was utilized for biological pathway analysis, and an enriched network of interacting genes was generated using GeneMANIA. Furthermore, PAJEK and the HITS algorithm were employed to identify important genes in the network according to betweeness centrality, hub, and authority node metrics., Results: Analyses revealed that CD56(dim)CD16(+) NK cells co-stimulated via the Fc receptor (FcR) and IL-12R led to the expression of a unique set of genes, including genes encoding cytotoxicity receptors, apoptotic proteins, intracellular signaling molecules, and cytokines that may mediate enhanced cytotoxicity and interactions with other immune cells within inflammatory tissues. Network analyses identified a novel set of connected key players, BATF, IRF4, TBX21, and IFNG, within an integrated network composed of differentially expressed genes in NK cells stimulated by various conditions (immobilized IgG, IL-12, or the combination of IgG and IL-12)., Conclusions: These results are the first to address the global mechanisms by which NK cells mediate their biological functions when encountering antibody-coated targets within inflammatory sites. Moreover, this study has identified a set of high-priority targets for subsequent investigation into strategies to combat cancer by enhancing the anti-tumor activity of CD56(dim)CD16(+) NK cells.

Interleukin-2 (IL-2) activates extracellular signal-regulated protein kinase (ERK) within immune cells. To examine the profile of phosphorylated ERK (p-ERK) in IL-2 stimulated immune cells of normal donors and patients receiving IL-2 therapy, we developed a dual parameter flow-cytometric assay. An analysis of PBMCs stimulated with IL-2 indicated that IL-2 exposure induced p-ERK in CD56bright NK cells and CD14+ monocytes, but not in CD3+ T cells or CD21+ B cells. CD3+ T cells that were induced to express functional high-affinity IL-2R did not exhibit enhanced p-ERK following IL-2 treatment. Measurement of p-ERK within PBMCs from cancer patients 1 h following their first dose of IL-2 revealed a complete absence of circulating NK cells, consistent with earlier observations. However, the total number of circulating CD14+ monocytes increased in these samples and 97% of these cells exhibited ERK activation. p-ERK was not observed in T cells post-IL-2 therapy. Analysis of PBMCs obtained 3 weeks post-IL-2 therapy revealed high-p-ERK levels in CD56bright NK cells in a subset of patients, while levels of p-ERK returned to baseline in monocytes. These studies reveal an effective method to detect ERK activation in immune cells and demonstrate that IL-2 activates ERK in a subset of NK cells and monocytes but not T cells.

Natural killer (NK) cells express an activating receptor for the Fc portion of IgG (FcgammaRIIIa) that mediates interferon (IFN)-gamma production in response to antibody (Ab)-coated targets. We have previously demonstrated that NK cells activated with interleukin-12 (IL-12) in the presence of immobilized IgG secrete 10-fold or more higher levels of IFN-gamma as compared with stimulation with either agent alone. We examined the intracellular signaling pathways responsible for this synergistic IFN-gamma production. NK cells costimulated via the FcR and the IL-12 receptor (IL-12R) exhibited enhanced levels of activated STAT4 and Syk as compared with NK cells stimulated through either receptor alone. Extracellular signal-regulated kinase (ERK) was also synergistically activated under these conditions. Studies with specific chemical inhibitors revealed that the activation of ERK was dependent on the activation of PI3-K, whose activation was dependent on Syk, and that sequential activation of these molecules was required for NK cell IFN-gamma production in response to FcR and IL-12 stimulation. Retroviral transfection of ERK1 into primary human NK cells substantially increased IFN-gamma production in response to immobilized IgG and IL-12, while transfection of human NK cells with a dominant-negative ERK1 abrogated IFN-gamma production. Confocal microscopy and cellular fractionation experiments revealed that FcgammaRIIIa and the IL-12R colocalized to areas of lipid raft microdomains in response to costimulation with IgG and IL-12. Chemical disruption of lipid rafts inhibited ERK signaling in response to costimulation and significantly inhibited IFN-gamma production. These data suggest that dual recruitment of FcgammaRIIIa and the IL-12R to lipid raft microdomains allows for enhanced activation of downstream signaling events that lead to IFN-gamma production.