Cytokine-activated NK cells display distinct gene expression programs in response to cytokine withdrawal. genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15Cinduced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15Cstimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy. Introduction Natural killer (NK)-cellCbased immunotherapy is a potential therapeutic modality in patients with advanced cancers as transfer of haploidentical NK cells induces beneficial responses in patients with hematological malignancies; and leukemia clearance correlates with persistence and in vivo expansion of NK cells after infusion.1-3 Thus, sustained NK-cell activity in vivo MM-102 likely represents a therapy performance-limiting factor. The type I cytokine family members interleukin-2 (IL-2) and IL-15 are essential in controlling homeostasis of innate and adaptive immunity.4 Despite their different protein sequences, IL-2 and IL-15 bind to shared (IL-2/IL-15R) and (c) subunits, but engage separate -chain receptors (IL-2/IL-15R).5 Although IL-2/IL-15 receptor complexes activate similar signal transduction cascades, they display distinct activities in vivo. Although IL-2 preferentially expands regulatory T cells and CD4+ helper T cells,6 IL-15 supports development of central MM-102 memory T cells7,8 and NK cells.9,10 Furthermore, sustained persistence of infused NK cells is linked to increased MM-102 levels of endogenous IL-15 following treatment with high-dose cyclophosphamide and fludarabine.1 Thus, NK-cell activation with IL-15 may have beneficial effects on their postinfusion activity. The molecular mechanisms underlying distinct effects from IL-2 and IL-15 on NK-cell proliferation and persistence are, however, unknown. Recently, studies of translatomes (ie, the pool of translated messenger RNAs [mRNAs]) using polysome or ribosome profiling have highlighted mRNA translation as a key mechanism controlling gene expression and influencing a wide range of functions in immune cells.11-13 Changes in translational efficiency affect protein levels without changes in steady-state mRNA levels, thereby enabling rapid adaptation to environmental changes essential for a functional immune system.13 Thus, profiling mRNA translation may be essential to efficiently link observed immune cell phenotypes to underlying gene expression programs. Here, we uncover IL-15Cmediated improved post-cytokine-withdrawal functions of NK cells associated with augmented mammalian target of rapamycin (mTOR) signaling and an IL-15Cprimed gene expression MM-102 program. Such detailed mechanistic and functional understanding of IL-15s impact on human NK cells supports implementation of IL-15 in adoptive NK-cell therapy. Materials and methods Patient MM-102 gene expression data sets We used a recent data set14 to investigate the role of IL-15 in patients (supplemental Methods, available on the Web site). Cell cultures K562 (short tandem repeat fingerprint in supplemental Table 1) and Epstein-Barr virus (EBV)-transformed B cells were maintained in Iscove modified Dulbecco medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen). Prior to NK-cell isolation, Rabbit Polyclonal to IRAK2 T cells were removed by CD3 depletion (RosetteSep kit from StemCell Technologies) during Ficoll gradient centrifugation. Primary human NK cells were subsequently isolated by magnetic-activated cell sorting purification (purity 98%; Miltenyi Biotec). Cytokine activation and expansion of NK cells To activate NK cells, 18.3 ng/mL recombinant human IL-2 (50% effective dose 0.1 ng/mL; Peprotech) or recombinant human IL-15 (50% effective dose 0.1 ng/mL; Biological Resources Branch, National Cancer Institute) was added to 1 mL of Iscove modified Dulbecco medium supplemented with 10% human AB serum containing 2 106 NK cells in 24-well plates for 48 hours. To evaluate the molecular mechanisms, dimethyl sulfoxide (DMSO), the mTOR inhibitor torin-1 (1 M; Tocris Biosciences), the STAT-3Cselective inhibitor S3I-201 (100 M; Sigma-Aldrich), or the STAT-5Cselective inhibitor CAS285986-31-4 (400 M; Merck) was added during the activation. For in vitro expansion of human primary NK cells, a clinically applicable protocol with minor modifications was used15 (see supplemental Methods). NK-cell function The cytolytic activity of NK cells.