title: Engagement of TLR3, TLR7, and NKG2D regulate IFN-gamma secretion but not NKG2D-mediated cytotoxicity by human NK cells stimulated with suboptimal doses of IL-12.
abstract: NK cells express different TLRs, such as TLR3, TLR7, and TLR9, but little is known about their role in NK cell stimulation. In this study, we used specific agonists (poly(I:C), loxoribine, and synthetic oligonucleotides containing unmethylated CpG sequences to stimulate human NK cells without or with suboptimal doses of IL-12, IL-15, or IFN-alpha, and investigated the secretion of IFN-gamma, cytotoxicity, and expression of the activating receptor NKG2D. Poly(I:C) and loxoribine, in conjunction with IL-12, but not IL-15, triggered secretion of IFN-gamma. Inhibition of IFN-gamma secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN-gamma was dependent on MEK1/ERK, p38 MAPK, p70(S6) kinase, and NF-kappaB, but not on calcineurin. IFN-alpha induced a similar effect, but promoted lesser IFN-gamma secretion. However, cytotoxicity (51Cr release assays) against MHC class I-chain related A (MICA)- and MICA+ tumor targets remained unchanged, as well as the expression of the NKG2D receptor. Excitingly, IFN-gamma secretion was significantly increased when NK cells were stimulated with poly(I:C) or loxoribine and IL-12, and NKG2D engagement was induced by coculture with MICA+ tumor cells in a PI3K-dependent manner. We conclude that resting NK cells secrete high levels of IFN-gamma in response to agonists of TLR3 or TLR7 and IL-12, and this effect can be further enhanced by costimulation through NKG2D. Hence, integration of the signaling cascades that involve TLR3, TLR7, IL-12, and NKG2D emerges as a critical step to promote IFN-gamma-dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.
authors.affiliation: Laboratorio de Inmunogenética, Hospital de Clínicas José de San Martín, Buenos Aires, Argentina.
authors.forename: María V
authors.initials: MV
authors.lastname: Girart
authors.forename: Mercedes B
authors.initials: MB
authors.lastname: Fuertes
authors.forename: Carolina I
authors.initials: CI
authors.lastname: Domaica
authors.forename: Lucas E
authors.initials: LE
authors.lastname: Rossi
authors.forename: Norberto W
authors.initials: NW
authors.lastname: Zwirner
mesh_terms: D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D002999:Clone Cells; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology; D004306:Dose-Response Relationship, Immunologic; D015972:Gene Expression Regulation, Neoplastic / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D016898:Interferon-alpha / Q000502:physiology; D007371:Interferon-gamma / Q000378:metabolism*; D018664:Interleukin-12 / Q000494:pharmacology / Q000502:physiology*; D007694:Killer Cells, Natural / Q000276:immunology* / Q000378:metabolism; D008213:Lymphocyte Activation / Q000276:immunology*; D008545:Melanoma / Q000276:immunology / Q000473:pathology / Q000628:therapy; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011971:Receptors, Immunologic / Q000096:biosynthesis / Q000378:metabolism* / Q000502:physiology; D055607:Receptors, Natural Killer Cell; D051196:Toll-Like Receptor 3 / Q000819:agonists / Q000276:immunology / Q000378:metabolism*; D051199:Toll-Like Receptor 7 / Q000819:agonists / Q000276:immunology / Q000378:metabolism*; D051217:Toll-Like Receptor 9 / Q000819:agonists