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Engagement of TLR3, TLR7, and NKG2D Regulate IFN- Secretion but Not NKG2D-Mediated Cytotoxicity by Human NK Cells Stimulated with Suboptimal Doses of IL-12¹

Laboratorio de Inmunogenética, Hospital de Clínicas "José de San Martín," and Departamento de Microbiología, Facultad de Medicina, Universidad de Buenos Aires (UBA), Buenos Aires, Argentina

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-, and investigated the secretion of IFN-, 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-. Inhibition of IFN- secretion by chloroquine suggested that internalization of the TLR agonists was necessary. Also, secretion of IFN- was dependent on MEK1/ERK, p38 MAPK, p70^S6 kinase, and NF-B, but not on calcineurin. IFN- induced a similar effect, but promoted lesser IFN- 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- 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- 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--dependent NK cell-mediated effector functions, which could be a strategy to promote Th1-biased immune responses in pathological situations such as cancer.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was funded with grants from National Agency for Promotion of Science and Technology, Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina, and Universidad de Buenos Aires (all to N.W.Z.). We also thank Fundación Sales for additional financial support. M.V.G., M.B.F., and C.I.D. are fellows of Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina. L.E.R. holds a fellowship of ANPCYT. N.W.Z. is a member of the Researcher Career of Consejo Nacional de Investigaciones Científicas y Técnicas de Argentina.

² Address correspondence and reprint requests to Dr. Norberto W. Zwirner, Laboratorio de Inmunogenética, Hospital de Clínicas "José de San Martín," Av. Córdoba 2351, 3^er piso., C1120AAF Buenos Aires, Argentina. E-mail address: nwz{at}sinectis.com.ar

³ Abbreviations used in this paper: NKG2DL, NKG2D ligand; CsA, cyclosporine; DC, dendritic cell; IC, isotype-matched negative control; MICA, MHC class I-chain related A; ODN, synthetic oligonucleotide containing unmethylated CpG sequences; Sz, sulfasalazine.

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