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NKp30 Ligation Induces Rapid Activation of the Canonical NF-B Pathway in NK Cells¹

Rahul Pandey^*, Christine M. DeStephan^*, Lisa A. Madge, Michael J. May and Jordan S. Orange^2,*

^* Division of Allergy and Immunology, The Joseph Stokes Jr. Research Institute, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, and Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA 19104

Studies of patients with congenital immunodeficiency due to mutation of the NF-B essential modulator (NEMO) gene have demonstrated that NEMO integrity is required for NK cell cytotoxicity. Thus, we have studied the physiology of NF-B activation in NK cells during the cytolytic program. In resting ex vivo human NK cells or cell lines, IB was degraded after 10 min exposure to PMA and ionomycin, or TNF and was maximally degraded by 30 min. Ligation of several NK cell activation receptors including NKp30 induced a similar response and was blocked by pretreatment with the proteosome inhibitor MG132. There was no short-term effect on p100 processing, the signature of noncanonical NF-B activation. NK cell IB degradation corresponded to increases in nuclear NF-B as detected by EMSA. Supershift of stimulated NK cells and fluorescence microscopy of individual NK cells in cytolytic conjugates demonstrated that the p65/p50 heterodimer was the primary NF-B used. NF-B function was evaluated in NK92 cells transduced with a B GFP reporter, and their conjugation with K562 cells or ligation of NKp30 ligation resulted in rapid GFP accumulation. The latter was prevented by the Syk inhibitor piceatannol. Thus, NK cell activation signaling specifically induces transcriptional activation and synthesis of new NF-B dependent proteins during the initiation of cytotoxicity.

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 supported by Grant N01 AI-22070 from the U.S. Immunodeficiency Network (to J.S.O. and M.J.M.), Grant AI-067946 from the National Institutes of Health, from The Pennsylvania Department of Health, and by a career development award from the American Academy of Allergy, Asthma and Immunology (to J.S.O.). The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions from this study.

² Address correspondence and reprint requests to Dr. Jordan S. Orange, The Children’s Hospital of Philadelphia, Abramson Research Center 1016H, 3615 Civic Center Boulevard, Philadelphia, PA 19104. E-mail address: Orange{at}mail.med.upenn.edu

³ Abbreviations used in this paper: IKK, IB kinase; NEMO, NF-B essential modulator; MFI, mean fluorescence intensity.