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Proteolytic Activation of the Cytotoxic Phenotype during Human NK Cell Development¹

Josephine L. Meade^*, Erica B. Wilson^*, Tim D. Holmes^*, Erika A. de Wynter^*, Peter Brett, Liz Straszynski^*, Paul A. S. Ballard, Joseph A. Trapani, Michael F. McDermott^* and Graham P. Cook^2,*

^* Leeds Institute of Molecular Medicine, Wellcome Trust Brenner Building, University of Leeds, St. James’s University Hospital, Leeds, United Kingdom; Division of Biomaterials and Tissue Engineering, University College London Eastman Dental Institute, London, United Kingdom; Department of Obstetrics and Gynaecology, Friarage Hospital, Northallerton, United Kingdom; and Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrew’s Place, East Melbourne, Australia

NK cells induce apoptosis in target cells via the perforin-mediated delivery of granzyme molecules. Cytotoxic human NK cells can be generated by IL-15-mediated differentiation of CD34⁺ cells in vitro and these cultures have been used extensively to analyze the development of the NK cell surface phenotype. We have used NK cell differentiation in vitro together with protease-deficient human NK cells to analyze the acquisition of the cytotoxic phenotype. Granzymes are synthesized as inactive zymogens and are proteolytically activated by the cysteine protease cathepsin C. Cathepsin C is also synthesized as a zymogen and activated by proteolysis. We show that human NK cells generated in vitro undergo granule exocytosis and induce the caspase cascade in target cells. IL-15 and stem cell factor (IL-15 plus SCF) induced the expression of the granzyme B and perforin genes and the activation of cathepsin C and granzyme B zymogens. Perforin activation is also mediated by a cysteine protease and IL-15 plus SCF-mediated differentiation was accompanied by perforin processing. However, cathepsin C-deficient human NK cells revealed that perforin processing could occur in the absence of cathepsin C activity. The combination of IL-15 plus SCF is therefore sufficient to coordinate the development of the NK cell surface phenotype with the expression and proteolytic activation of the cytotoxic machinery, reflecting the central role of IL-15 in NK cell development.

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 Candlelighter’s, Yorkshire Cancer Research and The Laura Crane Trust.

² Address correspondence and reprint requests to Dr. Graham Cook, University of Leeds, Wellcome Brenner Building, St. James’s University Hospital, Leeds, U.K. E-mail address: g.p.cook{at}leeds.ac.uk

³ Abbreviations used in this paper: SCF, stem cell factor; FHL, familial hemophagocytic lymphohistiocytosis; DNAM-1, DNAX accessory molecule-1.