Letters to the Editor 192 2 Department of Immunology, Erasmus MC, Erasmus University selected children with hypoplastic refractory cytopenia. Haematologica 2007; 92: Medical Center, Rotterdam, The Netherlands; 397–400. 3Department of Pediatrics and Adolescent Medicine, Division of 4 Dunn DE, Tanawattanacharoen P, Boccuni P, Nagakura S, Green SW, Kirby MR Pediatric Hematology and Oncology, University of Freiburg, et al. Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow Freiburg, Germany; failure syndromes. Ann Intern Med 1999; 131: 401–408. 4Department of Pathology, Clinical Centre South West, Bo¨blingen 5 Maciejewski JP, Follmann D, Nakamura R, Saunthararajah Y, Rivera CE, Simonis T et al. Increased frequency of HLA-DR2 in patients with paroxysmal nocturnal Clinics, Bo¨blingen, Germany; 5 hemoglobinuria and the PNH/aplastic anemia syndrome. Blood 2001; 98: Department of Clinical Genetics, Erasmus MC, Erasmus University 3513–3519. 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Supplementary Information accompanies this paper on the Leukemia website (http://www.nature.com/leu) Efficient generation of human natural killer cell lines by viral transformation Leukemia (2014) 28, 192–195; doi:10.1038/leu.2013.188 Most of these NK-like cell lines were derived from NK cell leukemia or lymphoma patients, lack important features of natural NK cells, are difficult to cultivate and require supplementation with various The short lifespan of human natural killer (NK) cells is one cytokines such as interleukin (IL)-2, IL-7, IL-12.2–6 Several of these of the major obstacles in human NK cell research and application. NK-like cell lines, like YT and NK-92, are latently infected with Here we describe the in vitro growth transformation of Epstein–Barr virus;7 the contribution of viral effectors to cell functional human NK cells by Herpesvirus saimiri (HVS), a primate proliferation and survival has not been studied. rhadinovirus. The primate gammaherpesvirus HVS strain C488 is able to NK cells are non-B, non-T lymphocytes that account for 2–18% efficiently transform human T lymphocytes of healthy donors to of lymphocytes in normal human peripheral blood. They are continuously proliferating T-cell lines in vitro.8 HVS strain C488 distinguished by a CD3 À CD56 þ phenotype and they commu- expresses two oncoproteins StpC (for saimiri transformation- nicate through interferon-g and tumor necrosis factor secretion associated protein of subgroup C) and Tip (for tyrosine kinase- with dendritic cells, macrophages, T cells and endothelial cells. The interacting protein).9 In vitro transformation with HVS is routinely natural lifespan for NK cells in the human body is 2 weeks.1 In performed by many research groups and used to establish model vitro, the lifespan of NK cells can be extended to a few weeks. Only cell lines.10–12 In order to detect further target cell populations for few human permanent NK-like cell lines are available for research. HVS infection, we studied early infection of human leukocytes Accepted article preview online 21 June 2013; advance online publication, 19 July 2013 Leukemia (2014) 179 – 238 & 2014 Macmillan Publishers Limited Letters to the Editor 193 Table 1. HVS-transformed NK cell lines express different NK-specific markers MJ23 WU36 LB33 DR32 KT09 CD2 (LFA-2) þþ þþþ ND þþ þþþ CD3 (TCR) ÀÀÀÀÀ CD4 ÀÀÀÀÀ CD8 þþ þþ þþ þþ þþ CD14 ÀÀÀÀÀ CD19 ÀÀÀÀÀ CD11b (ITGAM) ÀÀÀÀÀ CD16 (FCGR3A) þþþþþ CD25 (IL-2Ra) þþþ þþþ þþþ þþþ þþþ CD56 (NCAM1) þþþ þþþ þþþ þþþ þþþ CD57 (B3GAT1) ÀÀÀÀÀ CD58 (LFA3) þþþ þþþ ND þþþ þþþ CD69 (CLEC2C) þþþ þþþ þþþ þþþ þþþ CD83 (BL11) ND þþþ ND ND þþþ CD94 (KLRD1) þþþ þþþ þþþ þþþ þþþ CD122 (IL-2R) þþþ þþþ þþþ þþþ þþþ CD158a (KIR2DL1/S1) ÀÀþþÀ CD158b (KIR2DL2/DL3) ND þ ND ND À CD158e (KIR3DL1) ND þ ND ND À CD158f (KIR2DL5) ÀÀÀÀÀ CD160 (SLAMF7) ÀþþþÀ CD181 (IL-8R) ÀþþþÀ À CD244 (2B4) þþþ þþþ þþþ þþþ þþþ CD314 (NKG2D) þþþ þþþ þþþ þþþ þþþ CD319 (CS1) À À þþþ þþþ þþþ CD328 (Siglec-7) þþÀþþþþþ CD335 (NKp46) þþþ þþþ þþþ þþþ þþþ CD336 (NKp44) þþþ þþþ þþþ þþþ þþþ CD337 (NKp30) ND þþþ ND ND þþþ Siglec-9 ÀÀÀÀÀ IL-15Ra ÀÀÀÀÀ Granzyme B þþþ þþþ þþþ þþþ þþþ Perforin þþþ þþþ þþþ þþþ þþþ Granulysin þþþ þþþ þþþ þþþ þþþ Abbreviations: CD, cluster of differentiation; HVS, Herpesvirus saimiri; IL, interleukin; ND, no data; NK, natural killer. Flow cytometric analysis of five different HVS-transformed NK cell lines. A total of 34 extra- and intracellular markers were investigated. þ marker is expressed in 10–49%, þþin 50–89% and þþþ in 90–100% of NK cells; À symbolizes no expression; ND means no data, the cell line was not investigated for this marker. The NK cell lines have in common that they all express CD2, CD25, CD56, CD58, CD69, CD83, CD94, CD122, CD244, CD335, CD336, CD337, Granzyme B, Perforin and Granulysin. CD3, CD4, CD11b, CD14, CD19, CD57, CD158f, IL-15Ra and Siglec-9 were not observed on the surface of HVS-transformed NK cell lines. HVS transformation is a polyclonal event, resulting in mixed populations with different marker expression; CD8, CD16, CD158a, CD158b, CD158e, CD160, CD181, Cd319 and Siglec-7 are found on fractions, but not on all NK cells within the transformed cell lines. using a recombinant HVS harboring a PGK promoter-driven H-2Kk and NK-specific cluster of differentiation (CD) markers, and H-2Kk-IRES-bleoR cassette. Virus stocks from infected owl monkey eventually by fluorescence microscopy. This led to our discovery kidney cell viral supernatants were desalted and concentrated that HVS is able to efficiently in vitro transform human NK cells, through a Minimate Tangential Flow Filtration system (PALL, Dreieich, to stable cell lines with different NK phenotypes. Germany) with a MWCO 300 kDa filter cassette. In all, 500 ml HVS Infection of both CD8-positive and -negative, CD16-positive PGK-H2Kk-IRES-bleoR virus supernatant was concentrated to 15 ml and -negative NK cells could be observed. Non-infected (3.3 Â 107 genomes per ml). Cells infected with this recombinant control cells that were cultivated in parallel usually ceased HVS present murine H-2Kk on their cell surface and can be growing after 1 week. Even at 4 months after infection, flow detected and enriched with specific antibodies starting cytometric analysis of five HVS-NK cell lines indicated that a 1 day after infection.