NK Cell Activity During Human Cytomegalovirus Infection Is Dominated by US2–11-Mediated HLA Class I Down-Regulation 1 Christine S. Falk,* Michael Mach, † Dolores J. Schendel,* Elisabeth H. Weiss, ‡ Ivan Hilgert, § and Gabriele Hahn 2¶ A highly attractive approach to investigate the influence and hierarchical organization of viral proteins on cellular immune responses is to employ mutant viruses carrying deletions of various virus-encoded, immune-modulating genes. Here, we introduce a novel set of deletion mutants of the human CMV (HCMV) lacking the UL40 region either alone or on the background of a deletion mutant devoid of the entire US2–11 region. Deletion of UL40 had no significant effect on lysis of infected cells by NK cells, indicating that the expected enhancement of HLA-E expression by specific peptides derived from HCMV-encoded gpUL40 leader sequences was insufficient to confer target cell protection. Moreover, the kinetics of MHC class I down-regulation by US2–11 genes observed at early and late phases postinfection with wild-type virus correlated with increased susceptibility to NK lysis. Thus, the influence of HCMV genes on NK reactivity follows a hierarchy dominated by the US2–11 region, which encodes all viral genes capable of down-modulating expression of classical and non-classical MHC class I molecules. The insights gained from studies of such virus mutants may impact on future therapeutic strategies and vaccine development and incorporate NK cells in the line of defense mechanisms against HCMV infection. The Journal of Immunology, 2002, 169: 3257–3266. A n impressive variety of interactions between viral pro- teins and components of the host immune system has developed through evolutionary processes that maintain the survival of both the adapted virus and the host. Members of the herpesvirus family, in particular the human CMV (HCMV), 3 have acquired defense mechanisms to interfere with virus-specific im- mune responses (reviewed in Ref. 1– 4). Viral infectivity and im- mune surveillance as well as the balance between viral latency and reactivation are key determinants of pathogenesis (5). The conse- quences of a dysbalance between viral activity and immune re- sponse become evident in life-threatening HCMV infections in congenitally infected newborns, transplant recipients, and immu- nocompromised patients. Endemic infection of populations with extended strain variations, life-long persistent infection, and con- trol of reactivation from latency demonstrates the importance of understanding the immunological control of HCMV infection (6 – 8). Some HCMV immune-modulating proteins affect the core of cellular immunity through their interference with expression of MHC molecules (reviewed in Refs. 1 and 2). The impact of MHC class Ia and Ib modulation by these viral genes on NK cell-medi- ated responses is controversial, since many studies relied on model systems using cells transfected with individual selected genes. Four proteins encoded by the US2–11 region were found to down- regulate class I surface expression by different mechanisms, i.e., relocation of MHC heavy chains to the cytoplasm by US2 and US11 (9, 10), retention of class I molecules in the ER by US3 (11), and prevention of peptide transport through TAP by US6 (12, 13). Deletion of the entire region containing the US2–11 genes from the viral genome has previously been shown to prevent down- regulation of MHC class I molecules, indicating that virtually all MHC down-regulating genes are located within this region (14). US2–11-mediated class I modulation has been postulated to func- tion as a viral defense mechanism, allowing infected cells to es- cape recognition by class I-restricted, HCMV-specific CTL (15). Thus, the role of class I down-regulation by US2–11 genes as well as the influence of additional class I modulating genes, such as gpUL40, with respect to NK cell regulation have not been evalu- ated under conditions of natural HCMV infection. NK cell activity is mainly regulated by class I molecules in a negative fashion via receptor-mediated inhibition (reviewed in Refs. 16 –18). Therefore, an HCMV-mediated class I down-regu- lation may affect NK recognition of infected cells. A high density of MHC molecules results in a turning off of signals for NK cells that are triggered by interactions between class I molecules and inhibitory receptors (IR). The specificity of each NK cell is char- acterized by its individual expression of one or more IR that me- diate inhibition following contact with corresponding class I ligands. According to the missing self hypothesis, in situations of loss or sub- stantial reduction of total class I expression or of individual allelic *Institute of Molecular Immunology, GSF National Research Center for the Envi- ronment and Health, Munich, Germany; † Institute of Clinical and Molecular Virol- ogy, Friedrich Alexander University of Erlangen-Nurnberg, Erlangen, Germany; ‡ In- stitute of Anthropology and Human Genetics, Ludwig Maximilians University, Munich, Germany; § Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic; ¶ Max von Pettenkofer Institute, Depart- ment of Virology, Ludwig Maximilians University, Munich, Germany Received for publication December 14, 2001. Accepted for publication July 11, 2002. 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 grants from the Wilhelm Sander-Stiftung (to G.H.), the Deutsche Forschungsgemeinschaft (to G.H. and M.M.), and the SFB571 (to D.J.S and E.H.W.). 2 Address correspondence and reprint requests to Dr. Gabriele Hahn, Max von Petten- kofer Institute, Department of Virology, Ludwig Maximilians University, Pettenkofer- strasse 9a, D-80336 Munich, Germany. E-mail address: ghahn@m3401.mpk.med.uni- muenchen.de 3 Abbreviations used in this paper: HCMV, human CMV; ADCC, Ab-dependent cell- mediated cytotoxicity; BAC, bacterial artificial chromosome; EGFP, enhanced green fluorescence protein; HF, human foreskin fibroblast; IR, inhibitory receptor; KIR, killer cell Ig-like receptor; LIR, leukocyte Ig-like receptor; MOI, multiplicity of in- fection; p.i., postinfection; tn, transposon; RCR, relative cytotoxic response; WT, wild type. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00