Vimentin Expressed on Mycobacterium tuberculosis-Infected Human Monocytes Is Involved in Binding to the NKp46 Receptor 1 Ankita Garg,* † Peter F. Barnes,* †‡ Angel Porgador, ¶ Sugata Roy,* † Shiping Wu,* † Jagpreet S. Nanda,* † David E. Griffith, § William M. Girard, § Nenoo Rawal, ‡ Sreerama Shetty, ‡ and Ramakrishna Vankayalapati 2 * † We previously showed that human NK cells used the NKp46 receptor to lyse Mycobacterium tuberculosis H37Ra-infected mono- cytes. To identify ligands on H37Ra-infected human mononuclear phagocytes, we used anti-NKp46 to immunoprecipitate NKp46 from NK cells bound to its ligand(s) on H37Ra-infected monocytes. Mass spectrometry analysis identified a 57-kDa molecule, vimentin, as a putative ligand for NKp46. Vimentin expression was significantly up-regulated on the surface of infected monocytes, compared with uninfected cells, and this was confirmed by fluorescence microscopy. Anti-vimentin antiserum inhibited NK cell lysis of infected monocytes, whereas antiserum to actin, another filamentous protein, did not. CHO-K1 cells transfected with a vimentin construct were lysed much more efficiently by NK cells than cells transfected with a control plasmid. This lysis was inhibited by mAb-mediated masking of NKp46 (on NK cells) or vimentin (on infected monocytes). ELISA and Far Western blotting showed that recombinant vimentin bound to a NKp46 fusion protein. These results indicate that vimentin is involved in binding of NKp46 to M. tuberculosis H37Ra-infected mononuclear phagocytes. The Journal of Immunology, 2006, 177: 6192– 6198. N atural killer cells can kill autologous-infected cells with- out prior sensitization and are believed to play a central role in innate immunity to microbial pathogens (1–5). The lytic capacity of NK cells is controlled by complex interac- tions of inhibitory and activating receptors with specialized sig- naling machinery (6 –9). Some of the principal activating NK cell receptors on human NK cells are the natural cytotoxicity receptors, NKp30, NKp44 and NKp46, NKG2D, 2B4, and DNAX accessory molecule 1. For the natural cytotoxicity receptors, viral hemagglu- tinins have been identified as ligands that bind NKp46 and NKp44 (10), triggering lysis of infected cells. Some studies have shown that membrane-associated heparan sulfate proteoglycans (HSPG) 3 on tumor cells are involved in the recognition of cellular targets by NKp46 and NKp30 (11), whereas others have found that HSPG do not bind to NKp30 (12). Therefore, the ligands for natural cyto- toxicity receptors on human cells infected with bacteria and fungi remain unidentified. Previously, we demonstrated that NKp46 and NKG2D play im- portant roles in the lysis of Mycobacterium tuberculosis-infected monocytes (13, 14). Of five NKG2D ligands, only UL16-binding protein 1 was involved in lysis of M. tuberculosis-infected mono- cytes and alveolar macrophages. Characterization of the mecha- nisms by which NKp46 binds to M. tuberculosis H37Ra-infected mononuclear phagocytes would be a significant advance in our understanding of innate immunity to intracellular bacteria. In the current study, we found that vimentin contributed to binding of NKp46 to H37Ra-infected monocytes. Vimentin is a type III cytoskeletal protein that maintains the architecture of cytoplasm. It is also involved in cell adhesion, tran- scellular migration (15), wound healing (16), and cellular signaling (17). Recent studies suggest that vimentin plays a role during in- tracellular infection. For example, vimentin interacts with viruses during virion assembly and facilitates virion transport (18, 19). In addition, activated human macrophages secrete vimentin, which contributes to bacterial killing through generation of oxidative me- tabolites (20). Immunoprecipitation, followed by mass spectrometry analysis, identified vimentin as a putative ligand for NKp46. Furthermore, flow cytometry and confocal microscopy showed that vimentin was expressed on the surface of monocytes and up-regulated by M. tuberculosis H37Ra infection. NK cells lysed CHO-K1 cell trans- fectants that expressed vimentin, and lysis was inhibited by mAb- mediated masking of NKp46 on NK cells or vimentin on CHO-K1 cells. In addition, binding of vimentin to NKp46 was confirmed by ELISA, radio-iodinated vimentin cell binding assay, and Far West- ern blotting. Materials and Methods Patient population Heparinized venous blood was obtained from 18 tuberculin-negative healthy donors to eliminate effects due to M. tuberculosis-reactive T cells. *Center for Pulmonary and Infectious Disease Control, † Department of Microbiology and Immunology, § Department of Medicine, and ‡ Department of Biochemistry, Cen- ter for Biomedical Research, University of Texas Health Center, Tyler, TX 75708; and ¶ Department of Microbiology and Immunology, Faculty of Health Sciences, and Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva, Israel Received for publication February 16, 2006. Accepted for publication August 15, 2006. 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 National Institutes of Health Grants AI054629, AI044935, and A1063514, the Cain Foundation for Infectious Disease Research, and the Center for Pulmonary and Infectious Disease Control. 2 Address correspondence and reprint requests to Dr. Ramakrishna Vankayalapati, Center for Pulmonary and Infectious Disease Control, University of Texas Health Center, 11937 U.S. Highway 271, Tyler, TX 75708-3154. E-mail address: Krishna.vankayalapati@uthct.edu 3 Abbreviations used in this paper: HSPG, heparan sulfate proteoglycan; MOI, mul- tiplicity of infection; MFI, mean fluorescence intensity. The Journal of Immunology Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00