Critical Role for the Chemokine Receptor CXCR6 in Homeostasis and Activation of CD1d-Restricted NKT Cells 1 Elitza Germanov, 2 * Linnea Veinotte, 2 * Robyn Cullen, † Erin Chamberlain,* Eugene C. Butcher, § and Brent Johnston 3 * †‡ NK T (NKT) cells play important roles in the regulation of diverse immune responses. However, little is known about the mechanisms that regulate homeostasis and activation of these cells. Thymic NKT cells up-regulated the chemokine receptor CXCR6 following positive selection and migrated toward CXCL16 in vitro. However, CXCR6 was not essential for thymic development or maturation. In contrast, liver and lung NKT cells were depleted in CXCR6 / and CXCR6 / mice. The reduction in liver and lung NKT cells coincided with an increase in bone marrow NKT cells, suggesting a redistribution of NKT cells in CXCR6 / animals. In wild-type mice, CXCL16 neutralization reduced accumulation of mature NK1.1  , but not imma- ture NK1.1  NKT cell recent thymic emigrants in the liver. Given that thymic NKT cells are preferentially exported as NK1.1  cells, this suggests an additional role for CXCR6/CXCL16 in maturation or survival of immature liver NKT cells. CXCL16 blockade did not deplete resident NK1.1  NKT cells, indicating that CXCR6/CXCL16 are not required to retain mature NKT cells in the liver. Cytokine production by liver and spleen NKT cells was impaired in CXCR6 / mice following in vivo stimulation with -galactosylceramide, implicating a novel role for CXCR6 in NKT cell activation. Reduced IFN- production was not due to an intrinsic defect as production was normal following PMA and ionomycin stimulation. Preformed transcripts for IL-4, but not IFN-, were reduced in CXCR6 / liver NKT cells. These data identify critical roles for CXCR6/CXCL16 in NKT cell activation and the regulation of NKT cell homeostasis. The Journal of Immunology, 2008, 181: 81–91. T he NK T (NKT) 4 cells comprise a distinct lineage of lym- phocytes that share functional and phenotypic character- istics with NK cells and effector T lymphocytes (1, 2). Most NKT cells in mice express an invariant V14J18 TCR re- arrangement paired preferentially with V8.2, V7, or V2 (3), whereas human NKT cells express a homologous V24J18 re- arrangement coupled with V11 (3, 4). Instead of recognizing pep- tide Ags, these cells recognize glycolipids presented by the non- polymorphic MHC-like molecule CD1d and can be detected using CD1d tetramers loaded with the glycolipid -galactosylceramide (-GalCer) (5, 6). These prototypical NKT cells are often referred to as V14i T cells or invariant NKT cells to differentiate them from other rare NKT cell subsets and TCR diverse T cells that can up-regulate NK cell markers following activation (1, 2). NKT cells develop in the thymus and are thought to arise from uncommitted precursors that randomly rearrange the V14J18 gene segments (3, 7, 8). Following TCR rearrangement, developing NKT cells transit through a CD4  CD8  double-positive (DP) step (9 –11) and are selected via CD1d expressed on other DP thymocytes (12, 13). Positively selected NKT cells expand and undergo a maturation process characterized by sequential up-regulation of CD44 and NK1.1 (14, 15). Immature CD44  NK1.1  NKT cells are exported prefer- entially from the thymus and colonize peripheral tissues where they undergo final maturation into NK1.1  cells (14, 15). In contrast, most NKT cells that up-regulate NK1.1 in the thymus are retained as a mature resident population (16). In the periphery, NKT cells localize preferentially to the liver, spleen, and bone marrow where they respond rapidly to activating stimuli by secreting an array of immunoregulatory cytokines includ- ing IFN- and lL-4 (6, 17–19). Consequently, NKT cells have been implicated in a number of diverse immunological processes, including tumor responses, clearance of pathogens, regulation of autoimmunity, and tolerance induction (20 –23). Treatment with -GalCer or -Gal- Cer-loaded dendritic cells (DC) induces a potent antitumor response by stimulating IFN- production (24 –26), while -GalCer has also been shown to ameliorate autoimmunity through the induction of Th2 responses (27–29). In contrast to the ability of -GalCer to generate both Th1 and Th2 cytokines, OCH, a truncated derivative of -Gal- Cer, induces a preferential Th2 response that can suppress experimen- tal autoimmune encephalitis and collagen-induced arthritis (30, 31), whereas the C-glycoside derivative -C-GalCer induces a selective Th1 response and is very effective at preventing tumor metastasis (32). Although the nature of the glycolipid Ag influences the NKT cell response, costimulatory molecules are also important. CD40/ CD40L interactions are critical for inducing IFN- production (33, 34), while IL-4 production is regulated by the integrin LFA-1 (35), *Department of Microbiology and Immunology, † Department of Pathology, and ‡ De- partment of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada; and § Laboratory of Immunology and Vascular Biology, Department of Pathology, Stan- ford University School of Medicine, Stanford, CA 94305; and the Center for Molec- ular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304 Received for publication September 20, 2007. Accepted for publication April 21, 2008. 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 a grant from the Terry Fox Foundation awarded to B.J. by the National Cancer Institute of Canada. B.J. holds the Canada Research Chair in Inflammation and Immunity. L.V. is the recipient of a fellowship from the Killam Trusts. E.G. is the recipient of a studentship from the Nova Scotia Health Research Foundation. R.C. is the recipient of a Cancer Research Training Program Award with funding from the Dalhousie Cancer Research Program. 2 E.G. and L.V. contributed equally to this work. 3 Address correspondence and reprint requests to Dr. Brent Johnston, Department of Microbiology and Immunology, Dalhousie University, Sir Charles Tupper Medical Building, Room 7C, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. E-mail address: Brent.Johnston@Dal.Ca 4 Abbreviations used in this paper: NKT cell, NK T cell; -GalCer, -galactosylce- ramide; DC, dendritic cell; DN, double-negative; DP, double-positive; eGFP, en- hanced GFP; FSC, forward light scatter. The Journal of Immunology www.jimmunol.org