Critical Role for the Chemokine Receptor CXCR6 in Homeostasis and Activation of CD1d-Restricted NKT Cells This information is current as Elitza Germanov, Linnea Veinotte, Robyn Cullen, Erin of September 27, 2021. Chamberlain, Eugene C. Butcher and Brent Johnston J Immunol 2008; 181:81-91; ; doi: 10.4049/jimmunol.181.1.81 http://www.jimmunol.org/content/181/1/81 Downloaded from References This article cites 62 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/181/1/81.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2008 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Critical Role for the Chemokine Receptor CXCR6 in Homeostasis and Activation of CD1d-Restricted NKT Cells1 Elitza Germanov,2* Linnea Veinotte,2* Robyn Cullen,† Erin Chamberlain,* Eugene C. Butcher,§ and Brent Johnston3*†‡ 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 Downloaded from 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 http://www.jimmunol.org and the regulation of NKT cell homeostasis. The Journal of Immunology, 2008, 181: 81–91. he NK T (NKT)4 cells comprise a distinct lineage of lym- from other rare NKT cell subsets and TCR diverse T cells that can phocytes that share functional and phenotypic character- up-regulate NK cell markers following activation (1, 2). T istics with NK cells and effector T lymphocytes (1, 2). NKT cells develop in the thymus and are thought to arise from Most NKT cells in mice express an invariant V␣14J␣18 TCR re- uncommitted precursors that randomly rearrange the V␣14J␣18 gene arrangement paired preferentially with V␤8.2, V␤7, or V␤2 (3), segments (3, 7, 8). Following TCR rearrangement, developing NKT whereas human NKT cells express a homologous V␣24J␣18 re- cells transit through a CD4ϩCD8ϩ double-positive (DP) step (9–11) by guest on September 27, 2021 arrangement coupled with V␤11 (3, 4). Instead of recognizing pep- and are selected via CD1d expressed on other DP thymocytes (12, tide Ags, these cells recognize glycolipids presented by the non- 13). Positively selected NKT cells expand and undergo a maturation polymorphic MHC-like molecule CD1d and can be detected using process characterized by sequential up-regulation of CD44 and NK1.1 CD1d tetramers loaded with the glycolipid ␣-galactosylceramide (14, 15). Immature CD44ϩ NK1.1Ϫ NKT cells are exported prefer- (␣-GalCer) (5, 6). These prototypical NKT cells are often referred entially from the thymus and colonize peripheral tissues where they ␣ ϩ to as V 14i T cells or invariant NKT cells to differentiate them 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). *Department of Microbiology and Immunology, †Department of Pathology, and ‡De- In the periphery, NKT cells localize preferentially to the liver, partment of Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada; and §Laboratory of Immunology and Vascular Biology, Department of Pathology, Stan- spleen, and bone marrow where they respond rapidly to activating ford University School of Medicine, Stanford, CA 94305; and the Center for Molec- stimuli by secreting an array of immunoregulatory cytokines includ- ular Biology and Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, ␥ CA 94304 ing IFN- and lL-4 (6, 17–19). Consequently, NKT cells have been implicated in a number of diverse immunological processes, including Received for publication September 20, 2007. Accepted for publication April 21, 2008. tumor responses, clearance of pathogens, regulation of autoimmunity, ␣ ␣ The costs of publication of this article were defrayed in part by the payment of page and tolerance induction (20–23). Treatment with -GalCer or -Gal- charges. This article must therefore be hereby marked advertisement in accordance Cer-loaded dendritic cells (DC) induces a potent antitumor response with 18 U.S.C. Section 1734 solely to indicate this fact. by stimulating IFN-␥ production (24–26), while ␣-GalCer has also 1 This work was supported by a grant from the Terry Fox Foundation awarded to B.J. been shown to ameliorate autoimmunity through the induction of Th2 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 responses (27–29). In contrast to the ability of ␣-GalCer to generate Trusts. E.G. is the recipient of a studentship from the Nova Scotia Health Research both Th1 and Th2 cytokines, OCH, a truncated derivative of ␣-Gal- Foundation. R.C. is the recipient of a Cancer Research Training Program Award with funding from the Dalhousie Cancer Research Program. Cer, induces a preferential Th2 response that can suppress experimen- 2 E.G. and L.V. contributed equally to this work. tal autoimmune encephalitis and collagen-induced arthritis (30, 31), whereas the C-glycoside derivative ␣-C-GalCer induces a selective 3 Address correspondence and reprint requests to Dr. Brent Johnston, Department of Microbiology and Immunology, Dalhousie University, Sir Charles Tupper Medical Th1 response and is very effective at preventing tumor metastasis Building, Room 7C, 5850 College Street, Halifax, Nova Scotia, Canada B3H 1X5. (32). Although the nature of the glycolipid Ag influences the NKT E-mail address: [email protected] cell response, costimulatory molecules are also important. CD40/ 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- CD40L interactions are critical for inducing IFN- production (33, hanced GFP; FSC, forward light scatter. 34), while IL-4 production is regulated by the integrin LFA-1 (35), www.jimmunol.org 82 CXCR6 MEDIATES NKT CELL HOMEOSTASIS AND ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021 FIGURE 1. Functional expression of CXCR6 on thymic NKT cells. A, NKT cells were stained with loaded CD1d tetramers and fluorochrome-conjugated Abs against CD4 and CD8 to distinguish CD4ϪCD8Ϫ (DN), CD4ϩCD8ϩ (DP), CD4ϩ, and CD8ϩ subsets. Expression of CXCR6 on NKT cells subsets was determined by eGFP expression in CXCR6-eGFP knock-in heterozygotes, or binding of a CXCL16-Fc chimera in wild-type mice. Nonspecific staining cells were excluded using unloaded tetramers and doublets were excluded by FSC-H vs FSC-A gating (data not shown). B and C, Chemotactic migration assays of thymic NKT cells to CXCL16. Fresh unfractionated thymocytes were allowed to migrate through Transwell inserts (5-␮m pore size) for 2 h. B, Wild-type NKT cells were stained with loaded CD1d tetramers and fluorochrome-conjugated Abs against TCR␤, CD4, and CD8 to distinguish subsets. The proportion of migrated cells in each population was calculated as a fraction of the input population (43, 55). C, NKT cell migration of age- and sex-matched CXCR6ϩ/ϩ, CXCR6ϩ/Ϫ, and CXCR6Ϫ/Ϫ littermates in response to CXCL16. D, NKT cells were stained with loaded CD1d tetramers, CXCL16-Fc, and fluorochrome-conjugated Abs against CD44 and NK1.1 to distinguish sequential stages of NKT cell development: CD44ϪNK1.1Ϫ, CD44ϩNK1.1Ϫ, and CD44ϩNK1.1ϩ. E, NKT cells in the liver, lung, and spleen were identified with loaded CD1d tetramers and Ab against TCR␤. CXCL16-Fc was used to examine CXCR6 expression. FACS plots are representative of at least three independent experiments. Chemotaxis assays show the mean Ϯ SEM of six .p Ͻ 0.05 compared with basal migration. †, p Ͻ 0.05 compared with CXCR6ϩ/ϩ ,ء .individual experiments and CD28-B7 interactions are important for generation of both IFN-␥ lymphocyte accumulation in several inflammatory diseases (47– and IL-4 (33, 36).