CUTTING EDGE IMMUNOLOGY THE OF JOURNAL Cutting Edge: Activation by Innate Cytokines or Microbial Antigens Can Cause Arrest of Natural Killer T Cell Patrolling of Liver Sinusoids 1 Peter Vela ´zquez,* Thomas O. Cameron,* Yuki Kinjo, † Niranjana Nagarajan, ‡ Mitchell Kronenberg, † and Michael L. Dustin 2 * Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of -linked glycolipids pre- sented by CD1d leads to the production of IL-4, IFN-, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN- production only. We pre- viously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in ar- rest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria- derived weak agonist, galacturonic acid-containing glyco- sphingolipid, or a synthetic agonist, -galactosyl cer- amide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cy- tokines and natural microbial TCR agonists trigger sinu- soidal NKT cell arrest and an effector response. The Journal of Immunology, 2008, 180: 2024 –2028. N atural killer T (NKT) 3 cells are innate lymphocytes with diverse influences and multiple pathways leading to their activation. NKT cells are activated directly by cytokines such as IL-12 and IL-18 in synergy, independently of exogenous Ag (1, 2), a property that distinguishes them from most conventional, naive lymphocytes (3). Furthermore, NKT cells can be activated when the semi-invariant TCR they ex- press, containing a V14J18 rearrangement, recognizes gly- colipid Ag in the context of CD1d (4). Activation via the TCR with strong agonists does not require costimulation, demonstrating an effector state of these lympho- cytes, and it can result in a robust production of IL-4 or IFN- (5, 6). IFN- but not IL-4 is induced in NKT cells via the syn- ergistic activation by IL-12 and IL-18 produced by LPS-stim- ulated macrophages and dendritic cells in vivo (1, 7). Although several NKT cell TCR agonists have been identi- fied, the best studied is the glycolipid -galactosyl ceramide (GalCer), originally purified from a marine sponge and opti- mized by medicinal chemistry. Recent studies have identified bacterial glycolipids derived from sphingomonads that are nat- ural NKT cell TCR agonists (2, 8, 9). In particular, galactu- ronic acid-containing glycosphingolipid (GalA-GSL) is a cer- amide-based glycolipid agonist produced by Sphingomonas yanoikuyae. GalA-GSL is at least 100-fold less potent than GalCer in vitro. Like GalCer, activation by GalA-GSL is characterized, in part, by an early rapid and robust production of IL-4 by NKT cells (8). Anatomically, NKT cells are most abundant in the liver and spleen of mice (10). Therefore, understanding the basis of NKT cell activation at these sites in vivo is of major importance. Un- like in the spleen, in the liver NKT cells comprise a major per- centage of the lymphocyte population (10 – 40%) (10). Also, 90% of the liver’s blood supply comes from the portal vein, which drains the gut mucosa, a major site of Ag entry. Using intravital laser scanning microscopy we have previously shown that resident liver NKT cells patrol liver sinusoids both with and against the blood flow and arrest their migration in re- sponse to the presentation of GalCer by CD1d (11). The aim of the current study was to gain insight into the re- lationship between NKT cell patrolling and the different modes of activation known for these cells. Using intravital laser scan- ning microscopy we examined NKT cell patrolling in response to Ag receptor agonists and innate cytokines. We tested a range of doses of a natural Ag, GalA-GSL, compared with the more potent synthetic strong agonist, GalCer. Activation in the ab- sence of foreign Ag also was studied using innate cytokines, *Molecular Pathogenesis Program, The Helen L. and Martin S. Kimmel Center for Biol- ogy and Medicine at Skirball Institute of Biomolecular Medicine, New York, NY 10016; † La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; and ‡ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 Received for publication June 29, 2007. Accepted for publication December 26, 2007. 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 Institute of Allergy and Infectious Diseases, Na- tional Institutes of Health Grants R01 AI055037-03 (to M.L.D.) and R37 AI 71922 (to M.K.), grants from the Irene Diamond Fund (to M.L.D.) and the Cancer Research Insti- tute (to Y.K.), and National Institute of Diabetes and Digestive and Kidney Disease Grant F32-DK078153 (to P.V.). 2 Address correspondence and reprint requests to Dr. Michael L. Dustin, Molecular Patho- genesis Program, The Helen L. and Martin S. Kimmel Center for Biology and Medicine at Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016. E-mail address: dustin@saturn.med.nyu.edu 3 Abbreviations used in this paper: NKT, natural killer T; GalA-GSL, galacturonic acid- containing glycosphingolipid; GalCer, -galactosyl ceramide; EGFP, enhanced green fluorescent protein. 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