Lysosomal Localization of Murine CD1d Mediated by AP-3 Is Necessary for NK T Cell Development 1 Manuela Cernadas,* Masahiko Sugita, ‡ Nicole van der Wel, § Xiaochun Cao, ¶ Jenny E. Gumperz, † Sergei Maltsev,  Gurdyal S. Besra,  Samuel M. Behar, † Peter J. Peters, § and Michael B. Brenner 2† The presentation of lipid and glycolipid Ags to T cells is mediated through CD1 molecules. In the mouse and rat only a single isoform, CD1d, performs these functions, while humans and all other mammals studied have members of both group I (CD1a, -b, and -c) and group II (CD1d) isoforms. Murine CD1d contains a cytoplasmic tyrosine-based sorting motif that is similar to motifs recognized by adaptor protein complexes that sort transmembrane proteins. Here we show that the adaptor protein complex, AP-3, directly interacts with murine CD1d and controls its targeting to lysosomes. AP-3 deficiency results in a redistribution of CD1d from lysosomes to the cell surface of thymocytes, B cell-depleted splenocytes, and dendritic cells. The altered trafficking of CD1d in AP-3-deficient mice results in a significant reduction of NK1.1  TCR-  and CD1d tetramer-positive cells, consistent with a defect in CD1d self-Ag presentation and thymocyte-positive selection. The AP-3 complex has recently been shown to associate with the human CD1b isoform, which has an intracellular distribution pattern similar to that of murine CD1d. We propose that lysosomal sampling may be so critical for efficient host defense that mice have evolved mechanisms to target their single CD1 isoform to lysosomes for sampling lipid Ags. Here we show the dominant mechanism for this trafficking is mediated by AP-3. The Journal of Immunology, 2003, 171: 4149 – 4155. C D1 molecules are composed of transmembrane MHC class I-like heavy chains that noncovalently associate with  2 -microglobulin and mediate the presentation of nonpeptide lipid and glycolipid Ags to T cells. They are separated into two groups, group I (CD1a, -b, and -c) and group II (CD1d) molecules, based on nucleotide and amino acid sequence similarity (1). Species differences in expression are also striking, since both group I and II CD1 molecules are present in humans, while CD1d is the only isoform expressed in muroid rodents. The group I CD1 molecules are best characterized for their ability to present foreign mycobacterial lipids (2– 4) to T cells with diverse TCRs. In con- trast, while the physiological lipids presented by CD1d remain enigmatic, both human and murine CD1d-restricted T cells are likely to recognize self-lipid Ags in vivo and recognize -galac- tosylceramide (-GalCer) 3 in experimental systems (5–9). CD1d- restricted T cells have proven functionally significant, since they secrete copious amount of IL-4, IL-10, and IFN- and also display cytotoxic effects (10–13). Besides differences in Ag-presenting capacity and T cell acti- vation, the CD1 isoforms follow discrete intracellular trafficking routes that correlate with their ability to intersect with the lipid Ags that each presents. The CD1b, CD1c, and CD1d isoforms contain a cytoplasmic tyrosine-based sorting motif, YXXZ (Y = tyrosine, X = any amino acid, Z = bulky hydrophobic amino acid), that mediates trafficking to appropriate intracellular compartments. In- teraction with AP-2 may be important in their internalization from the plasma membrane (14). The AP-3 complex has been shown to be important in the receptor-mediated trafficking of proteins such as lysosome-associated membrane protein 1 (LAMP-1) and CD63 that are targeted to lysosomes (15, 16). Recently, the tyrosine- based sorting motif of CD1b has been shown to interact with AP-3 and to be critical for the localization of CD1b to late endosomes and lysosomes (17, 18). Although CD1c has a similar cytoplasmic tyrosine motif, it does not associate with AP-3 and primarily lo- calizes to early recycling endosomes, with only a small fraction distributed in lysosomes (18 –21). Similarly, human CD1d (hCD1d) displays limited lysosomal localization and lacks the ability to bind AP-3 in yeast two-hybrid assays (18). CD1a, which does not contain a cytosolic sorting motif, traffics exclusively through early recycling endosomes (22). Consistent with the dif- ferences in CD1 isoform localization is the dependence of vesic- ular acidification for effective Ag presentation by CD1b, which traffics to lysosomes, but not for CD1a, which traffics through early endosomes (19, 22, 23). *Division of Pulmonary and Critical Care Medicine, † Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; ‡ De- partment of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan; § The Netherlands Cancer Institute, Amsterdam, The Netherlands; ¶ Department of Microbiology and Immunology, Department of Cellular Biochemistry, Max Plank Institute of Biochemistry, Martinsried/Munich, Germany; and  School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom Received for publication April 18, 2003. Accepted for publication August 14, 2003. 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 K08HL0445302 (to M.C.), R37AI128973 (to M.B.B.), and R01AI48932 (to M.B.B.); a grant from the Ministry of Education, Culture, Sports, Science, and Technology (Grant-in-Aid for Scientific Research on Priority Areas 14021122, to M.S.); and The Netherlands Lep- rosy Relief (IELP no. 702.02.20, to P.P. and N.v.d.W.). G.S.B., who is currently a Lister-Jenner Research Fellow, was supported by the Medical Research Council. 2 Address correspondence and reprint requests to Dr. Michael B. Brenner, Lympho- cyte Biology Section, Division of Rheumatology, Immunology and Allergy, Depart- ment of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Smith Building, Room 552, 1 Jimmy Fund Way, Boston, MA 02115. E-mail address: mbrenner@rics.bwh.harvard.edu 3 Abbreviations used in this paper: -GalCer, -galactosylceramide; DC, dendritic cells; h, human; LAMP-1, lysosome-associated membrane protein 1; m, murine. The Journal of Immunology Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00