The Journal of Immunology A Threonine-Based Targeting Signal in the Human CD1d Cytoplasmic Tail Controls Its Functional Expression Jianyun Liu,* Daniel Shaji,* Sungyoo Cho,* Wenjun Du,† Jacquelyn Gervay-Hague,† and Randy R. Brutkiewicz* CD1d molecules are MHC class I-like molecules that present lipids to a unique subpopulation of T cells called NKT cells. The cytoplasmic tail of human CD1d possesses a tyrosine-based endosomal targeting motif (YXXZ). As such, these molecules traffic through the endocytic pathway, where it is believed that they are loaded with the antigenic lipid that stimulates NKT cells. In the current study, it was found that the T322 residue in the human CD1d tail is a major signal controlling transport to the cell surface and thus its functional expression. Mimicking the phosphorylation of this residue or removal of the entire cytoplasmic tail negates its ability to regulate CD1d trafficking, resulting in lysosomal targeting and degradation. These results demonstrate an important role of a heretofore unknown signal in the cytoplasmic tail of CD1d that may have relevance to other type I integral membrane proteins that traverse through the endocytic pathway. The Journal of Immunology, 2010, 184: 4973–4981. D1 molecules are nonpolymorphic transmembrane gly- lipids, including phosphatidylinositol and phosphatidylcholine, coproteins encodedby geneslocatedoutside theMHC locus generally cannot activate NKT cells. Instead, they likely play an C (1). There are five members of the CD1 family: CD1a, analogous role to the invariant chain-derived CLIP peptide for CD1b, CD1c, CD1d, and CD1e. Like MHC class I, all of the CD1 MHC II (4). Either before or after lipid loading, the CD1d H proteins consist of three extracellular domains (a1, a2, and a3), chain binds to b2m and then is transported to the Golgi. In the a transmembrane domain, and cytoplasmic tail. The CD1 a3do- Golgi, the glycans on CD1d are processed further before being mains are nonconvalently associated with b2-microglobulin (b2m), transported to the plasma membrane (12, 13). From the surface, forming a heterodimer soon after translation in the endoplasmic due to the YXXZ motif (where Y is tyrosine, X is any amino acid, reticulum (ER) (2, 3). Unlike MHC class I and class II molecules and Z is bulky hydrophobic residue) in its cytoplasmic tail, surface that present peptide Ags, CD1 molecules present lipids to T cells. CD1d is internalized and traffics through compartments of the The CD1d molecule presents lipid Ags mostly to a unique sub- endocytic pathway (5, 14). CD1d-mediated lipid Ag presentation population of T cells that express an invariant TCR a-chain and requires lipid exchange in late endocytic compartments and re- surface markers also present on NK cells (4). These are thus named expression on the cell surface to activate NKT cells (15, 16). invariant NKT cells. Activated NKT cells secrete both Th1 (e.g., Sphingolipid activator proteins, especially saposin B, may facilitate IFN-g and GM-CSF) and Th2 (e.g., IL-4) cytokines, playing im- lipid binding to CD1d (or other CD1 molecules) throughout the en- portant roles in both innate and adaptive immunity (5), including docytic pathway (17–20). Among the lipid Ags that activate NKT cells, antitumor, autoimmune, and antimicrobial responses (6–8). some are natural cellular ligands, such as isoglobotrihexosylceramide, Microsomal triglyceride transfer protein, a protein involved in whereas others are microbial lipids such as a-glucuronosylceramide lipoprotein assembly (9), has been reported to be important for from Sphingomonas (21–23). a-Galactosylceramide (a-GalCer), CD1d function in vivo. It is likely required for the loading of self- a glycolipid extracted from marine sponges, is recognized by invariant lipids into the hydrophobic groove formed by the a1 and a2 NKT cells in a CD1d-dependent manner (4, 24). domains of CD1d when synthesized in the ER (10, 11). These self- CD1d a type I transmembrane protein and its cytoplasmic tail contains at least one signal for endocytic trafficking (Supplemental Fig. 1). Both human and mouse CD1d cytoplasmic tails contain *Department of Microbiology and Immunology, Indiana University School of Med- icine, Indianapolis, IN 46202; and †Department of Chemistry, University of Califor- a YXXZ motif, which is believed to be a binding motif for the nia at Davis, Davis, CA 95616 adaptor protein (AP) 2m1 subunit and AP3, respectively (25). Received for publication May 7, 2009. Accepted for publication March 1, 2010. Destroying this motif causes the accumulation of both human and This work was supported by National Institutes of Health Grants R01 AI46455 and mouse CD1d on the cell surface (15, 26). Interestingly, the YXXZ P01 AI056097 (to R.R.B.) and National Science Foundation Grant CHE-0194682 (to motif is also required for CD1d downregulation caused by a mi- J.G.-H.). crobial infection, such as HIV and Chlamydia (13, 27, 28). The Address correspondence and reprint requests to Dr. Randy R. Brutkiewicz, Depart- lysine in the CD1d cytoplasmic tail is also believed to be im- ment of Microbiology and Immunology, Indiana University School of Medicine, 950 West Walnut Street, Building R2, Room 302, Indianapolis, IN 46202. E-mail address: portant, because the monoubiquitination of lysines can also [email protected] function as a signal for endocytic trafficking (29, 30) and infection The online version of this article contains supplemental material. with Kaposi’s sarcoma herpesvirus downregulates surface CD1d Abbreviations used in this paper: a-GalCer, a-galactosylceramide; AP, adaptor pro- through ubiquitination of the K326 residue, a presumed means of tein; b2m, b2-microglobulin; EE, early endosome; Endo H, endoglycosidase H; ER, immune evasion (31). endoplasmic reticulum; HC, H chain; hCD1d, human CD1d; LE, late endosome; MFI, In the current study, we have identified contrasting signals in the mean fluorescence intensity; MFIchl, MFI from chloroquine-treated cells; MFImon, MFI from monensin-treated cells; MFIveh, MFI from vehicle-treated cells; PNGase cytoplasmic tail of human CD1d important for its intracellular F, peptide:N-glycosidase F; SE, sorting endosome; TGN, trans-Golgi network; WT, distribution, endocytic trafficking, and ability to present Ag. Our wild-type. data strongly suggest that there are two major signals in the cy- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 toplasmic tail that are important for lysosomal targeting. One www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901448 4974 A THREONINE-BASED TARGETING SIGNAL FOR HUMAN CD1d directly targets the CD1d molecule to lysosomes and is Y331- CA). A PE-labeled goat anti-mouse Ig antiserum was purchased from based; the second is T322-based and, under normal conditions, DakoCytomation (Carpenteria, CA). A Texas Red-conjugated donkey anti- permits cell surface expression. When altered to mimic a phos- rabbit Ig antiserum was from Jackson ImmunoResearch Laboratories (West Grove, PA). A Texas Red-conjugated goat anti-mouse Ig antiserum phorylated form (T322D), CD1d is directed to lysosomes for and Hoechst stain were purchased from Molecular Probes (Portland, OR). degradation. The T322-based signal is dominant over that which is The HB95 hybridoma (pan-HLA class I-specific mAb) was a kind gift Y331-based and therefore controls the functional expression of from J. Yewdell and J. Bennink (Laboratory of Viral Diseases, National CD1d. We speculate that this type of threonine-based signal for Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). Abs against the ER marker Sec61b were from Millipore targeting lysosomes also exists in many other type I transmembrane (Billerica, MA), whereas the anti-CD1d free H chain (HC)-specific mAb proteins and probably has a broad application in their intracellular (C3D5, for immunoprecipitation and Western analysis) was from Santa distribution and endocytic trafficking. Cruz Biotechnology (Santa Cruz, CA). A FITC-conjugated rabbit anti-PE Ab was obtained from Rockland Immunochemicals (Gilbertsville, PA). The CD1d-binding lipid a-GalCer was generated as described (35) or Materials and Methods purchased from Alexis Biochemicals (San Diego, CA). Recombinant hu- CD1d and mutant constructs man IL-2, IL-4, and GM-CSF were from PeproTech (Rocky Hill, NJ), whereas Ab pairs for the human IL-4 and GM-CSF ELISA assays (de- The wild-type (WT) human CD1d (hCD1d) cDNA was excised from scribed below) were obtained from BD Biosciences and Biolegend (San pBSKII-hCD1d (kindly provided by S. Balk, Harvard Medical School, Diego, CA), respectively. Peptide:N-glycosidase F (PNGase F) and en- Cambridge, MA) by XhoI/BamHI digestion (NEB, Ipswich, MA) and doglycosidase H (Endo H) were purchased from NEB. Monensin (BD inserted into the pcDNA3.1-neo vector (Invitrogen, Carlsbad, CA) to GolgiStop) was from BD Biosciences. generate pcDNA3.1-neo-hCD1d WT. The CD1d Y331A and TD-6 mutants were generated as described (13, 27) and also subcloned into pcDNA3.1- Culture of human NKT cells neo. The T322A and T322D mutants were generated by site-directed mutagenesis of pcDNA3.1-neo-hCD1d WT. The forward and reverse pri- Human NKT cells were generated following the protocol described by mers for T322A were 59-c att gtg ggc ttt gcc tcc cgg ttt aag-39 and 59-ctt Exley et al. (36). Briefly, human PBMCs were isolated from deidentified aaa ccg gga ggc aaa gcc cac aat g-39, respectively. The primers for T322D donated whole human blood (Indiana Blood Center, Indianapolis, IN) by were 59-c att gtg ggc ttt gac tcc cgg ttt aag-39 (forward) and 59-ctt aaa ccg density gradient centrifugation on Ficoll-Hypaque (GE Healthcare, Piscat- gga gtc aaa gcc cac aat g-39 (reverse). away, NJ). Va24-Ja18+ NKT cells were purified by positive selection using To generate hCD1d mutants in which the last 10 or all 14 cytoplasmic the 6B11 mAb followed by goat anti-mouse Ig coupled to magnetic beads tail amino acids were deleted (TD-10 and TD-14, respectively), two reverse (Miltenyi Biotec, Auburn, CA).