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 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 , 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). After magnetic bead separation, the NKT cells primers were designed either lacking the C-terminal 10 (59-t ata gga tcc tca were expanded by stimulation with irradiated allogeneic human PBMCs in aaa ccg gga ggt aaa gcc-39) or 14 aas (59-t ata gga tcc tca aaa gcc cac aat the presence of 100 ng/ml a-GalCer and 10 ng/ml recombinant human IL-2. gag gag g-39). Both primers were flanked with BamHI sites as underlined. These two primers were paired with a T7 forward primer for PCR am- T cell stimulation assays plification of hCD1d. The PCR products were then digested with XhoI and 5 BamHI, purified, and subcloned into pcDNA3.1-neo. HEK293-hCD1d cells (WT and mutants; 1 3 10 cells per well) were CD25/CD1d fusion proteins (containing the CD1d transmembrane do- incubated with human NKT cells at an E:T ratio of 1:1 and with 1 ng/ml main with WT or mutant [T322D or TD-14] cytoplasmic tails) were recombinant human IL-2 in the presence or absence of a-GalCer (100 ng/ generated by Ntsane Moleleki and Mark Peggie (Medical Research Council ml) for 48 h. Secreted IL-4 and GM-CSF levels were measured by ELISA. Protein Phosphorylation Unit, University of Dundee, Dundee, Scotland, The anti-CD1d–b2m mAb (42.1; 500 ng/ml) was used to block CD1d- U.K.) and based in the pcDNA3.1-neo vector. mediated Ag presentation in some experiments. To generate CD1a/CD1d tail fusion proteins, CD1a cDNA was first amplified from C1R.CD1a cells (kindly provided by S. Balk) by PCR using Confocal microscopy two primers designed to flank the amplified PCR product with XhoI and Staining for confocal microscopy was performed as described previously BamHI sites. After XhoI/BamHI digestion, the amplified fragment was (15). Briefly, HEK293 cells were plated in sterile glass-bottom 35-mm purified and inserted into the pcDNA3.1-neo vector to generate pcDNA3.1- dishes coated with collagen (MatTek, Ashland, MA). After the cells be- 9 a neo-CD1a. There is a common XbaI site in the 3 end of the 3 domain of came 50–80% confluent, they were washed, fixed, and then permeabilized both CD1d and CD1a molecules (32–34). Thus, the pcDNA3.1-neo- by 0.1% saponin in HBSS with 0.1% BSA (HBSS/BSA). To stain the hCD1d vector was digested with XbaI to remove the CD1d extracellular CD1d–b m complex, cells were incubated with the CD1d–b m complex- domain, and the extracellular domain of CD1a was excised from 2 2 specific 42.1 mAb followed by a Texas Red-conjugated donkey anti-mouse pcDNA3.1-neo-CD1a with XbaI and subcloned to generate a CD1a ex- Ig antiserum. After the free reactive sites were blocked with normal mouse tracellular domain fused to the CD1d transmembrane and cytoplasmic tail. serum (Sigma-Aldrich, St. Louis, MO), the cells were incubated with This construct was used as a template to generate T322D and TD-14 cy- a FITC-conjugated anti-human LAMP-1 mAb. To stain the free CD1d HC, toplasmic tail mutants as described above. the cells were incubated with the anti-CD1d free HC mAb C3D5 followed by a FITC-conjugated anti-mouse Ig antiserum. ER-specific staining was per- Cell lines formed by incubating the cells with a rabbit anti-Sec61b polyclonal anti- HEK293 cells (kindly provided by Prof. Philip Cohen, Medical Research serum followed by a Texas Red-conjugated anti-rabbit Ig antiserum. For the Council Protein Phosphorylation Unit, University of Dundee) were cultured nucleus staining, cells were immersed in HBSS/BSA with 0.1% saponin in DMEM supplemented with 10% FBS and 2 mM L-glutamine. These cells containing Hoechst (1:2000) for 5 min. Just prior to confocal analysis, the were transfected with the pcDNA3.1-neo–based vectors containing hCD1d cells were placed in mounting medium (10 mM Tris [pH 8.5] and 2% 1,4- with the WT or indicated cytoplasmic tail mutants or CD1a extracellular diazabicyclo[2.2.2]octane). The cells were viewed on a LSM-510 laser domain fused to the CD1d WT (or mutant) cytoplasmic domain, using scanning confocal microscope (Zeiss, Thornwood, NY) modified for one- polyethylenimine following standard techniques. C1R cells (a kind gift photon microscopy using an oil immersion lens at 3100. Analysis of the from S. Balk) were transfected by electroporation with pcDNA3.1-neo relative level of CD1d colocalization with LAMP-1 was performed using containing WT and cytoplasmic tail mutants of hCD1d (or CD1a/CD1d MetaMorph software (version 5; Molecular Devices, Sunnyvale, CA). fusion proteins). To generate stable transfectants, the cells were selected and maintained in DMEM medium supplemented with 10% FBS, 2 mM L- Flow cytometry m glutamine, and 500 g/ml G418. Aliquots of cells were washed three times in PBS and then fixed in 1% Abs and related reagents paraformaldehyde. For surface staining, the cells were washed three times in HBSS/BSA and incubated with the appropriate mAb followed by a PE- Purified, unlabeled stocks of the CD1d-specific mAbs 51.1.3 (for immu- conjugated rabbit anti-mouse Ig antiserum or FITC-labeled anti-CD1a (or noprecipitation) and 42.1 (for flow cytometry and confocal microscopy) anti-CD25) mAb. For intracellular staining, the cells were permeabilized in were generous gifts from M. Exley (Brigham and Women’s Hospital, HBSS/BSA with 0.1% saponin and then incubated with the appropriate Harvard University, Boston, MA). Purified 6B11 (anti-human invariant mAb followed by a PE-conjugated rabbit anti-mouse Ig antiserum in the NKT cells), FITC conjugates of mAb against human LAMP-1, CD1a, and presence of saponin. Analysis was performed by flow cytometry as pre- CD25, and PE-conjugated 42.1 were from BD Biosciences (San Diego, viously described (15). The Journal of Immunology 4975

Coimmunoprecipitation was some degree of colocalization with the lysosomal/late endo- Cells were lysed in lysing buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 0.5 somal marker LAMP-1 (Fig. 1C), as has been previously de- mM EDTA, and 2% CHAPS), and the lysates were incubated with the scribed (37). The overlap between the anti-CD1d HC and anti- CD1d–b2m complex-specific (51.1; IgG2b) or anti-CD1d HC (C3D5 [Ig- CD1d–b2m mAb staining was quite limited (Fig. 1C), suggesting G2a]) mAb coupled to protein G-conjugated Sepharose beads (Pierce, the existence of two different forms of CD1d inside the cell. Thus, Rockford, IL) overnight at 4˚C. The lysates were then washed four times in the free CD1d HC appears to reside mainly in the ER, whereas the PBS containing 0.02% azide, resuspended in SDS loading buffer (4% SDS, 100 mM Tris-HCl [pH 6.8], 20% glycerol, 2% 2-ME, and 0.1% bromo- CD1d–b2m heterodimer is found on the cell surface and in en- phenol blue), run on a 10% SDS-PAGE gel, and then transferred to docytic subcellular organelles. a polyvinylidene difluoride membrane (Millipore). The blot was then In addition to the flow cytometry and confocal microscopic processed using the anti-CD1d HC mAb and developed using chem- analyses, human CD1d expressed by HEK293 was also charac- iluminescence before exposure on film. terized by coimmunoprecipitation with the anti-CD1d HC and anti- Statistical analysis CD1d–b2m heterodimer mAbs. CD1d that was pulled down by the ∼ An unpaired two-tailed Student t test was performed by Prism software anti-CD1d–b2m heterodimer mAb showed slower mobility ( 42 (version 5.00 for Windows; GraphPad, San Diego, CA). A p value ,0.05 kDa) than that immunoprecipitated with the CD1d HC-specific was considered significant. The error bars in the bar graphs show the SD mAb (∼38 kDa). The band corresponding to the CD1d–b2m from the mean. complex was almost undetectable in the total cell lysate (Fig. 1D). Our results suggest that the CD1d–b2m heterodimer is more Results glycosylated than the free CD1d HC, as has been previously re- Identification of two physical forms of CD1d in HEK293-CD1d ported (12, 13). Thus, the free CD1d HC is not (or poorly) gly- cells cosylated, and the CD1d–b2m heterodimer is at least partially (if To study the subcellular distribution and intracellular trafficking of not fully) glycosylated (Fig. 1D). Further, the HC is much more CD1d, a stable HEK293 cell line expressing WT human CD1d was abundant than the heterodimer. CD1d has four potential N-linked generated. Three human CD1d-specific mAbs, C3D5, 42.1, and glycosylation sites, and one of the glycans (glycan 2 at N42) is 51.1, were used to characterize this stable CD1d-expressing cell particularly important for CD1d–b2m association (38). Human line. It has been reported that the C3D5 mAb binds only to the epithelial cells and some cell lines express nonglycosylated CD1d CD1d HC, whereas the 42.1 and 51.1 mAbs bind to the CD1d–b2m (12, 39, 40), whereas other cell lines express a glycosylated form heterodimer (12). In the current study, an analysis by flow cy- (12). To determine whether the CD1d molecules expressed in tometry indicated that the HEK293-CD1d cells express no de- HEK293 cells are glycosylated, immunoprecipitants of CD1d tectable free CD1d HC on the surface but a high level of the using the free HC- and CD1d–b2m-specific mAbs were digested CD1d–b2m heterodimer (Fig. 1A). Intracellular staining of the with Endo H or PNGase F. The free CD1d HC was partially same cells revealed substantial CD1d HC (Fig. 1B). By confocal glycosylated and Endo H-sensitive (Fig. 1E), suggesting that it is microscopy, it was apparent that the free HC (as detected by the mainly located in the ER. However, the CD1d–b2m heterodimer C3D5 mAb) was restricted to the perinuclear area and colocalized was Endo H-resistant (Fig. 1E), indicating that this complex had with the Sec61b translocon, an ER marker (Fig. 1C). In contrast, been processed in the Golgi, whereas the high-mannose glycans the CD1d–b2m complex-specific mAb mostly stained molecules on CD1d were modified (reviewed in Ref. 41). We also found that on the cell surface and areas distal from the cell nucleus. There b2m was coimmunoprecitated with CD1d molecules pulled down

FIGURE 1. Identification of two physical forms of CD1d in HEK293 cells. HEK293 cells stably transfected with CD1d were fixed and stained with the

CD1d HC- or CD1d–b2m-specific mAb (solid line) or isotype control mAb (dotted line) followed by a PE-conjugated second Ab in the absence (A)or presence (B) of saponin. C, HEK293-CD1d cells were fixed and stained with anti-CD1d–b2m (red), anti–LAMP-1 (green), and Hoechst (blue) (upper panel), anti-CD1d HC (red), anti-Sec61b (green), and Hoechst (blue) (middle panel), or anti-CD1d–b2m (red), anti-CD1d HC (green) and Hoechst (blue) (lower panel). Magnification 31000. D, HEK293-CD1d and vector control lysates were immunoprecipitated with the CD1d HC- or CD1d–b2m-specific mAb and run on a 10% SDS-PAGE under reducing conditions. The proteins were then transferred onto a polyvinylidene difluoride membrane and probed with anti-CD1d (C3D5) for Western blot analysis. The same membrane was then stripped and reprobed with an anti-GAPDH Ab. The solid arrow points to glycosylated CD1d, whereas the dashed arrow points to the unglycosylated form. E, HEK293-CD1d lysates were immunoprecipitated with the anti-CD1d

HC or anti-CD1d–b2m mAb, and the immunoprecipitants were digested with Endo H or PNGase F. The proteins were resolved on 10% SDS-PAGE followed by Western blot analysis with the anti-CD1d HC mAb as described above. Total lysates of HEK293-CD1d were loaded on the gel as a control. The top arrow points to glycosylated CD1d, the middle arrow points to partially glycosylated CD1d, and the bottom arrow points to unglycosylated CD1d. The blot below shows that only the CD1d–b2m-specific (but not CD1d free HC-specific) mAb recognizes the CD1d–b2m heterodimer. 4976 A THREONINE-BASED TARGETING SIGNAL FOR HUMAN CD1d

using the anti-CD1d–b2m mAb (but not CD1d HC-specific mAb; shown) but expressed the same level of CD1d HC as the WT in- Fig. 1E). These data further confirmed that there are two different tracellularly as determined by flow cytometry (Fig. 2B, Supple- forms of CD1d: the free HC is located mainly in the ER, whereas mental Fig. 3). Interestingly, when the cells were stained with the the glycosylated CD1d–b2m heterodimer is processed in the Golgi anti-CD1d–b2m mAb for flow cytometry analysis, the T322D before its expression on the cell surface. mutant was expressed at a very low level on the cell surface (Fig. 2B), as was the mutant with the entire cytoplasmic tail deleted Destroying the YXXZ motif results in increased surface (TD-14; Fig. 2B). In contrast, the T322A mutant was expressed at expression of the CD1d–b m heterodimer, whereas T322D or 2 a comparable level as WT CD1d (Fig. 2B). Notably, the T322D deletion of the entire CD1d tail reduces its surface expression and TD-14 mutants could be detected as a CD1d–b2m hetero- The cytoplasmic tail of CD1d contains a YXXZ motif, which is dimer, but likely only in lysosomes, as determined by colocali- believed to be used for binding to APs involved in clathrin- zation with LAMP-1 (Fig. 2C). The Y331A mutant was expressed dependent endocytosis, such as AP2 (25). It has been shown at a significantly higher level on the surface (Fig. 2B) and showed previously that a Y331A mutation in the CD1d tail reduces its rate less colocalization with LAMP-1 as compared to that with WT of endocytosis (26). For other glycoproteins, it has been reported (Fig. 2C,2D). As expected (15, 26), the TD-6 mutant, lacking the that the phosphorylation of threonine, serine, and/or tyrosine YXXZ motif (Fig. 2A), was also expressed at a higher level on the residues in the cytoplasmic tail affects their intracellular traffick- surface (Fig. 2B) and showed less colocalization with LAMP-1 ing (42, 43). To determine whether the phosphorylation of the than WT (Fig. 2C,2D). Although the TD-10 mutant also lacks the most N-terminal amino acids (threonine and serine) in the CD1d YXXZ motif, it actually showed more colocalization with LAMP- cytoplasmic tail and/or destroying the YXXZ motif would affect 1, with a surface level below that of WT (albeit not statistically its trafficking, a broad series of mutants were generated by site- significant; Fig. 2B–D). This suggests that besides the YXXZ directed mutagenesis (Supplemental Fig. 1). We then compared motif there are other lysosomal-targeting signals in the cytoplas- the expression of CD1d from mutants to that of the WT by FACS mic tail of CD1d. These signals would only become dominant and confocal microscopy. We found that there was almost no when the last 10 aas are absent. difference of CD1d HC expression but great difference of Chloroquine rescues CD1d–b2m heterodimer expression in the CD1d–b2m heterodimer expression among the mutant cell lines (Supplemental Fig. 2A). We also found some difference in the T322D and TD-14 mutants colocalization of CD1d–b2m with the late endosomal/lysosomal Due to the fact that the CD1d–b2m heterodimer was mostly ob- marker LAMP-1 among different mutant cell lines (Supplemental served in LAMP-1+ compartments with the T322D and TD-14 Fig. 2B). For simplicity, the data from six representative mutants, mutants, it was hypothesized that changes in lysosomal-targeting T322A, T322D, Y331A, and those with a deletion of the last six signals in these mutants would result in their degradation in lyso- (TD-6), 10 (TD-10), or all 14 (TD-14) C-terminal amino acids, are somes. To test this hypothesis, WT CD1d and the six mutants presented here (Fig. 2), with the results from the other mutants described above were incubated with the lysosomotropic inhibitor also presented in the Supplemental Material. The replacement of chloroquine to prevent acidification of this compartment. Chloro- a threonine with aspartate is commonly used to mimic its phos- quine treatment increased the intracellular levels of the T322D, TD- phorylated form (44). None of the six mutants, similar to WT, was 10, and TD-14 mutants as CD1d–b2m heterodimers (Fig. 3B, detected by the anti-CD1d HC mAb on the surface (data not Supplemental Fig. 4) but had no effect on the HC overall. Chloroquine

FIGURE 2. CD1d cytoplasmic tail mutants show different surface and intracellular expression patterns. A, Amino acid sequence of the cytoplasmic tail mutants used in the experiments shown in this figure. B, WT- and mutant CD1d-expressing HEK293 cells were fixed and stained with the anti-CD1d–b2m mAb (white bars) or the pan–anti-HLA class I-specific anti-MHC I mAb (black bars) followed by a PE-conjugated anti-mouse Ig antiserum. For staining with the anti-CD1d HC mAb (gray bars), the cells were permeabilized with 0.1% saponin. Analysis was by flow cytometry. The data are displayed as the mean fluorescence intensity (MFI) relative to WT (WT = 1). Each bar represents the mean of the MFI in six experiments 6 SD. ppp , 0.01. C, HEK293-

CD1d WT and the indicated mutants were fixed, permeabilized, and stained with the anti-CD1d–b2m (red) and anti–LAMP-1 (green) mAb and Hoechst (blue). The stained cells were then analyzed by confocal microscopy. Magnification 31000. D, The level of CD1d (anti-CD1d–b2m-reactive) and LAMP-1 colocalization in the graph below was determined by MetaMorph analysis. pp , 0.05. The Journal of Immunology 4977

FIGURE 3. Chloroquine prevents the intracellular loss of the T322D and TD-14 CD1d mutants. A, HEK293-CD1d WT cells were treated with vehicle (solid line) or

20 mM chloroquine (dotted line) overnight. The cells were then fixed, permeablized, and stained with the anti-CD1d–b2m mAb for flow cytometry analysis. B, HEK293 cells expressing CD1d WTor the CD1d mutants were treated with or without 20 mM chloroquine overnight. The cells were then fixed, permeablized, and stained with anti-CD1d HC (white bars), anti-CD1d–b2m (black bars), or anti-MHC I (gray bars) mAbs for flow cytometry analysis. The increase in MFI after overnight treatment of chloroquine was calculated using this formula: change of MFI = 100% 3(MFIchl 2MFIveh)/MFIveh. The data shown in the figure are the mean change of the MFI 6SD of two independent experiments. C, CD1d WT-, T322D-, and TD-14-expressing HEK293 cells were treated with or without 20 mM chloroquine overnight and lysed, and then the lysates were immunoprecipitated with the anti-CD1d HC or anti-CD1d–b2m mAb. Total lysates of each treatment were loaded onto the gel as a control. The membrane was probed with the CD1d HC-specific mAb for Western blot analysis. The same membrane was stripped and reprobed with anti-GAPDH. The arrows point to an anti-CD1d–b2m-reactive band in the T322D and TD-14 mutants after chloroquine treatment. HEK293-CD1d WT (D), T322D (E), and TD-14 (F) cells were treated overnight with 20 mM chloroquine (or vehicle). The cells were then fixed, permeabilized, and stained with the anti-CD1d–b2m mAb (red), anti–LAMP-1 (green), and Hoechst (blue). The stained cells were then analyzed by confocal microscopy. Magnification 31000. G,ThelevelofCD1d(CD1d–b2m complex) and LAMP-1 colocalization was determined by MetaMorph analysis. pp , 0.05; ppp , 0.01. MFIchl, MFI from chloroquine-treated cells; MFIveh, MFI from vehicle-treated cells. did not alter the intracellular level of WT CD1d (Fig. 3A). These ated stable C1R (human B lymphoblastoid) cell lines expressing results were supported by coimmunoprecipitation (Fig. 3C)and CD1d WT, T322D, and TD-14. C1R cells also expressed less CD1d confocal microscopy analyses (Fig. 3D–G). For the latter experi- on the surface with the T322D and TD-14 mutants as compared with ments, chloroquine treatment generally increased the colocalization WT (Supplemental Fig. 5). This result is further support for the of CD1d and LAMP-1 in cells expressing WT CD1d as well as the existence of a lysosomal-targeting signal besides the YXXZ motif. T322D and TD-14 mutants (Fig. 3D–G). Therefore, these data sug- To determine whether only the CD1d cytoplasmic tail contains gest that the T322D mutant or a total deletion of the cytoplasmic tail signals that control trafficking to lysosomes, chimeric constructs of (TD-14) causes lysosomal targeting and, consequently, likely degra- the CD25 extracellular domain fused to the transmembrane domain dation of the CD1d–b2m heterodimer in that compartment. of CD1d and WT (or mutated) cytoplasmic tail of CD1d were generated. The chimeric constructs were used to transfect HEK293 Threonine-based signals in the CD1d cytoplasmic tail control cells, and stable transfectants were characterized by confocal the intracellular trafficking of fusion proteins microscopy. The CD25/CD1d fusion protein could be observed on We have shown that the T322D or TD-14 mutations cause less ex- the plasma membrane and in the cytosol, with some level of pression of CD1d on the surface in HEK293 cells. To test whether colocalization with LAMP-1 (Fig. 4A,4B). When the cytoplasmic this phenomenon is only limited to fibroblast cells, we also gener- tail was deleted (CD25/CD1d TD-14), CD25 was only detected in 4978 A THREONINE-BASED TARGETING SIGNAL FOR HUMAN CD1d

FIGURE 4. The cytoplasmic tail from the T322D and TD-14 CD1d mutants alters the surface expression of CD25 or CD1a fusion proteins. A, HEK293 cells were transfected with the CD25 extracellular domain fused to WT, T322D, or TD-14 CD1d tail. The cells were stained with an anti-CD25 (green) and anti–LAMP-1 (red) mAb and Hoechst (blue) and analyzed by confocal microscopy. Magnification 31000. B, The level of CD25 and LAMP-1 colocal- ization in the graph below was determined by MetaMorph analysis. C, Constructs encoding the CD1a extracellular domain fused to the WT, T322D, or TD- 14 CD1d tail mutants were transfected to HEK293 cells. The cells were stained with anti-CD1a for flow cytometry analysis. The data are shown as the MFI relative to the WT (WT as 100%). Each bar corresponds to the MFI mean 6 SD. pp , 0.05. the perinuclear area and in lysosomes (Fig. 4A), suggesting that due to less CD1d surface expression or a defect in the Ag- the CD1d cytoplasmic tail is required for its targeting to the presenting capacity of mutant CD1d molecules; the addition of plasma membrane (16, 26, 45). Thus, as was observed with the a-GalCer to these cultures resulted in NKT activation by the CD1d TD-14 construct (Fig. 3), deletion of the entire cytoplasmic mutants at comparable levels to that induced by WT CD1d (Fig. tail of CD1d caused lysosomal targeting of the CD25/CD1d (TD- 5B), suggesting that the CD1d mutants are indeed functional. 14) fusion protein. Surprisingly, the T322D mutation in the CD25/ Threonine-based (T322) signals in the CD1d cytoplasmic tail CD1d fusion protein showed similar intracellular distribution and regulate direct trafficking from the trans-Golgi network to colocalization with LAMP-1 as the WT chimeric protein in lysosomes HEK293 cells (Fig. 4A,4B). However, one explanation could be that the T322D residue is a signal for lysosomal targeting, whereas Our data suggest that there are two lysosomal-targeting signals in the other 13 aas of the cytoplasmic tail contains recycling signals the cytoplasmic tail of CD1d: threonine- (T322) and tyrosine-based for trafficking back to the plasma membrane. As a single-chain (YXXZ motif). To determine which signal is dominant, we gen- protein, CD25 is likely much more easily recycled back to the erated T322A/Y331A and T322D/Y331A double mutants and plasma membrane than the CD1d–b2m heterodimer. Thus, to test analyzed their surface expression by flow cytometry. The level of this hypothesis, chimeric proteins with the CD1a extracellular the T322A/Y331A double mutant on the cell surface was com- domain fused to the transmembrane and cytoplasmic domains of parable to that of the Y331A single mutant. In contrast, the T322D/ WT (or mutated) CD1d were generated. Like CD1d, CD1a is Y331A double mutant was expressed at a level below that of the a b2m-associated molecule (46). As observed with the intact WT (Fig. 6A, Supplemental Fig. 7), but not as low as the T322D CD1d molecule, the T322D and TD-14 mutations in the CD1a single mutant, suggesting that the T322-based signal acts up- chimeric construct resulted in reduced cell surface expression of stream of the YXXZ motif and is dominant. Most likely, the CD1a, whereas when the WT CD1d tail was used much higher T322D and TD-14 mutants are directly targeted from the trans- levels of CD1a could be detected (Fig. 4C), consistent with our Golgi network (TGN) to lysosomes. To further address this hy- data above with intact WT CD1d. pothesis, WT CD1d and the T322D and TD-14 mutants were treated with monensin overnight to block protein transport in the The cytoplasmic tail of CD1d is required for Ag presentation to Golgi. This treatment caused a reduction in intracellular WT human NKT cells CD1d but raised the levels of both the T322D and TD-14 mutants CD1d molecules present lipid Ags to NKT cells for their activation, (Fig. 6B). Therefore, these results strongly suggest that two sig- and these Ags are likely acquired in a late endocytic compartments nals in the cytoplasmic tail of CD1d regulate its trafficking to (4). Because it was shown above that the CD1d cytoplasmic tail lysosomes; the T322D mutation or the deletion of the entire cy- contains two different signals for its intracellular trafficking, it is toplasmic tail of CD1d causes direct targeting of CD1d to lyso- reasonable to postulate that mutations in those signals would af- somes for degradation. fect Ag presentation to NKT cells. To test this hypothesis, HEK293 cells expressing WT CD1d or those with mutations in Discussion their cytoplasmic tail were treated with vehicle or a-GalCer [a Of the CD1 isoforms that are Ag-presenting molecules, only CD1a synthetic glycolipid that strongly stimulates NKT cells in a CD1d- lacks the AP-binding motif YXXZ in its cytoplasmic tail (5). As restricted manner (4)] and cocultured with human NKT cells a likely consequence, CD1a is mostly expressed on the cell sur- generated from normal PBMCs. HEK293 cells expressing WT face (47). In contrast, the other CD1 family members CD1b, CD1d were able to activate human NKT cells to secrete cytokines CD1c, and CD1d all possess the YXXZ motif, resulting in their that can be blocked by an anti-CD1d Ab (Supplemental Fig. 6), localization both on the surface and in early and late endocytic suggesting that the NKT cell activation is CD1d-specific. In the compartments (37). It has been suggested that the cytoplasmic absence of a-GalCer, the T322A mutant activated NKT cells to tails of these CD1 molecules contain signals for their intracellular the same level as WT CD1d (Fig. 5A). In contrast, the mutants distribution and endosomal trafficking (reviewed in Ref. 5). The lacking the lysosomal-targeting motif YXXZ (i.e., Y331A, TD-6, data presented in the current study suggest that the cytoplasmic TD-10, and TD-14) were less effective at activating NKT cells. Ag tail is also important for the stability of the CD1d–b2m hetero- presentation by the T322D mutant, which was expressed at dimer. We speculate that the cytoplasmic tail of CD1d contains a lower surface level than WT, was also reduced (Fig. 5A). signals for directing the molecule to other compartments. When these However, the decrease in NKT cell stimulation was not simply signals are missing or altered, the molecule traffics to lysosomes for The Journal of Immunology 4979

FIGURE 5. WT CD1d and the cytoplasmic tail mutants differ in their Ag presentation abilities. CD1d WT- and the indicated CD1d tail mutant-expressing cells were cocultured with PBMC-derived human NKT cells (E:Tratio of 1:1) for 48 h in the presence of vehicle (PBS + 0.02% Tween 20; A)ortheindicated concentrations of a-GalCer (B). Supernatants were harvested, and the production of GM-CSF was mea- sured by ELISA. Each bar is the mean of triplicate samples 6 SD. ppp , 0.01. The data shown are representative of three independent experiments. its degradation (by a default pathway). Thus, our data show that the is expressed at a higher level than WT and has a reduced ability to T322D mutant causes a substantial decrease in the cell surface level activate NKT cells. In TD-10, CD1d shows reduced surface ex- of CD1d. Rather than an alteration of the threonine-based signal, it is pression and more colocalization with LAMP-1+ compartments, possible that the T322D mutation simply caused conformational suggesting that the KRQT sequence present in TD-6 (but not in TD- changes in the extracellular domain of CD1d that reduced its ability 10; Supplemental Fig. 1) may also play a role in efficient plasma to be detected by the CD1d–b2m complex-specific mAb. However, membrane trafficking. In fact, the K326R mutant of CD1d is ex- were that to be the case, it is unlikely that we would have been able pressed on the surface at reduced levels (Supplemental Fig. 2A). It is to detect an increase in the T322D and TD-14 mutants in lysosomes well known that lysines are targets for different types of ubiq- in chloroquine- and monensin-treated cells. uitylation, and each type can have distinct effects on such proteins, On the basis of our results, we propose a model to explain how including directing their degradation by proteasomes (49), and the two signals in the cytoplasmic tail influence the intracellular monoubiquitination has also been shown to be an endocytic traf- distribution and the trafficking of the CD1d–b2m heterodimer and ficking signal (29). Of immunological importance, the CD1d K326 its surface expression (Fig. 7). After its synthesis in the ER, CD1d residue also seems to be an important target for immune evasion by molecules quickly associate with b2m and are transported to the KSHV (31). TGN. There, they are further glycosylated and transported to the We and other investigators have clearly shown that the YXXZ plasma membrane with the help of the T322 residue in the cyto- motif in mouse CD1d1 is required for mouse CD1d-mediated Ag plasmic tail. The T322D mutant [equivalent to its phosphorylation presentation to invariant NKT cells (15, 50). In this report, our data (48)] or deletion of the entire cytoplasmic tail would result in the also suggest that the YXXZ motif is required for human CD1d- loss of signals that target the CD1d–b2m complex to the plasma mediated Ag presentation. However, this conclusion appears to be membrane and thereby diverts this complex to lysosomes. Here, in conflict with some prior publications, suggesting that this motif b2m dissociates from the CD1d HC, and the molecule is degraded. is less important with human CD1d (32, 51). The difference in our From the cell surface, CD1d is endocytosed via clathrin-dependent study could be because we used endothelial cells (HEK293) rather vesicles due to its YXXZ motif. In late endosomes and/or lyso- than B lymphoblastoid cells such as C1R cells (32) or cells de- somes, CD1d meets and binds the lipid Ags that are required for rived from PBLs (51). Of further relevance to our study, Colgan NKT cell activation (4). CD1d is then returned to the cell surface for et al. (52) found that in epithelial cells the YXXZ motif is required recognition by NKT cells. When the Y331 residue is replaced with for CD1d autocrine signaling. We also observed that CD1d mol- an alanine or the YXXZ motif is deleted (e.g., TD-6), the ecules lacking this motif activate NKT cells the same as WT does CD1d–b2m complex remains on the cell surface rather than tra- versing through endocytic compartments. As a result, this molecule

FIGURE 6. The T322 signal in the CD1d cytoplasmic tail is dominant. A, HEK293 cells were transfected with WT CD1d, the T322D and Y331A single mutants, or T322A/Y331A and T322D/Y331A double mutants to generate stable transfectants. The cells were stained with an anti-

CD1d–b2m mAb for flow cytometry analysis. The data are shown as the percentage of the MFI relative to WT. Each bar represents the mean of the FIGURE 7. Model illustrating how threonine- and tyrosine-based sig- MFI 6 SD. B, CD1d WT-, T322D-, and TD-14-expressing HEK293 cells nals in the cytoplasmic tail of CD1d control its functional expression. The were treated with or without monensin (GolgiStop, 1:1500) overnight. The black lines and arrows show the typical pathway for newly synthesized cells were then fixed, permeablized, and stained with anti-CD1d HC (white CD1d, whereas the red lines and arrow show the effect of specific muta- bars), anti-CD1d–b2m (black bars), or anti-MHC I (gray bars) mAb for tions of these signals on CD1d intracellular trafficking. Details are pro- flow cytometry analysis. The increase in MFI after monensin treatment vided in Discussion. 1) CD1d is synthesized in the ER. 2) After association was calculated using this formula: increase of MFI = 100% 3 (MFImon 2 with b2m, CD1d is transported to the TGN, where it undergoes glyco- MFIveh)/MFIveh. The data shown in the graph represent the mean increase sylation modification. 3) CD1d is endocytosed from the cell surface of MFI 6 SD of two independent experiments combined. pp , 0.05. through clathrin-dependent vesicles. EE, early endosome; LE, late endo-

MFImon, MFI from monensin-treated cells. some; SE, sorting endosome. 4980 A THREONINE-BASED TARGETING SIGNAL FOR HUMAN CD1d in C1R cells (data not shown), suggesting that distinct APCs detect phosphorylation of threonine residues in CD1d molecules, differentially activate NKT cells. This could be due to cell (or suggesting that phosphorylated T322 may be below the level of tissue)-specific lipid Ags presented by CD1d. This is not un- detection by Western blot analyses under the conditions that we precedented (53). Nevertheless, we can most certainly draw the used (data not shown). However, we have found that an HSV conclusion that the YXXZ motif is important for human CD1d- infection causes less degradation of the T322A/S323A double mediated Ag presentation in epithelial cells. mutant as compared with CD1d WT (data not shown). This sug- Two forms of CD1d have been shown to exist in both primary gests that the decrease in CD1d surface expression observed fol- human intestinal epithelium and a number of cell lines (12, 13, 40, lowing an HSV-1 infection (54 and J. Liu and R.R. Brutkiewicz, 52). They are either associated with b2m or exist as free H chains. unpublished observations) may be due to phosphorylation of the Usually, the b2m-associated form is glycosylated, whereas the T322 residue and consequent faster degradation of CD1d. b2m-independent form can be glycosylated in some cell lines such In summary, our results show that a threonine-based signal in the as C1R and some epithelial cell lines such as FO-1 (12, 13) but cytoplasmic tail controls the functional expression of CD1d. It is appears to be nonglycosylated in most epithelial cell lines, in- highly likely that phosphorylation of this residue (or complete loss cluding HEC, HeLa, HT-29, and T84 cells (39, 40, 52, 54). The of the cytoplasmic domain) results in a default program to direct the relationship between these two forms of CD1d has remained un- CD1d molecule to lysosomes for degradation. Such regulation of clear, although it is known that the group 1 CD1b molecule is the intracellular trafficking of a glycoprotein that is thus normally trapped in the ER when b2m is absent (3). Our confocal microscopy in a nonphosphorylated state has many applications to a number of data indicate that the CD1d HC is also retained in the ER in other molecules that also traverse through the endocytic pathway HEK293 cells. Thus, consistent with a prior report (55), the results with critical relevance to the function of that cell. of the current study suggest that the CD1d HC accumulates in the ER possibly due to a shortage of b m and association of the HC 2 Acknowledgments with b2m likely serves as a potential “release” signal to permit CD1d to begin its exit from the ER. Small amounts of free CD1d We thank the Indiana Center for Biological Microscopy for important help in these studies as well as Ntsane Moleleki and Mark Peggie for generating HC may be targeted to the plasma membrane as has been suggested the CD25/CD1d fusion constructs. We also thank the Flow Cytometry Re- (12, 13, 55). Another remaining question is whether the b2m- source Facility, Indiana University School of Medicine, for assistance. Philip independent form of CD1d is functional in NKT cell activation. Cohen and Mark Exley generously provided the HEK293 cell line and 51.1 2/2 One study reported that spleen cells from b2m mice are able to and 42.1 mAbs, respectively. We also thank Renukaradhya Gourapura for activate CD1d-specific T cells, suggesting that the free CD1d HC helpful discussions and Claire Willard for expert technical assistance. alone can activate NKT cells (56). 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