The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cernadas, M.
Right arrow Articles by Brenner, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cernadas, M.
Right arrow Articles by Brenner, M. B.
The Journal of Immunology, 2003, 171: 4149-4155.
Copyright © 2003 by The American Association of Immunologists

Lysosomal Localization of Murine CD1d Mediated by AP-3 Is Necessary for NK T Cell Development 1

Manuela Cernadas*, Masahiko Sugita{ddagger}, Nicole van der Wel§, Xiaochun Cao, Jenny E. Gumperz{dagger}, Sergei Maltsev||, Gurdyal S. Besra||, Samuel M. Behar{dagger}, Peter J. Peters§ and Michael B. Brenner2,{dagger}

* Division of Pulmonary and Critical Care Medicine, {dagger} Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115; {ddagger} Department 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{beta}+ 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD1 molecules are composed of transmembrane MHC class I-like heavy chains that noncovalently associate with {beta}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, 3, 4) to T cells with diverse TCRs. In contrast, 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 {alpha}-galactosylceramide ({alpha}-GalCer) 3 in experimental systems (5, 6, 7, 8, 9). CD1d-restricted T cells have proven functionally significant, since they secrete copious amount of IL-4, IL-10, and IFN-{gamma} and also display cytotoxic effects (10, 11, 12, 13).

Besides differences in Ag-presenting capacity and T cell activation, 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. Interaction 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 localizes to early recycling endosomes, with only a small fraction distributed in lysosomes (18, 19, 20, 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 differences in CD1 isoform localization is the dependence of vesicular acidification for effective Ag presentation by CD1b, which traffics to lysosomes, but not for CD1a, which traffics through early endosomes (19, 22, 23).

Like human CD1b (hCD1b), murine CD1d (mCD1d) molecules are prominently expressed in LAMP-1+ lysosomes, and redistribution from lysosomes to the plasma membrane had been observed for cytoplasmic tail-truncated forms of both molecules. Furthermore, cytosolic tail modifications that alter trafficking of hCD1b and mCD1d impair their ability to present foreign microbial Ags. CD1b and CD1d tail deletion resulted in loss of recognition by foreign Ag-reactive T cells and the majority of invariant CD1d-reactive T cells, respectively (5, 17, 24, 25). These findings led us to speculate that the intracellular distribution of mCD1d appeared to more closely resemble that of hCD1b than that of hCD1d. This suggested a possible mechanism for rodents to compensate for the absence of CD1a, -b, and -c molecules. Here, we show that mCD1d, like hCD1b, interacts with the adaptor protein AP-3 via its cytoplasmic tail tyrosine-based sorting motif that controls the localization of mCD1d to lysosomes. This interaction is critical for the development and maintenance of CD1d specific NK T (NKT) cells. Pearl mice, which have a 125-aa deletion in the gene encoding the {beta}3A subunit of the AP-3 complex (26, 27, 28), and mice deficient in the {beta}3A subunit of the AP-3 complex by homologous recombination (AP-3b1LN) (16) were found to have significant defects in CD1d intracellular localization and NKT cell number. AP-3 deficiency causes redistribution of mCD1d from lysosomes resulting in increased cell surface expression on thymocytes, dendritic cells (DC), and B cell-depleted splenocytes. Despite a higher level of cell surface expression, the altered CD1 trafficking appeared to impair CD1d Ag presentation and positive selection in the thymus, since a significant reduction of NKT and CD1d tetramer-positive cells was noted in spleen, thymus, and liver. Thus, the murine group II CD1d isoform accomplishes critical trafficking to and sampling of lysosomes by adopting adaptor protein interactions noted for the human group I CD1b isoform.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Pearl mice on the C57BL/6 background and age- and sex-matched, wild-type C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME). AP-3b1LN-deficient mice on the C57BL/6 background were provided and previously characterized by Dr. S. Mansour (16). Heterozygotes were mated to generate homozygous AP-3b1LN mutants identified by PCR (16) and coat color and were compared with their heterozygote, age- and sex-matched littermates. Mice were used between 8–20 wk of age. Mice were maintained according to institutional animal care and use committee guidelines.

Flow cytometry and Abs

Single-cell suspensions were prepared from thymus, spleen, and liver (after perfusion with 30 cc of PBS). Splenocyte, thymocyte, and liver cell suspensions in RPMI medium were centrifuged over a layer of Histopaque (Sigma-Aldrich, St. Louis, MO). Mononuclear cells were harvested from the interface. To obtain splenic DC, teased spleen tissue was digested with collagenase (1 mg/ml; type IV, Sigma-Aldrich) for 1 h at 37°C. Splenic DC were isolated using CD11c magnetic microbeads (Miltenyi Biotec, Germany) as per manufacturer’s instructions. Bone marrow derived DC were generated as previously described (29). Purity of the DC populations was assessed by flow cytometry using hamster anti-mouse CD11c (HL3)-FITC and rat anti-mouse I-A/I-E (M5/114.15.2)-PE Abs (Abs) partially quenched with unconjugated rat anti-mouse I-A/I-E Abs (BD PharMingen, San Diego, CA). B cells were depleted from splenocytes with CD19 magnetic microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) according to the manufacturer’s instructions.

To block Fc{gamma}Rs, cells were blocked with 2.4G2-purified mAb. Cells were subsequently stained with optimal concentrations of conjugated mAbs and their appropriate isotype controls for 20 min at 4°C in flow cytometry buffer (PBS with 1 mg/ml of BSA (Sigma-Aldrich), 2 mM EDTA, and 0.05% NaN3), washed, and analyzed using a FACScan flow cytometer and CellQuest software (BD Biosciences, San Jose, CA). Fluorochrome-conjugated Abs to pan-TCR-{beta}-FITC, NK1.1-PE, CD8{alpha}-PE, CD1d-PE, CD3-CyChrome, CD22-FITC, rat IgG2b-PE control, and hamster IgG-CyChrome control were obtained from BD PharMingen. Abs to CD19-FITC and mouse IgG2a-PE control were obtained from Caltag (Burlingame, CA) and eBioscience (San Diego, CA), respectively. Abs to CD3 (2C11), CD4 (GK1.5), and CD8 (53.6.7) were generated from American Type Culture Collection (Manassas, VA) hybridomas, purified and conjugated to Alexa 488 (Molecular Probes, Eugene, OR) according to the manufacturer’s instructions.

CD1d-{alpha}-GalCer tetramer staining

Murine CD1d tetramers complexed to Alexa 488 (Molecular Probes) were constructed as previously described (6, 30). {alpha}-GalCer was synthesized from commercially available D-lyxose according to a modification of the previously reported procedures (31), employing different protecting groups and removal and coupling strategies. The complete synthesis will be reported elsewhere (G. S. Besra and S. Maltsev, unpublished observations). The key features were the use of tert-butyldiphenyl silyl ether and benzoate protecting groups and removal therein. The sugar-coupling procedure employed the intermediate protected azido-sphingosine, followed by reduction, N-acylation, and final removal of protecting groups. The final material {alpha}-GalCer was in excellent agreement with 1H-nuclear magnetic resonance, 13C-nuclear magnetic resonance, and mass spectral data previously reported (31). Lyophilized lipid was dissolved in DMSO at a concentration of 100 µg/ml at 37°C by agitation and was sonicated before Ag loading. {alpha}-GalCer-loaded or DMSO mock-loaded CD1d tetramers and fluorochrome-conjugated mAbs were incubated with cells after blocking with 2.4G2 mAb.

Yeast two-hybrid assays

The GAL4ad-µ3A and GAL4bd-human CD1a, CD1b, and CD1d two-hybrid constructs were generated as previously described (18). GAL4bd-murine CD1d was generated similarly by ligation of the GAL4bd construct with synthetic dsDNA encoding the entire cytoplasmic domain of murine CD1d. The constructs were confirmed by DNA sequencing. Saccharomyces cerevisiae strain HF7c was transformed with the GAL4ad-µ3A construct together with each of the GAL4bd-CD1 tail constructs. Subsequently, the transformed cells were inoculated in histidine-deficient medium, and their growth was assessed by measuring OD600 at various time points as previously described (15).

Immunogold-labeled transmission electron microscopy and quantitation

Wild-type and pearl splenic and bone marrow-derived DC, purified as described above, were fixed with 2% paraformaldehyde (Polysciences, Warrington, PA). Samples were prepared for ultrathin cryosectioning as previously described (32). Cryosections were labeled with rat anti-mouse CD1d mAb (19G11, gift from Dr. A. Bendelac) for 60 min and secondarily incubated with rabbit anti-rat Ab (DAKO, Carpinteria, CA) and protein A conjugated to 15 nm of gold (EM Laboratory, Utrecht University, Utrecht, The Netherlands). Sections were analyzed on a Technai electron microscope (Philips Electronic Instruments, Mahwah, NJ). Quantitation of immunogold-labeled lysosomes and membranes was performed as previously described (33, 34).

Generation of chimeric CD1d molecule plasmid constructs

A cDNA encoding a chimeric mCD1d molecule with the hCD1d tail was generated by PCR using mouse CD1d in pSR{alpha}-neo as template DNA. The primers used were 5'-GGC GGC GGC CGC TAT GCG GTA CCT ACC ATG GCT GTT-3' (sense) and 5'-GCG CTC GAG TCA CAG GAC GCC CTG ATA GGA AGT TTG CCT CTT CCA GAT ATA GTA GAC TAC-3' (antisense). Following digestion with NotI and XhoI, the PCR product was cloned into pBluescript KS (Stratagene, La Jolla, CA) and sequenced. The inserted cDNA construct was then cut out by digestion with NotI and KpnI, and recloned into pcDNA3.1- for transfection.

Confocal microscopy and immunofluorescence labeling

HeLa cells grown on coverslips were transfected with plasmids encoding wild-type mCD1d or the chimeric construct by calcium phosphate precipitation as previously described (35). Two days after transfection the cells were fixed, permeabilized, and stained intracellularly as previously described (18). Staining with Abs against murine CD1d (15F7; American Type Culture Collection) and LAMP-1 (36) (a gift of Dr. Minoru Fukuda, The Burnham Institute, La Jolla, CA) was followed by incubation with FITC-conjugated donkey anti-rat IgG and Texas Red-conjugated donkey anti-rabbit IgG Abs (Jackson ImmunoResearch Laboratories, West Grove, PA). Confocal microscopy was performed on the labeled cells with a TCS-NT confocal laser scanning microscope (Leica, Deerfield, IL) fitted with krypton and argon lasers as previously described (22).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Murine, but not human, CD1d binds the AP-3 µ3A subunit and mediates lysosomal localization

One mechanism by which differential protein sorting occurs involves the selective recognition of nearly similar tyrosine-based sorting signals in the cytoplasmic tails of transmembrane proteins. Recently, we showed that the AP-3 complex selectively recognizes hCD1b, but not other hCD1 isoforms (hCD1a, -c, or -d). This interaction with the AP-3 complex plays a critical role in the lysosomal localization and function of hCD1b (14, 18). In contrast, hCD1d fails to bind the AP-3 complex, resulting in a much smaller lysosomal pool of hCD1d molecules. Since mCD1d, unlike hCD1d, has been demonstrated to colocalize extensively with LAMP-1 (5) we sought to determine the mechanism by which mCD1d might accomplish its critical lysosomal trafficking. Therefore, a yeast two-hybrid system was employed to determine whether the tyrosine-based motif in the cytoplasmic tail of mCD1d was capable of interacting with AP-3. The GAL4 transcription activation domain was fused to the µ3A subunit of human AP-3, which is 99% homologous to murine AP-3, and was coexpressed in yeast cells with the GAL4 DNA binding domain fused to the cytoplasmic tyrosine based sequences derived from either human or mouse CD1d. Specific interactions were identified by growth of the transformed yeast cells in histidine-deficient medium. As previously demonstrated, no growth was detected in histidine-deficient medium for yeast cells expressing the hCD1d tail (18). Surprisingly, yeast cells expressing the mCD1d tail showed efficient growth in histidine-deficient medium, suggesting a specific interaction of the µ3A subunit of AP-3 with murine, but not human, CD1d tail (Fig. 1).



View larger version (12K):
[in this window]
[in a new window]
  		FIGURE 1. Murine CD1d, but not hCD1d, binds to the adaptor complex AP-3. Yeast cells were transformed with the GAL4ad-µ3A construct together with GAL4bd-mCD1d, GAL4bd-hCD1d, GAL4bd-hCD1b as a positive control, or GAL4bd-hCD1a as a negative control. Cells were inoculated in histidine-deficient medium. Specific interactions between the constructs were detected by the ability of the transformed cells to grow in the absence of histidine, as determined by measuring the OD600 at the indicated time points. Murine CD1d and hCD1b specifically bound to AP-3, whereas hCD1d did not.

 
To directly evaluate the role of the CD1d tail in intracellular localization in vivo, wild-type mCD1d and a chimeric mCD1d molecule in which the cytoplasmic tail was swapped with the hCD1d tail were expressed in HeLa cells by transfection, and their localization was compared using confocal microscopy. Wild-type mCD1d molecules were prominent in peripherally distributed vesicles, as was LAMP-1, an endogenous marker for lysosomes. Electronic merging of the images revealed that the mCD1d-containing vesicles and the LAMP-1-containing vesicles were substantially colocalized, confirming the prominent localization of wild-type mCD1d molecules in lysosomes (Fig. 2C). In contrast, the chimeric mCD1d:hCD1d tail molecules showed markedly reduced colocalization with LAMP-1 (Fig. 2F) and were expressed more prominently on the cell surface than wild-type mCD1d. Thus, the efficient targeting of mCD1d to lysosomes required the sorting motif present in the mCD1d tail and was significantly impaired by replacement with the hCD1d tail. These findings suggest that the ability of mCD1d molecules to bind to AP-3 and the lack of interaction with AP-3 by hCD1d molecules may be the main molecular explanation for the differential intracellular localization of the CD1d molecules in these two species.



View larger version (67K):
[in this window]
[in a new window]
  		FIGURE 2. Replacement of murine with human CD1d tail fails to deliver murine CD1d to the lysosomal compartment. HeLa cells transfected with wild-type mCD1d (top panels) or chimeric mCD1d with hCD1d tail (bottom panels) were labeled intracellularly with Abs against mCD1d (A and D, green) and the lysosomal marker LAMP-1 (B and E, red) and examined by confocal microscopy. The two images were superimposed to detect the intracellular compartments expressing both CD1d and LAMP-1 (C and F, yellow). Scale bars (5 µm) are in the lower left of the merge panel.

 
CD1d expression at the cell surface is increased in AP-3-deficient mice

Functional experiments have shown that the lysosomal trafficking of hCD1b and mCD1d are important determinants of their ability to present Ags to T cells. Given that the data presented here indicated a role for AP-3 in localization of mCD1d, we next examined the expression and function of CD1d in AP-3-deficient pearl mice. Importantly, we noted a marked increase in the cell surface expression of mCD1d on pearl thymocytes, splenic DC, and splenic T cells (Fig. 3). The effect of AP-3 deficiency on mCD1d expression was most pronounced on DC and B cell-depleted splenocytes, where the fold increases, respectively, in the mean fluorescence intensity over that in wild-type cells were 2.8 ± 0.2 and 2.6 ± 0.1. Further, there was a 1.8 ± 0.2-fold increase in CD1d mean fluorescence intensity in pearl thymocytes over that in wild-type cells. Interestingly, there was no increase in the cell surface expression of mCD1d on B cells, including splenic mCD1dhigh B cells (data not shown). The basis for B cells differing from DC, T cells, and thymocytes in altered mCD1d expression is not known. The marked increase in CD1d cell surface expression on DC, T cells, and thymocytes is consistent with a redistribution of CD1d from intracellular lysosomal compartments to the plasma membrane as a result of the absence of their interaction with the AP-3 complex (see below). These findings were confirmed in another AP-3-deficient strain, AP-3b1LN knockout mice generated by homologous recombination, which were compared with their heterozygote littermates (data not shown).



View larger version (34K):
[in this window]
[in a new window]
  		FIGURE 3. Increased cell surface expression of CD1d in AP-3-deficient mice. Cell surface expression of CD1d was analyzed by flow cytometry. Freshly isolated splenic CD11c+ DC (A), B cell-depleted splenocytes (B), thymocytes (C), and CD19+ B cells (D) from wild-type (filled) and pearl (open) mice were cell surface stained with CD1d Abs. The negative control profile is indicated in hatched lines. Representative histograms are shown. AP-3 deficiency resulted in increased cell surface expression of CD1d in all cell types except B cells. Results are representative of five mice per group.

 
Altered trafficking of CD1d molecules in AP-3-deficient mice

Previous studies have shown that the lysosome-related organelle proteins, LAMP-1, LAMP-2, CD63, tyrosinase, and hCD1b, have altered trafficking patterns in AP-3-deficient cells (15, 16, 18). The altered trafficking of these proteins in AP-3-deficient cells resulted in their redistribution to the plasma membrane. Given the difficulties involved in using lysosomal markers such as LAMP-1, which is misrouted in AP-3 deficiency (16), in colocalization experiments, transmission electron microscopy was used to assess the cellular distribution of CD1d in wild-type and pearl splenic CD11c+ and bone marrow-derived DC. Immunogold-labeled mCD1d was observed on plasma membrane, lysosomes, recognized by characteristic morphology, and, in some small vesicles, probably endocytic recycling vesicles. Consistent with the increase in cell surface expression observed by flow cytometry, there was a 2.3-fold increase in mCD1d plasma membrane staining in pearl bone marrow-derived DC compared with wild-type cells. There was a reciprocal 2.9-fold reduction in mCD1d lysosomal labeling consistent with redistribution of mCD1d to the plasma membrane in AP-3-deficient mice (Fig. 4 and Table I). In splenic CD11c+ DC, which have higher levels of cell surface mCD1d by flow cytometry than bone marrow-derived DC, the redistribution of mCD1d to the plasma membrane was even more dramatic, with a 5.8-fold increase in mCD1d plasma membrane labeling in pearl mice compared with wild-type mice, respectively (1.56 ± 0.315 vs 0.27 ± 0.045 gold particles/µm plasma membrane).



View larger version (143K):
[in this window]
[in a new window]
  		FIGURE 4. Murine CD1d is redistributed in AP-3-deficient mice. The cellular distribution of CD1d in cryosections of bone marrow-derived DC from wild-type C57BL/6 (A) and pearl mice (B) was analyzed by electron microscopy. The white arrowheads indicate lysosomal labeling, the black arrowheads indicate labeling of the plasma membrane, and the black arrows point to labeling of small endocytic vesicles. Bar = 200 nm. PM, plasma membrane; M, mitochondria; L, lysosomes.

 

View this table:
[in this window]
[in a new window]
  		Table I. Distribution of mCD1d molecules on plasma membrane and lysosomesa

 
Reduction of NKT number in AP-3-deficient mice

Next we examined the possibility that interactions with AP-3 might impair CD1d function despite its higher cell surface expression. Since CD1d expression is required for the development of NKT cells, as demonstrated in CD1d-deficient mice (37, 38), we determined whether the alteration in CD1d trafficking observed in AP-3-deficient cells resulted in impaired NKT cell development. Thus, NKT cell numbers were determined by staining with both anti-NK1.1 Ab- and {alpha}-GalCer-loaded CD1d tetramers. NK1.1+TCR{beta}+ cells in pearl mice were reduced in thymus and spleen, consistent with a defect in positive selection and self-Ag presentation (Fig. 5A). There was, on the average, a 60% reduction in NK1.1+TCR{beta}+ thymocytes in pearl compared with wild-type mice. NK1.1+TCR{beta}+ splenocytes were reduced, on the average, by 41% in pearl mice. Liver NK1.1+TCR{beta}+ cells in pearl mice were reduced, on the average, by 30% compared with those in wild-type mice. There was a corresponding reduction in {alpha}-GalCer-loaded CD1d tetramer-stained cells, which are a major subset of NKT cells in thymus and spleen (Fig. 5B). Tetramer-positive cells in thymus and spleen in pearl mice were reduced, on the average, by 55 and 43%, respectively, compared with wild-type mice. These findings are consistent with an important role for the adaptor complex AP-3 in CD1d intracellular trafficking that alters the ability of CD1d self-Ags to be presented for positive selection of CD1d-reactive T cells in the thymus or to sustain their maintenance in peripheral compartments.



View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 5. NKT and CD1d tetramer-positive cells are reduced in AP-3 deficiency. Thymocytes, B cell-depleted splenocytes, and liver mononuclear cells from C57BL/6 and pearl mice were stained with NK1.1, CD3, and TCR-{beta} Abs and {alpha}-GalCer-loaded CD1d tetramers and analyzed by flow cytometry. Representative flow cytometry profiles gated on lymphocytes are shown. A, NK1.1+TCR-{beta}+ cells were significantly reduced in thymus, spleen, and liver of AP-3-deficient mice compared with wild-type C57BL/6 mice. The percentage of NK1.1+TCR-{beta}+ cells is indicated in the upper right quadrant. B, V{alpha}14/J{alpha}281 TCR{alpha}-expressing T cells were identified by staining with {alpha}-GalCer-loaded CD1d tetramers. {alpha}-GalCer-loaded CD1d tetramer + T cells were similarly reduced in pearl mice. The percentage of tetramer-positive cells is indicated in the upper right quadrant. Results are representative of at least five mice per group.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The efficient processing and presentation of many foreign and self-Ags by Ag-presenting molecules such as MHC class II and CD1d requires their intersection in specialized lysosomal compartments. Murine CD1d contains a tyrosine-based sorting motif and the MHC class II{alpha}{beta}Ii complex contains di-leucine-based sorting motifs that are targeting signals recognized by adaptins to direct intracellular trafficking (39, 40). Differences within the sorting motif as well as the amino acid residues adjacent to the motif in the cytoplasmic tail are important in conferring specificity for adaptor protein interactions (41, 42, 43). Secondary effects on CD1d localization may involve association between human and murine CD1d with MHC class II and invariant chain complexes in the late endosomal pathway. This interaction was independent of the cytoplasmic tail of CD1d and may represent a secondary trafficking pathway into the endocytic system (44, 45, 46). The alteration of CD1d expression, localization, and function in AP-3-deficient mice would be independent of invariant chain and MHC class II trafficking as trafficking of both these molecules have been shown to be AP-3 independent (47, 48). Here, we identify and delineate the critical role of the adaptor complex AP-3 in mCD1d trafficking and function.

The AP-3 adaptor complex is an important regulator of trans-Golgi network and lysosomal trafficking. Localization of prototypic lysosomal proteins such as LAMP-1 and CD63 to lysosomes has been shown to be AP-3 dependent (15, 16). Recent studies have demonstrated a role for AP-3 in the intracellular trafficking of hCD1b, while no role was observed in hCD1d trafficking (14, 18). Murine CD1d is the only isoform found in the mouse for which two CD1d genes, designated CD1D1 and CD1D2, have been identified. In the C57BL/6 strain the CD1D2 gene, which has not been shown to have a role in NKT cell development, is a pseudogene secondary to a frameshift mutation (49, 50). Murine CD1d has a similar intracellular distribution pattern to hCD1b, raising the question of whether AP-3 may play a role in its trafficking. In pearl and AP-3b1LN mice, CD1d cell surface expression was significantly elevated in thymocytes, B cell-depleted splenocytes, and splenic DC consistent with an important role for AP-3 in CD1d trafficking. Interestingly, however, mCD1d expression was not increased on B cells. In wild-type mice, B cells have significantly higher cell surface expression levels of mCD1d than thymocytes or T cells. B cell mCD1d cell surface expression levels are approached in AP-3 deficiency by thymocytes and T cells. These findings suggest that AP-3 does not play a significant role in CD1d trafficking in B cells and that intracellular trafficking pathways and adaptins may play different roles in different cell types. Alternatively, AP-3 may be expressed at lower levels in murine B cells vs other cell types. These data also raise the possibility that the repertoire of Ags that mCD1d in B cells encounters may be different from that in other cell types such as DC or thymocytes.

Consistent with the increase in cell surface CD1d expression observed by flow cytometry in pearl cells, pearl DC had increased plasma membrane and reduced CD1d localization in lysosomes noted by immunogold-labeled transmission electron microscopy analyses. The redistribution of CD1d to the plasma membrane, however, was only partial, suggesting that there are additional AP-3-independent pathways for lysosomal targeting of CD1d, as also noted for LAMP-1 and CD63. Recent studies have demonstrated an association between CD1d and invariant chain, raising this as a potential mechanism for the lysosomal targeting of a subset of CD1d molecules. Pearl and AP-3b1LN mice, which have intact MHC class II expression and function, may provide a tool for refined dissection of the role of invariant chain in CD1d trafficking and function.

We suggest that lysosomal Ag sampling may be essential for the Ag presentation function of mCD1d. In humans, CD1b reveals the most dramatic lysosomal trafficking. Since mice lack the group I CD1 isoforms (CD1a, -b, and -c), it is possible that they compensate, at least partially, for this deficiency. Here, we show that mCD1d interacts with AP-3, thus providing mice the capacity for lysosomal sampling by a single CD1 isoform. The functional impact in vivo of altered trafficking of mCD1d was illustrated in pearl mice. Murine CD1d-reactive T cell selection, as reflected by NK1.1+TCR-{beta}+ and CD1d tetramer-positive cells, was markedly impaired. These results are consistent with the importance of endosomal processing of self-Ags for V{alpha}14/J{alpha}281+ NKT cell development and reactivity that were previously noted (5, 25). CD1d cytoplasmic tail-deleted knockin mice were also found to have reduced NKT cell numbers, consistent with a defect in positive selection (25). Our findings provide a mechanistically significant role for the AP-3 complex in the targeting of mCD1d to self or foreign Ags with lysosomal processing requirements. In addition, by using specific adaptins such as AP-3, which target late endosomal compartments, mCD1d can function independently of MHC class II trafficking and respond dynamically to stimuli that may not affect MHC class II function. Similarly, there is evidence that hCD1b and hCD1c trafficking, for example, is independent of the MHC class II reorganization that occurs during DC maturation (51, 52). Together, our results underscore the immense importance of intracellular trafficking to the function of Ag-presenting molecules.


   Acknowledgments
 
We thank Carrie Morehouse, Jean Lai, and Gerald Watts for their technical assistance, and Dr. Christopher Dascher for helpful suggestions.


   Footnotes
 
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 Leprosy 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. Back

2 Address correspondence and reprint requests to Dr. Michael B. Brenner, Lymphocyte Biology Section, Division of Rheumatology, Immunology and Allergy, Department 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{at}rics.bwh.harvard.edu Back

3 Abbreviations used in this paper: {alpha}-GalCer, {alpha}-galactosylceramide; DC, dendritic cells; h, human; LAMP-1, lysosome-associated membrane protein 1; m, murine. Back

Received for publication April 18, 2003. Accepted for publication August 14, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Calabi, F., C. Milstein. 2000. The molecular biology of CD1. Semin. Immunol. 12:503.[Medline]
  2. Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, M. B. Brenner. 1994. Recognition of a lipid antigen by CD1-restricted {alpha}{beta}+ T cells. Nature 372:691.[Medline]
  3. Beckman, E. M., A. Melian, S. M. Behar, P. A. Sieling, D. Chatterjee, S. T. Furlong, R. Matsumoto, J. P. Rosat, R. L. Modlin, S. A. Porcelli. 1996. CD1c restricts responses of mycobacteria-specific T cells: evidence for antigen presentation by a second member of the human CD1 family. J. Immunol. 157:2795.[Abstract]
  4. Sieling, P. A., D. Chatterjee, S. A. Porcelli, T. I. Prigozy, R. J. Mazzaccaro, T. Soriano, B. R. Bloom, M. B. Brenner, M. Kronenberg, P. J. Brennan, et al 1995. CD1-restricted T cell recognition of microbial lipoglycan antigens. Science 269:227.[Abstract/Free Full Text]
  5. Chiu, Y. H., J. Jayawardena, A. Weiss, D. Lee, S. H. Park, A. Dautry-Varsat, A. Bendelac. 1999. Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments. J. Exp. Med. 189:103.[Abstract/Free Full Text]
  6. Gumperz, J. E., C. Roy, A. Makowska, D. Lum, M. Sugita, T. Podrebarac, Y. Koezuka, S. A. Porcelli, S. Cardell, M. B. Brenner, et al 2000. Murine CD1d-restricted T cell recognition of cellular lipids. Immunity 12:211.[Medline]
  7. Kawano, T., J. Cui, Y. Koezuka, I. Toura, Y. Kaneko, K. Motoki, H. Ueno, R. Nakagawa, H. Sato, E. Kondo, et al 1997. CD1d-restricted and TCR-mediated activation of v{alpha}14 NKT cells by glycosylceramides. Science 278:1626.[Abstract/Free Full Text]
  8. Spada, F. M., Y. Koezuka, S. A. Porcelli. 1998. CD1d-restricted recognition of synthetic glycolipid antigens by human natural killer T cells. J. Exp. Med. 188:1529.[Abstract/Free Full Text]
  9. Matsuda, J. L., O. V. Naidenko, L. Gapin, T. Nakayama, M. Taniguchi, C. R. Wang, Y. Koezuka, M. Kronenberg. 2000. Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers. J. Exp. Med. 192:741.[Abstract/Free Full Text]
  10. Hayakawa, K., B. T. Lin, R. R. Hardy. 1992. Murine thymic CD4+ T cell subsets: a subset (Thy0) that secretes diverse cytokines and overexpresses the V{beta}8 T cell receptor gene family. J. Exp. Med. 176:269.[Abstract/Free Full Text]
  11. Arase, H., N. Arase, K. Nakagawa, R. A. Good, K. Onoe. 1993. NK1.1+ CD4+ CD8- thymocytes with specific lymphokine secretion. Eur. J. Immunol. 23:307.[Medline]
  12. Yoshimoto, T., A. Bendelac, C. Watson, J. Hu-Li, W. E. Paul. 1995. Role of NK1.1+ T cells in a TH2 response and in immunoglobulin E production. Science 270:1845.[Abstract/Free Full Text]
  13. Eberl, G., H. R. MacDonald. 2000. Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells. Eur. J. Immunol. 30:985.[Medline]
  14. Briken, V., R. M. Jackman, S. Dasgupta, S. Hoening, S. A. Porcelli. 2002. Intracellular trafficking pathway of newly synthesized CD1b molecules. EMBO J. 21:825.[Medline]
  15. Dell’Angelica, E. C., V. Shotelersuk, R. C. Aguilar, W. A. Gahl, J. S. Bonifacino. 1999. Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the {beta}3A subunit of the AP-3 adaptor. Mol. Cell. 3:11.[Medline]
  16. Yang, W., C. Li, D. M. Ward, J. Kaplan, S. L. Mansour. 2000. Defective organellar membrane protein trafficking in Ap3b1-deficient cells. J. Cell Sci. 113:4077.[Abstract]
  17. Sugita, M., R. M. Jackman, E. van Donselaar, S. M. Behar, R. A. Rogers, P. J. Peters, M. B. Brenner, S. A. Porcelli. 1996. Cytoplasmic tail-dependent localization of CD1b antigen-presenting molecules to MIICs. Science 273:349.[Abstract]
  18. Sugita, M., X. Cao, G. F. Watts, R. A. Rogers, J. S. Bonifacino, M. B. Brenner. 2002. Failure of trafficking and antigen presentation by CD1 in AP-3-deficient cells. Immunity 16:697.[Medline]
  19. Briken, V., R. M. Jackman, G. F. Watts, R. A. Rogers, S. A. Porcelli. 2000. Human CD1b and CD1c isoforms survey different intracellular compartments for the presentation of microbial lipid antigens. J. Exp. Med. 192:281.[Abstract/Free Full Text]
  20. Schaible, U. E., K. Hagens, K. Fischer, H. L. Collins, S. H. Kaufmann. 2000. Intersection of group I CD1 molecules and mycobacteria in different intracellular compartments of dendritic cells. J. Immunol. 164:4843.[Abstract/Free Full Text]
  21. Sugita, M., N. van Der Wel, R. A. Rogers, P. J. Peters, M. B. Brenner. 2000. CD1c molecules broadly survey the endocytic system. Proc. Natl. Acad. Sci. USA 97:8445.[Abstract/Free Full Text]
  22. Sugita, M., E. P. Grant, E. van Donselaar, V. W. Hsu, R. A. Rogers, P. J. Peters, M. B. Brenner. 1999. Separate pathways for antigen presentation by CD1 molecules. Immunity 11:743.[Medline]
  23. Sugita, M., P. J. Peters, M. B. Brenner. 2000. Pathways for lipid antigen presentation by CD1 molecules: nowhere for intracellular pathogens to hide. Traffic 1:295.[Medline]
  24. Jackman, R. M., S. Stenger, A. Lee, D. B. Moody, R. A. Rogers, K. R. Niazi, M. Sugita, R. L. Modlin, P. J. Peters, S. A. Porcelli. 1998. The tyrosine-containing cytoplasmic tail of CD1b is essential for its efficient presentation of bacterial lipid antigens. Immunity 8:341.[Medline]
  25. Chiu, Y. H., S. H. Park, K. Benlagha, C. Forestier, J. Jayawardena-Wolf, P. B. Savage, L. Teyton, A. Bendelac. 2002. Multiple defects in antigen presentation and T cell development by mice expressing cytoplasmic tail-truncated CD1d. Nat. Immunol. 3:55.[Medline]
  26. Feng, L., A. B. Seymour, S. Jiang, A. To, A. A. Peden, E. K. Novak, L. Zhen, M. E. Rusiniak, E. M. Eicher, M. S. Robinson, et al 1999. The {beta}3A subunit gene (Ap3b1) of the AP-3 adaptor complex is altered in the mouse hypopigmentation mutant pearl, a model for Hermansky-Pudlak syndrome and night blindness. Hum. Mol. Genet. 8:323.[Abstract/Free Full Text]
  27. Zhen, L., S. Jiang, L. Feng, N. A. Bright, A. A. Peden, A. B. Seymour, E. K. Novak, R. Elliott, M. B. Gorin, M. S. Robinson, et al 1999. Abnormal expression and subcellular distribution of subunit proteins of the AP-3 adaptor complex lead to platelet storage pool deficiency in the pearl mouse. Blood 94:146.[Abstract/Free Full Text]
  28. Peden, A. A., R. E. Rudge, W. W. Lui, M. S. Robinson. 2002. Assembly and function of AP-3 complexes in cells expressing mutant subunits. J. Cell Biol. 156:327.[Abstract/Free Full Text]
  29. Villadangos, J. A., M. Cardoso, R. J. Steptoe, D. van Berkel, J. Pooley, F. R. Carbone, K. Shortman. 2001. MHC class II expression is regulated in dendritic cells independently of invariant chain degradation. Immunity 14:739.[Medline]
  30. Gumperz, J. E., S. Miyake, T. Yamamura, M. B. Brenner. 2002. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J. Exp. Med. 195:625.[Abstract/Free Full Text]
  31. Higa, T., T. Natozi, Y. Koezuka, and K. Motok. 1988. U.S. Patent 5,780,441.
  32. Peters, P. J., V. W. Hsu, C. E. Ooi, D. Finazzi, S. B. Teal, V. Oorschot, J. G. Donaldson, R. D. Klausner. 1995. Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments. J. Cell Biol. 128:1003.[Abstract/Free Full Text]
  33. Griffiths, G. M., S. Isaaz. 1993. Granzymes A and B are targeted to the lytic granules of lymphocytes by the mannose-6-phosphate receptor. J. Cell Biol. 120:885.[Abstract/Free Full Text]
  34. Mayhew, T. M., J. M. Lucocq, G. Griffiths. 2002. Relative labelling index: a novel stereological approach to test for non-random immunogold labelling of organelles and membranes on transmission electron microscopy thin sections. J. Microsc. 205:153.[Medline]
  35. Sugita, M., M. B. Brenner. 1995. Association of the invariant chain with major histocompatibility complex class I molecules directs trafficking to endocytic compartments. J. Biol. Chem. 270:1443.[Abstract/Free Full Text]
  36. Carlsson, S. R., J. Roth, F. Piller, M. Fukuda. 1988. Isolation and characterization of human lysosomal membrane glycoproteins, h-lamp-1 and h-lamp-2: major sialoglycoproteins carrying polylactosaminoglycan. J. Biol. Chem. 263:18911.[Abstract/Free Full Text]
  37. Chen, Y. H., N. M. Chiu, M. Mandal, N. Wang, C. R. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6:459.[Medline]
  38. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6:469.[Medline]
  39. Boehm, M., J. S. Bonifacino. 2001. Adaptins: the final recount. Mol. Biol. Cell. 12:2907.[Abstract/Free Full Text]
  40. Hiltbold, E. M., P. A. Roche. 2002. Trafficking of MHC class II molecules in the late secretory pathway. Curr. Opin. Immunol. 14:30.[Medline]
  41. Owen, D. J., Y. Vallis, M. E. Noble, J. B. Hunter, T. R. Dafforn, P. R. Evans, H. T. McMahon. 1999. A structural explanation for the binding of multiple ligands by the {alpha}-adaptin appendage domain. Cell 97:805.[Medline]
  42. Ohno, H., M. C. Fournier, G. Poy, J. S. Bonifacino. 1996. Structural determinants of interaction of tyrosine-based sorting signals with the adaptor medium chains. J. Biol. Chem. 271:29009.[Abstract/Free Full Text]
  43. Stephens, D. J., G. Banting. 1998. Specificity of interaction between adaptor-complex medium chains and the tyrosine-based sorting motifs of TGN38 and lgp120. Biochem. J. 335:567.
  44. Jayawardena-Wolf, J., K. Benlagha, Y. H. Chiu, R. Mehr, A. Bendelac. 2001. CD1d endosomal trafficking is independently regulated by an intrinsic CD1d-encoded tyrosine motif and by the invariant chain. Immunity 15:897.[Medline]
  45. Kang, S. J., P. Cresswell. 2002. Regulation of intracellular trafficking of human CD1d by association with MHC class II molecules. EMBO J. 21:1650.[Medline]
  46. Riese, R. J., G. P. Shi, J. Villadangos, D. Stetson, C. Driessen, A. M. Lennon-Dumenil, C. L. Chu, Y. Naumov, S. M. Behar, H. Ploegh, et al 2001. Regulation of CD1 function and NK1.1+ T cell selection and maturation by cathepsin S. Immunity 15:909.[Medline]
  47. Sevilla, L. M., S. S. Richter, J. Miller. 2001. Intracellular transport of MHC class II and associated invariant chain in antigen presenting cells from AP-3-deficient mocha mice. Cell. Immunol. 210:143.[Medline]
  48. Caplan, S., E. C. Dell’Angelica, W. A. Gahl, J. S. Bonifacino. 2000. Trafficking of major histocompatibility complex class II molecules in human B-lymphoblasts deficient in the AP-3 adaptor complex. Immunol. Lett. 72:113.[Medline]
  49. Park, S. H., J. H. Roark, A. Bendelac. 1998. Tissue-specific recognition of mouse CD1 molecules. J. Immunol. 160:3128.[Abstract/Free Full Text]
  50. Chen, Y. H., B. Wang, T. Chun, L. Zhao, S. Cardell, S. M. Behar, M. B. Brenner, C. R. Wang. 1999. Expression of CD1d2 on thymocytes is not sufficient for the development of NK T cells in CD1d1-deficient mice. J. Immunol. 162:4560.[Abstract/Free Full Text]
  51. Cao, X., M. Sugita, N. Van Der Wel, J. Lai, R. A. Rogers, P. J. Peters, M. B. Brenner. 2002. CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation. J. Immunol. 169:4770.[Abstract/Free Full Text]
  52. van der Wel, N., M. Sugita, D. M. Fluitsma, X. Cao, G. Schreibelt, M. B. Brenner, P. J. Peters. 2003. CD1 and major histocompatibility complex II molecules follow a different course during dendritic cell maturation. Mol. Biol. Cell 14:3378.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
E. A. Leadbetter, M. Brigl, P. Illarionov, N. Cohen, M. C. Luteran, S. Pillai, G. S. Besra, and M. B. Brenner
NK T cells provide lipid antigen-specific cognate help for B cells
PNAS, June 17, 2008; 105(24): 8339 - 8344.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
X. Chen, X. Wang, J. M. Keaton, F. Reddington, P. A. Illarionov, G. S. Besra, and J. E. Gumperz
Distinct Endosomal Trafficking Requirements for Presentation of Autoantigens and Exogenous Lipids by Human CD1d Molecules
J. Immunol., May 15, 2007; 178(10): 6181 - 6190.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
S. K. Dougan, P. Rava, M. M. Hussain, and R. S. Blumberg
MTP regulated by an alternate promoter is essential for NKT cell development
J. Exp. Med., March 19, 2007; 204(3): 533 - 545.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Cheng, A. Ueno, S. Cho, J. S. Im, S. Golby, S. Hou, S. A. Porcelli, and Y. Yang
Efficient Activation of V{alpha}14 Invariant NKT Cells by Foreign Lipid Antigen Is Associated with Concurrent Dendritic Cell-Specific Self Recognition
J. Immunol., March 1, 2007; 178(5): 2755 - 2762.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
J. Jung, G. Bohn, A. Allroth, K. Boztug, G. Brandes, I. Sandrock, A. A. Schaffer, C. Rathinam, I. Kollner, C. Beger, et al.
Identification of a homozygous deletion in the AP3B1 gene causing Hermansky-Pudlak syndrome, type 2
Blood, July 1, 2006; 108(1): 362 - 369.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
S. Fontana, S. Parolini, W. Vermi, S. Booth, F. Gallo, M. Donini, M. Benassi, F. Gentili, D. Ferrari, L. D. Notarangelo, et al.
Innate immunity defects in Hermansky-Pudlak type 2 syndrome
Blood, June 15, 2006; 107(12): 4857 - 4864.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
S. C. Yue, A. Shaulov, R. Wang, S. P. Balk, and M. A. Exley
CD1d ligation on human monocytes directly signals rapid NF-{kappa}B activation and production of bioactive IL-12
PNAS, August 16, 2005; 102(33): 11811 - 11816.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Geng, P. Laslo, K. Barton, and C.-R. Wang
Transcriptional Regulation of CD1D1 by Ets Family Transcription Factors
J. Immunol., July 15, 2005; 175(2): 1022 - 1029.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. P. Lawton, T. I. Prigozy, L. Brossay, B. Pei, A. Khurana, D. Martin, T. Zhu, K. Spate, M. Ozga, S. Honing, et al.
The Mouse CD1d Cytoplasmic Tail Mediates CD1d Trafficking and Antigen Presentation by Adaptor Protein 3-Dependent and -Independent Mechanisms
J. Immunol., March 15, 2005; 174(6): 3179 - 3186.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Cernadas, M.
Right arrow Articles by Brenner, M. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Cernadas, M.
Right arrow Articles by Brenner, M. B.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS