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4 cd1a reciclagem

  1. 1. Early Recycling Compartment Trafficking of CD1a Is Essential for Its Intersection and Presentation of Lipid AntigensThis information is current as Manuela Cernadas, Marco Cavallari, Gerald Watts, Luciaof April 18, 2012 Mori, Gennaro De Libero and Michael B. Brenner J Immunol 2010;184;1235-1241; Prepublished online 21 December 2009; doi:10.4049/jimmunol.0804140 http://www.jimmunol.org/content/184/3/1235 Downloaded from www.jimmunol.org on April 18, 2012 References This article cites 33 articles, 12 of which can be accessed free at: http://www.jimmunol.org/content/184/3/1235.full.html#ref-list-1 Subscriptions Information about subscribing to The Journal of Immunology is online at http://www.jimmunol.org/subscriptions Permissions Submit copyright permission requests at http://www.aai.org/ji/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at http://www.jimmunol.org/etoc/subscriptions.shtml/The Journal of Immunology is published twice each month byThe American Association of Immunologists, Inc.,9650 Rockville Pike, Bethesda, MD 20814-3994.Copyright ©2010 by The American Association ofImmunologists, Inc. All rights reserved.Print ISSN: 0022-1767 Online ISSN: 1550-6606.
  2. 2. The Journal of ImmunologyEarly Recycling Compartment Trafficking of CD1a IsEssential for Its Intersection and Presentation ofLipid AntigensManuela Cernadas,*,1 Marco Cavallari,†,1 Gerald Watts,‡ Lucia Mori,† Gennaro De Libero,†,2and Michael B. Brenner‡,2A major step in understanding differences in the nature of Ag presentation was the realization that MHC class I samples peptidestransported to the endoplasmic reticulum from the cytosol, whereas MHC class II samples peptides from lysosomes. In contrast toMHC class I and II molecules that present protein Ags, CD1 molecules present lipid Ags for recognition by specific T cells. Each ofthe five members of the CD1 family (CD1a–e) localizes to a distinct subcompartment of endosomes. Accordingly, it has been widelyassumed that the distinct trafficking of CD1 isoforms must also have evolved to enable them to sample lipid Ags that traffic viadifferent routes. Among the CD1 isoforms, CD1a is unusual because it does not have a tyrosine-based cytoplasmic sorting motifand uniquely localizes to the early endocytic recycling compartment. This led us to predict that CD1a might have evolved to focus Downloaded from www.jimmunol.org on April 18, 2012on lipids that localize to early endocytic/recycling compartments. Strikingly, we found that the glycolipid Ag sulfatide alsolocalized almost exclusively to early endocytic and recycling compartments. Consistent with colocalization of CD1a and sulfatide,wild-type CD1a molecules efficiently presented sulfatide to CD1a-restricted, sulfatide-specific T cells. In contrast, CD1a:CD1b tailchimeras, that retain the same Ag-binding capacity as CD1a but traffic based on the cytoplasmic tail of CD1b to lysosomes, failedto present sulfatide efficiently. Thus, the intracellular trafficking route of CD1a is essential for efficient presentation of lipid Agsthat traffic through the early endocytic and recycling pathways. The Journal of Immunology, 2010, 184: 1235–1241.T he CD1 family of Ag-presenting molecules is unique in its d isoforms is determined by their unique cytoplasmic tail tyrosine- ability to present lipid, glycolipid, and lipopeptide Ags to based sorting motif (2). The intracellular localization of the CD1b CD1-restricted T cells. In contrast to MHC-restricted Ag isoform, for example, is almost exclusively in the lysosomal com-presentation, CD1 molecules are functionally nonpolymorphic and partment. This CD1b localization is mediated by the adaptor proteinhave been shown to present both exogenous microbial Ags as well as AP-3 through interactions with the cytoplasmic tyrosine-basedendogenous lipid ligands (1). Similar to MHC class I molecules, CD1 motif of CD1b (3).molecules are noncovalently associated with b2-microglobulin. The CD1a isoform is almost exclusively expressed on pro-CD1a, b, and c molecules are primarily expressed on professional fessional APC, including Langerhans cells (LCs) and other myeloidAPCs and have been implicated in adaptive immunity against mi- dendritic cell (DC) subsets (2). CD1a is distinct among the CD1crobial lipids. In contrast, CD1d-restricted NK T cells are innate- isoforms in that it does not contain a tyrosine-based cytoplasmiclike lymphocytes that may bridge the innate and adaptive immune motif. CD1a has been shown to localize intracellularly to the en-system. Each of the five members of the human CD1 family—CD1a, docytic recycling compartment (ERC) (4, 5). In contrast to CD1b,b, c, d, and e—has a distinct cellular distribution and intracellular CD1a internalization into endosomes is independent of clathrin ortrafficking pattern. The intracellular localization of the CD1b, c, and dynamin. Once internalized, CD1a molecules follow a Rab22a- and ADP ribosylation factor 6-dependent recycling pathway (5). Under*Division of Pulmonary and Critical Care Medicine and ‡Division of Rheumatology, steady-state conditions, CD1a is not found to localize to late en-Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, dosomes (LEs) or lysosomes (LYs) (4). Moreover, CD1a moleculesBoston, MA 02115; and †Experimental Immunology, Department of Biomedicine,University Hospital Basel, Basel, Switzerland have also been shown to localize in Birbeck granules, which are1 intracellular compartments unique to LCs that have been demon-M.C. and M.C. contributed equally to this work.2 strated to be subdomains of the endocytic system (6).G.D.L. and M.B.B. contributed equally to this work. Several T cell Ags presented by CD1a have been described in-Received for publication December 11, 2008. Accepted for publication November17, 2009. cluding didehydroxymycobactin, a lipopeptidic Ag isolated from Mycobacterium tuberculosis, and sulfatide (7, 8). Sulfatide (39-This work was supported by grants from the National Institutes of Health (AI 028973to M.B.B.), the Swiss National Science Foundation (3100AO-109918), and Roche sulfated b1-D-galactosylceramide) is an endogenous glycolipidResearch Foundation (to G.D.L.). highly expressed in neuronal cells, kidney, and pancreas (9–11), andAddress correspondence and reprint requests to Dr. Michael B. Brenner, Division of its synthesis is upregulated in DCs upon bacterial infection (12).Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Sulfatide can be presented by all group 1 CD1 molecules includingMedical School, Smith Building, Room 552, 1 Jimmy Fund Way, Boston, MA 02115.E-mail address: mbrenner@rics.bwh.harvard.edu mouse and human CD1d (8, 13, and M. Cavallari and G. De Libero,Abbreviations used in this paper: DC, dendritic cell; EEA-1, early endosome Ag-1; ERC, unpublished results). DC pulsed with sulfatide maintained theendocytic recycling compartment; LAM, lipoarabinomannan; LAMP-1, lysosome- ability to stimulate CD1a-restricted T cells over a 3 d period. Inassociated membrane protein 1; LC, Langerhans cell; LE, late endosome; LY, lysosome; contrast, the ability of the sulfatide-pulsed DC to stimulate CD1b-Trf, transferrin; WT, wild-type. and CD1c-restricted T cells was reduced by ∼75% over the sameCopyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 time period. These findings suggest that CD1a has a unique abilitywww.jimmunol.org/cgi/doi/10.4049/jimmunol.0804140
  3. 3. 1236 CD1a TRAFFICKING IS ESSENTIAL FOR LIPID ANTIGEN PRESENTATIONto present sulfatide. However, these studies cannot distinguish be- Hercules, CA). Stable clones were isolated by limiting dilution under se-tween higher affinity of CD1a binding for sulfatide versus more lection with 1 mg/ml G418 sulfate (Invitrogen) in DMEM complete media. Transient transfections were performed with FuGENE 6 (Roche, Basel,extensive colocalization of the Ag-presenting molecule with the Ag. Switzerland) as per manufacturer’s instructions. CD1a cell surface ex- In this study, we show that the intracellular localization of the pression was confirmed by flow cytometric staining and intracellular traf-CD1a isoform to the early endocytic system allows it to intersect with ficking by confocal microscopy as detailed below. Monocyte-derived DCslipids that are primarily or, in the case of sulfatide, almost exclusively, were generated as previously described (15). Briefly, CD14+ monocyteslocalized in the early endocytic system. This colocalization was were isolated from buffy coats obtained from normal blood donors as per institutional guidelines. Mononuclear cells were isolated with Ficoll-Paqueshown to be critical for the sustained ability of CD1a molecules to PLUS (GE Healthcare, Buckinghamshire, U.K.) and monocytes positivelystimulate CD1a-restricted T cells. When the same CD1a extracellular selected with CD14 MicroBeads (MACS; Miltenyi Biotec, Auburn, CA).domain was fused to the CD1b tail, redirecting it to LYs, efficient and DCs were differentiated in RPMI 1640 complete media (Life Technologies,prolonged presentation of CD1a lipid Ag was abrogated. This finding Carlsbad, CA), 10% heat-inactivated FCS (HyClone, Logan, UT), 2 mM L- glutamine, 10 mM HEPES, and 100 U/ml penicillin-streptomycin (Lifehas significant implications for the efficient and sustained pre- Technologies) supplemented with 300 U/ml GM-CSF (Immunex, Thousandsentation of other CD1a-binding lipid Ags by DC in vivo. This work Oaks, CA) and 200 U/ml IL-4 (PeproTech, Rocky Hill, NJ). WT CD1a-also highlights the importance of understanding lipid trafficking as (16), CD1aab- (14), or CD1abb-transfected T2 cells were used as APCs.a critical factor in the study of CD1 Ag presentation and function. Briefly, stable transfectants were achieved by electroporation and selection with G418 sulfate (0.8 mg/ml; Calbiochem, La Jolla, CA). Surface ex- pression of CD1a was confirmed by flow cytometry, and intracellular lo-Materials and Methods calization studies were performed by confocal microscopy. The CD1a-Chimeric CD1a:CD1b constructs restricted, sulfatide-specific human T cell clone K34B9.1 was derived and cultivated as previously described (8).Three types of CD1a:CD1b hybrid constructs containing sequencesencoding the CD1a extracellular domain fused to three different CD1b Flow cytometrychimeric tails were generated by PCR using human CD1a and CD1b cDNA Downloaded from www.jimmunol.org on April 18, 2012as the templates. The first CD1a:b tail chimeric construct contains the HeLa transfectants and monocyte-derived DCs were analyzed by flowtyrosine-based motif of CD1b and two additional 39 amino acids from cytometry. In the case of the HeLa transfectants, the cells were detached byCD1b wild-type (WT) sequence (highlighted in boldface) swapped with brief incubation with 0.5% trypsin-EDTA (Life Technologies/Invitrogen)the terminal sequence (last three amino acids) of CD1a [i.e., WT CD1a (… and washed prior to staining. The cells were stained with PE-conjugatedFRKRCFC) was changed to (… FRKRRSYQNIP)]. The CD1a:CD1b anti-CD1a or isotype control mouse IgG1 mAb (BD Biosciences, San Jose,chimeric tail construct was cloned between the BamHI and XhoI sites of CA) on ice. The cells were analyzed using a FACScan or Canto flow cy-pcDNA3 vector (Invitrogen, Carlsbad, CA) and sequenced before trans- tometer (BD Biosciences).fection. This construct is referred to in the text as CD1aab, indicating the Lipid Agsorigin of the extracellular domain (first letter), transmembrane (secondletter), and cytoplasmic sequences (CD1b). The second construct is es- Bovine sulfatide and ceramide (C24) were obtained from Matreya (Pleasantsentially identical to the first with the exception of one amino acid. In the Gap, PA) and lipoarabinomannan (LAM) from Colorado State University,second CD1a:CD1b tail chimeric construct, the WT CD1a (…FRKRCFC) Fort Collins, CO.tail was changed to (…FRRRSYQNIP) as previously described (14). Thisconstruct, which was used to transfect the T2 cells, will also be referred to Confocal microscopyCD1aab. The CD1aab constructs were independently generated, account-ing for the one amino acid difference. Each laboratory included additional Laser confocal microscopy was performed on the HeLa transfectants andamino acids from the transmembrane region of CD1b because replacement monocyte-derived DCs as previously described (3, 15). Briefly, HeLa trans-of the tyrosine motif alone was not sufficient to fully redirect CD1a to the fectants were cultured on coverslips and DC plated on fibronectin-coated cov-LY-associated membrane protein-1+ (LAMP-1+) lysosomal compartments. erlips overnight. After washing, the cells were fixed with paraformaldehyde,In the third construct called CD1abb, the extracellular CD1a region was permeabilized with 0.2% saponin, and stained with mAb against CD1a (10H3,joined to the transmembrane and cytoplasmic sequences of CD1b. The mouse IgG1), CD1b (BCD1, mouse IgG1), LAMP-1 (H4A3, mouse IgG1),latter two sequences were cloned into pBluescript II KS+ (Stratagene, La sulfatide (04, mouse IgM supernatants, generously provided by Dr. Joan Boggs,Jolla, CA), sequenced, and subcloned into the XhoI-NotI sites of the Hospital for Sick Children, Toronto, Ontario, Canada) (17), LAM (rabbit anti-BCMGSNeo vector for transfection. All PCR primer sequences used to LAM antisera was generously provided by Dr. Daniel Clemens, University ofgenerate the constructs are available upon request. California, Los Angeles, Los Angeles, CA), Rab5 (BD Biosciences), early en- dosome Ag-1 (EEA-1; BD Biosciences), and appropriate species and isotypeCells controls for 60 min at room temperature. The purified Abs were used at 10 mg/ml and the supernatants at 1:5 dilution. The cells were stained with the appropriateHeLa cells (American Type Culture Collection, Manassas, VA) were secondary Ab, Alexa 546- or Alexa 488-conjugated anti-mouse IgM, Alexa 488-transfected with WT CD1a (CD1aaa) and HeLa CD1aab chimeric con- conjugated F(ab9)2 fragment of goat anti-mouse IgG (Molecular Probes/In-structs by electroporation (Bio-Rad Gene Pulser II, 280V, 960 mF; Bio-Rad, vitrogen), and FITC-conjugated F(ab9)2 fragment of donkey anti-rabbit IgG AbsFIGURE 1. CD1a localization is redirected to LYs in the CD1aab tail chimera. HeLa CD1a WT, CD1b WT, and CD1aab tail chimera transfectants wereanalyzed by confocal microscopy. A, The CD1 transfectants were stained with Abs directed at their extracellular domains. CD1a WT transfectants show cellsurface and perinuclear staining. The CD1b WT and CD1aab chimera have similar intracellular staining patterns. B, Colocalization studies with anti–LAMP-1 mAbs demonstrated the localization of the CD1aab chimera to LAMP-1+ compartments similar to WT CD1b. There is no colocalization betweenCD1a and LAMP-1. The fluorescent label used for each protein is indicated by color.
  4. 4. The Journal of Immunology 1237(Jackson ImmunoResearch, West Grove, PA) for 60 min at room temperature. Sulfatide colocalizes to the early endocytic/recycling pathwayAdditional staining was performed with Alexa 488-conjugated anti–LAMP-1mAb (H4A3, BD Biosciences) and FITC-conjugated transferrin (Trf) (In- Despite extensive prior analyses of CD1a and CD1b trafficking, onlyvitrogen) at 10 mg/ml. In the case of the Trf experiments only, the HeLa trans- rarely has lipid Ag trafficking been directly visualized, because tag-fectants were serum-starved in DMEM media with 0.2% BSA for 30 min. The ging of lipid Ags often alters their trafficking, and few mAb againstcells were washed three times prior to fixation and confocal analysis. For the Ag lipids exist. No prior studies have examined the trafficking of CD1a-loading studies, the cells were incubated for 30 min, unless clearly indicatedotherwise as in the time course analysis, at 37˚C with sulfatide at 10 mg/ml, after presented lipid Ags. We developed a protocol to determine the in-which the cells were washed three times with PBS at room temperature prior to tracellular distribution of sulfatide using confocal microscopy andconfocal staining. In the case of the Trf experiments, 100 mg/ml sulfatide was the anti-sulfatide specific mAb (O4) in sulfatide-pulsed HeLa CD1used. The slides were analyzed with an inverted microscope (TE2000, Nikon, transfectant cells. Sulfatide was sonicated in complete media at 37˚CMelville, NY) with C1 confocal system and the Plan Apochromat 603 oil (NA1.4) objective lens using EZ-C1 software (Nikon) at room temperature. Only and then incubated with CD1a HeLa transfectants for the time pointsrepresentative images were acquired. indicated in Fig. 2A. After 30 min of incubation, sulfatide was taken up by the cells as revealed by well-defined, punctuate staining withPulse and chase Ag presentation assays anti-sulfatide mAb (Fig. 2A), whereas minimal to no backgroundCD1a WT and CD1aab HeLa transfectants (1 3 106) were irradiated (5000 staining was observed with the anti-sulfatide mAb in the unpulsedrads, [137Cs] source) and pulsed with 2 mg (pulse experiments) of sonicated cells. Strikingly, HeLa CD1a transfectants incubated with sulfatidesulfatide for indicated times. CD1a WT-, CD1aab-, and CD1abb-transfected demonstrated an almost exclusive localization of sulfatide to theT2 cells were pulsed for 3 h with 10 mg (chase experiments) of sonicated early endocytic and recycling pathways, identified using anti-Rab5sulfatide. For the these experiments, purified synthetic C24:1 (nervonoyl-)sulfatide (18) was used and was kindly provided by L. Panza (Universita `del Piemonte Orientale, Novara, Italy). After pulse, the cells were washedand chased or not for indicated times. At time zero, the cells for all theconditions were collected, counted, and plated (2.5 3 104/well) in RPMI Downloaded from www.jimmunol.org on April 18, 20121640 medium containing 10% FCS with or without the addition of freshlysonicated Ag (at the same concentration as pulse). T cells (1 3 105/well)were added in triplicate or quadruplicate. Supernatants were harvestedafter 24 h, and released cytokines were measured by ELISA.ELISAsMaxiSorp ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at4˚C with 1 mg/ml 8D4-8 (anti-human IL-4, BD Biosciences), 1 mg/ml MAb1(anti-human TNF-a, BD Biosciences), or 3 mg/ml HB-8700 (anti-human IFN-g,American Type Culture Collection) mAb, blocked with PBS 0.05% Tween-20and 10 mg/ml BSA, and incubated with the supernatants of the Ag presentationassays. For detection, 1 mg/ml MP4-25D2 (anti-human IL-4 biotin labeled, BDBiosciences), 0.5 mg/ml MAb11 (anti-human TNF-a biotin labeled, BD Bio-sciences), or 0.72 mg/ml g69 (anti-human IFN-g biotin labeled) (19) mAb wereused. The plates were developed colorimetrically, and concentrations were de-termined by comparison with standards of human IL-4 (human IL-4 secretingX63, kind gift of U. Grawunder, 4-Antibody AG, Basel, Switzerland), re-combinant human TNF-a (ImmunoKontact, Abingdon, U.K.), and recombinanthuman IFN-g (Bender MedSystems, Burlingame, CA).ResultsRelocation of CD1a to LY by providing residues of CD1bcytoplasmic tailIt has been previously shown that CD1a localizes to the ERC,whereas CD1b localizes to the LE/LY. To investigate the importanceof tyrosine-based motifs in CD1 molecule trafficking, HeLa CD1a:CD1b tail chimeric constructs containing the tyrosine-based motif ofCD1b were generated and intracellular localization of CD1 mole-cules analyzed by confocal microscopy. The CD1aab tail chimerashowed a different localization from CD1a WT and an identicalstaining pattern compared with WT CD1b (Fig. 1A). This was furtherdemonstrated by colocalization of the CD1aab chimera to LE/LYwith LAMP-1, as it was also demonstrated for WT CD1b. This is inagreement with the known important role for the tyrosine-basedsorting motif of CD1b in binding AP-3 and directing trafficking tothe LY. In contrast, as expected, there was no colocalization between FIGURE 2. Sulfatide localizes to the early endocytic and recyclingWT CD1a and LAMP-1 (Fig. 1B). Both WT and chimeric proteins compartments. A, The anti-sulfatide mAb (O4) specifically labels sulfa-were well expressed on the cell surface, as demonstrated by flow tide-pulsed HeLa CD1a transfectants. Numbers above panels indicate the sulfatide pulsing times. IgM control staining was performed at 60 min. B,cytometry. The CD1aab chimera transfectants consistently showed Colocalization of sulfatide with the early endocytic markers Rab5 andlower surface mean fluorescence intensity compared with WT CD1a EEA-1 was performed in HeLa CD1a WT cells pulsed with sulfatide foras determined by flow cytometry (data not shown). It should be 30 min. Individual Ab stainings and the merged images are presented. C,noted that there was significant anti-CD1a mAb intracellular stain- Colocalization pattern of CD1a and Trf in FITC-Trf–pulsed HeLa CD1aing with anti-CD1a mAb of the CD1aab chimera, indicating con- WT transfectants (top panels) and sulfatide and Trf in sulfatide- and FITC-formational stability of the CD1a extracellular domain in the late Trf–pulsed HeLa CD1a WT (bottom panels). Individual Ab stainings andendocytic system (Fig. 1A). the merged images are displayed.
  5. 5. 1238 CD1a TRAFFICKING IS ESSENTIAL FOR LIPID ANTIGEN PRESENTATIONand EEA-1–specific mAb and fluorescently-labeled Trf. There was CD1aab tail chimera, there was a marked reduction in the co-marked colocalization between sulfatide and the early endocytic localization of sulfatide with the CD1aab tail chimeric proteinmarkers Rab5 and EEA-1 (Fig. 2B). Sulfatide was also present in the (Fig. 3A). This suggests that the intracellular localization providedERC as demonstrated by its partial colocalization with Trf (Fig. 2C). by the CD1b tyrosine-based motif determines the degree of in-CD1a and Trf also demonstrated partial overlap (Fig. 2C) consistent teraction of the CD1aab tail chimera with sulfatide. The distri-with the previously published localization of CD1a in the ERC (4, 5). bution of sulfatide was very similar in cells expressing CD1a WTHowever, there was no colocalization with LAMP-1 (Fig. 1B), and CD1aab tail chimera as demonstrated by the almost completea marker of the late endocytic system. colocalization of sulfatide and Rab5. Furthermore, there was no redirection of sulfatide to the late endocytic system with theDifferential colocalization of CD1a and CD1b with sulfatide CD1aab tail chimera (Fig. 3B).Next, the colocalization pattern of CD1a and CD1b and the CD1aab Time course studies of sulfatide distribution were also performedchimera with sulfatide was examined. HeLa CD1a WT, CD1b WT, to investigate whether sulfatide traffics to LEs at late time points.and CD1aab chimera transfectants were pulsed with sulfatide for The transfectants were incubated with sulfatide for various time30 min and stained with anti-CD1a, anti-CD1b, and anti-sulfatide periods before fixation prior to confocal microscopy. These studiesmAb. Consistent with the above findings, there was marked show that there is no detectable colocalization of LAMP-1 withcolocalization of CD1a with sulfatide in the HeLa CD1a WT sulfatide up to 20 h after sulfatide pulse (Fig. 3B, 3C). This lack oftransfectant cells (Fig. 3A). On the other hand, although small colocalization was observed for both the CD1a WT and CD1aabamounts of punctate colocalization of CD1b with sulfatide were tail chimera transfectants. Minor colocalization was observed forobserved in some cells, the majority of sulfatide did not colocalize sulfatide and CD1b at late time points (data not shown). At all timewith CD1b in the HeLa CD1b WT transfectants (Fig. 3A). For the points, the vast majority of sulfatide staining remained in the early Downloaded from www.jimmunol.org on April 18, 2012FIGURE 3. Marked intracellularcolocalization between CD1a andsulfatide. HeLa CD1a WT, CD1bWT, and CD1aab chimera trans-fectants were pulsed with sulfatideand analyzed by confocal micros-copy. A, Colocalization betweeneach CD1 molecule and sulfatidewas determined for each trans-fectant. The fluorescent labels usedfor each protein are indicated bycolor. B, Colocalization betweensulfatide and early (Rab5) and lateendocytic (LAMP-1) markers wasdetermined in sulfatide-pulsedCD1a WT and CD1aab tail chimeratransfectants. C, The colocalizationpattern between CD1a and LAMP-1was analyzed in CD1a WT trans-fectants pulsed with sulfatide for 4 h(top panel) and 20 h (bottom panel).D, Confocal staining for CD1a (flu-orescently labeled in red) and sul-fatide (labeled in green) wasperformed on CD1a WT trans-fectants incubated with sulfatide(top panels) and C24 ceramidecontrol (bottom panels) at increasingconcentrations for 30 min.
  6. 6. The Journal of Immunology 1239endocytic and recycling compartments. The absence of sulfatide CD1a intracellular trafficking alters sulfatide Ag presentationdetection in LAMP-1+ compartments even at later time points is to specific T cellslikely due to its preferential sorting to the early endocytic and re- In order to determine the functional relevance of the intracellularcycling compartments and to the degradation of sulfatide by en- trafficking of CD1a in Ag presentation, we next examined thedogenous lysosomal arylsulfatases that are well described to be ability of CD1a WT and different CD1a:CD1b tail chimeras topresent in this compartment (20). Lastly, to rule out the possible activate CD1a-restricted sulfatide-specific T cells. First, to assesscontribution of sulfatide to changes in CD1a expression, in- the appearance of sulfatide:CD1a stimulatory complexes, wetracellular distribution, or cell morphology, confocal microscopy performed pulse experiments using HeLa CD1a WT and CD1aabwas performed with increasing concentrations of sulfatide or cer- chimera transfectants with matched cell surface expression ofamide (C24) as an additional control. No changes were observed by CD1a as determined by flow cytometry. The transfectants wereconfocal microscopy in the distribution or localization of CD1a or in pulsed with sulfatide for 0, 7, 20, 25, 45, and 60 min and washedthe transfectant morphology after culture with either lipid (Fig. 3D). before the addition of T cells. The ability of sulfatide-pulsedCD1a but not CD1b colocalizes with sulfatide in human transfectants to stimulate the sulfatide-specific T cell clonemonocyte-derived DCs K34B9.1 increased with increasing pulse period independently of the measured cytokine (IFN-g, TNF-a, or IL-4). CD1a WTThese findings were confirmed in primary cells, human monocyte- transfectants outperformed the CD1aab chimera at pulse periodsderived DCs that express all CD1 isoforms. Human monocyte- longer than 30 min (Fig. 5). CD1aab chimera transfectants evenderived DCs were pulsed with sulfatide as described above. showed a slight decrease in stimulatory capacity at pulse periodsConfocal microscopy of DCs demonstrated areas of strong longer than 30 min. Then, to assess the disappearance of sulfatide:colocalization between sulfatide and CD1a molecules but not CD1a stimulatory complexes, we performed chase experimentsbetween sulfatide and CD1b (Fig. 4A). These findings are in using T2 CD1a WT and CD1a:CD1b chimeras instead of HeLa Downloaded from www.jimmunol.org on April 18, 2012agreement with the known localization of CD1a and CD1b mol- transfectants to avoid potential effects of trypsinization requiredecules in monocyte-derived DCs to the ERC or to LE/LY com- for the release of the HeLa transfectants. The T2 transfectants werepartments, respectively (21, 22). pulsed with sulfatide for 3 h, washed, and chased for 0, 24, 48, and 72 h before addition of T cells and cytokine release measurement.Colocalization of CD1b with LAM in DC At each time point, control groups were represented by trans-Next, the colocalization of CD1a and CD1b with LAM, an Ag fectants incubated with sulfatide together with T cells to determinepresented by CD1b, was analyzed. Monocyte-derived DCs were the maximal Ag-presenting capacity. The ability of sulfatide-incubated with LAM for 30 min and analyzed by confocal mi- pulsed transfectants to stimulate the sulfatide-specific clonecroscopy. Strikingly, in contrast to the specific colocalization of K34B9.1 decayed with increasing chase period independently ofCD1a with sulfatide in DC, LAM strictly colocalized with CD1b the measured cytokine. WT CD1a transfectants showed halfbut not with CD1a (Fig. 4B). The localization of LAM with CD1b maximal stimulation at 40 h of chase, whereas CD1abb trans-is consistent with the known interaction of LAM with mannose fectants showed half maximal stimulation at 20 h. CD1aab trans-receptors that likely mediates the internalization and direct tar- fectants showed intermediate behavior, with half maximalgeting of LAM and similar Ags to the late endocytic system (23). stimulation at 30 h (Fig. 6). After 72 h, CD1a WT transfectantsThese findings suggest that CD1 molecules select the lipid Ag to were still capable of stimulating sulfatide-specific T cells, whereasbe presented according to their capacity to bind and form stable both chimera transfectants were no longer stimulatory.complexes and also according to the endosomal localization ofeach lipid Ag and its intersection with the relevant CD1 molecule. Discussion Successful Ag presentation involves an orchestrated series of steps that are critically dependent on the intersection of Ags with the relevant Ag-presenting molecules. Lessons from the MHC class I and II pathways have made it clear that access to the proper Ag loading compartment is a key feature for those presentation systems to succeed. The proteolytic environment present in LYs of DCs plays a critical role in their ability to process protein Ags for prolonged Ag presentation. APCs such as macrophages with high levels of lyso- somal proteases rapidly degrade internalized proteins, reducing their capacity for sustained Ag presentation compared with DCs (24). Although the ability of MHC class I and II molecules to survey different intracellular compartments and Ags is a hallmark of these systems, the role of CD1 trafficking in the presentation of exoge- nous and endogenous Ags has been examined in only a few cases. The unique intracellular distribution of each CD1 isoform has been appreciated for some time. It has been demonstrated for the CD1b and CD1c isoforms that their intracellular trafficking is directed by their respective tyrosine-based motifs using tail-deletedFIGURE 4. Reciprocal intracellular localization patterns of CD1a and constructs (3, 25). Tail-deleted CD1b molecules have been dem-sulfatide and CD1b and LAM. The pattern of CD1a and CD1b colocali- onstrated to have significant impairment in their Ag-presentingzation with sulfatide (A) and LAM (B) was analyzed by confocal mi- abilities in vitro (3). Glucose monomycolate possessing a longcroscopy in human monocyte-derived DCs pulsed with sulfatide. The lipid tail has been shown to require the acidic environment foundfluorescent labels used for each protein are indicated by color. in the late endocytic system to be loaded efficiently (26, 27).
  7. 7. 1240 CD1a TRAFFICKING IS ESSENTIAL FOR LIPID ANTIGEN PRESENTATIONFIGURE 5. WT CD1a transfectants present sulfatide more efficiently than CD1aab chimeric transfectants. WT CD1a (white diamonds) HeLa trans-fectants showed increased capacity to stimulate the CD1a-restricted sulfatide-specific T cell clone K34B9.1 as compared with CD1aab chimeric trans-fectants (gray diamonds) for pulse periods longer than 30 min. Both transfectants were pulsed with nervonoyl sulfatide for indicated times, then washed andcoincubated with T cells. Supernatants were harvested after 24 h, and the release of human IL-4 (A), human IFN-g (B), and TNF-a (C) was measured byELISA. Error bars represent SD of triplicate measurements. CD1a is the only CD1 isoform that does not have a tyrosine-based enzymatically or as a result of acidic pH in the late endocyticmotif and almost exclusively localizes to the early endocytic and system. This is supported by the enhanced ability of WT CD1a torecycling pathways. In this study, we demonstrate that the locali- present sulfatide compared with the CD1aab and CD1abb tailzation of CD1a to the early endosomal and recycling compartments chimeras and the sustained ability of sulfatide-pulsed DCs to ac-is critical for the efficient and sustained presentation of specific Ags tivate CD1a- compared with CD1b- and CD1c-restricted sulfatide- Downloaded from www.jimmunol.org on April 18, 2012that also predominantly localize to the same endosomal sites. CD1a specific T cells. Additional interactions or pathways contributing toand sulfatide are almost exclusively colocalized to early endocytic CD1a Ag presentation may also be contributory. For example, theand recycling compartments. The redirection of CD1a to the LE by colocalization of CD1a with the unique LC-specific C-type lectinthe addition of the transmembrane and/or cytoplasmic CD1b ty- required for Birbeck granule formation, langerin, in this endosomalrosine-based motif to the extracellular domain of CD1a resulted in subcompartment has been shown to augment CD1a-mediated Agloss of colocalization with sulfatide and a correspondingly signif- presentation (28). We cannot rule out the possibility that theicant reduction in the ability of these transfectants to stimulate CD1aab–sulfatide complexes dissociate in the LY. We should alsoCD1a-restricted sulfatide-specific T cells. Because the extracellular mention the possibility that the anti-sulfatide mAb may not be abledomains of the chimeric molecules are identical, the most likely to detect sulfatide in the LY secondary to changes to the antigenicexplanation for these findings is the difference in trafficking epitope recognized. However, similar sulfatide colocalization pat-imparted by the chimeric cytoplasmic tails. The activity and lo- terns have been observed with other anti-sulfatide mAbs, makingcalization of endogenous sulfatases suggests that even if some this less likely.sulfatide is delivered to LYs, it would be rapidly degraded. There are The differential localization of CD1a to the early endocytictwo classes of sulfatases, catabolic and synthetic. The catabolic compartment may be important in vivo. The efficient and sustainedsulfatases are localized in LYs and exert their activity at acidic pH on presentation of Ags such as sulfatide by CD1a+ DC during theira wide range of molecules, including sulfolipids and glyco- migration to regional lymph nodes is likely critical for maximiz-soaminoglycans. Accumulation of these molecules occurs in the ing T cell activation and proliferation. This may be even of greatersetting of lysosomal sulfatase deficiency and leads to human disease, importance in vivo, where Ag concentrations are likely lower thanincluding several mucopolysaccharidoses and metachromatic leu- our studies where higher concentrations of sulfatides were addedkodystrophy (20). The prolonged ability of CD1a molecules to to allow for Ab detection and localization by microscopy.present sulfolipids such as sulfatide may be critically dependent on The specific intracellular distribution of CD1 molecules and theirtheir localization to early endocytic compartments where the sul- Ags may also explain the evolutionary diversity of the CD1 iso-fatide Ag is not degraded by sulfatases. The absence of a tyrosine- forms (29). It is interesting to speculate that the distribution of thebased motif allows CD1a to traffic and recycle through the early different CD1 isoforms throughout the endocytic system may haveendocytic system, where it can most efficiently present lipid Ags developed to allow for the presentation of differentially distributedthat preferentially localize there or that would be destroyed either Ags or Ags that are destroyed either enzymatically or by the acidicFIGURE 6. CD1a:CD1b chimeras have shorter-lived ability to stimulate sulfatide-specific T cells than WT CD1a molecules. WT CD1a (white diamonds)T2 transfectants showed prolonged capacity to stimulate the CD1a-restricted sulfatide-specific human T cell clone K34B9.1 as compared with CD1aab(gray diamonds) or CD1abb (black diamonds). All transfectants were pulsed with nervonoyl sulfatide and chased for indicated times before addition ofT cells. In control wells, fresh Ag was added to chased T2 cells together with T cells. Supernatants were harvested after 24 h, and the release of human IL-4(A), human IFN-g (B), and human TNF-a (C) was measured by ELISA and expressed as percent of control at time zero 6 SD (of triplicates or quad-ruplicates) normalized at each time point for the maximal Ag-presenting capacity of the APC calculated as (chase/chase with fresh Ag)/(no chase/no chasefresh Ag) 3 100.
  8. 8. The Journal of Immunology 1241microenvironment of the late endocytic system. The CD1a, b, c, 10. Sandhoff, R., S. T. Hepbildikler, R. Jennemann, R. Geyer, V. Gieselmann, R. L. Proia, H. Wiegandt, and H. J. Grone. 2002. Kidney sulfatides in mouseand d isoforms may have developed to maximize the efficient and models of inherited glycosphingolipid disorders: determination by nano-electro-sustained Ag presentation of endogenous and exogenous lipids spray ionization tandem mass spectrometry. J. Biol. Chem. 277: 20386–20398.with differences in both intracellular distribution and half-life. ˆ 11. Fredman, P., J. E. Mansson, B. M. Rynmark, K. Josefsen, A. Ekblond, L. Halldner, T. Osterbye, T. Horn, and K. Buschard. 2000. The glycosphingolipid These studies also highlight the importance of the differential sulfatide in the islets of Langerhans in rat pancreas is processed through re-intracellular trafficking of lipid Ags in immunity. This was dem- cycling: possible involvement in insulin trafficking. Glycobiology 10: 39–50.onstrated by the specific intracellular localization of sulfatide to 12. De Libero, G., A. P. Moran, H. J. Gober, E. Rossy, A. Shamshiev, O. Chelnokova, Z. Mazorra, S. Vendetti, A. Sacchi, M. M. Prendergast, et al.early endocytic and recycling pathways and the receptor-mediated 2005. Bacterial infections promote T cell recognition of self-glycolipids. Im-internalization of LAM to LEs. The biophysical properties of lipid munity 22: 763–772.Ags, both the head group and acyl chains, have been demonstrated to 13. Zajonc, D. M., I. Maricic, D. Wu, R. Halder, K. Roy, C. H. Wong, V. Kumar, and I. A. Wilson. 2005. Structural basis for CD1d presentation of a sulfatide derivedbe important in Ag presentation. Interaction between the glycan head from myelin and its implications for autoimmunity. J. Exp. Med. 202: 1517–groups of CD1a- and CD1b-restricted Ags such as Mycobacterium 1526. 14. Manolova, V., M. Kistowska, S. Paoletti, G. M. Baltariu, H. Bausinger,leprae lipid Ags and LAM and the C-type lectins langerin and D. Hanau, L. Mori, and G. De Libero. 2006. Functional CD1a is stabilized bymannose receptor, respectively, have been demonstrated to play exogenous lipids. Eur. J. Immunol. 36: 1083–1092.important roles in Ag uptake and presentation (28, 30). The length 15. Hava, D. L., N. van der Wel, N. Cohen, C. C. Dascher, D. Houben, L. Leon, ´ S. Agarwal, M. Sugita, M. van Zon, S. C. Kent, et al. 2008. Evasion of peptide,and saturation of the acyl chain of the glycosphingolipid glucose but not lipid antigen presentation, through pathogen-induced dendritic cellmonomycolate and GM1 length have been demonstrated to be maturation. Proc. Natl. Acad. Sci. USA 105: 11281–11286.important in CD1b-mediated Ag presentation (27, 31). Lipid 16. Manolova, V., Y. Hirabayashi, L. Mori, and G. De Libero. 2003. CD1a and CD1b surface expression is independent from de novo synthesized glycosphingolipids.length, saturation, and head groups have also been demonstrated to Eur. J. Immunol. 33: 29–37.contribute to the endocytic sorting of lipids and the preferential 17. Sommer, I., and M. Schachner. 1981. Monoclonal antibodies (O1 to O4) to ol-retention of certain lipids in organelles (32, 33). igodendrocyte cell surfaces: an immunocytological study in the central nervous system. Dev. Biol. 83: 311–327. Downloaded from www.jimmunol.org on April 18, 2012 CD1-mediated sulfatide Ag presentation may be of increased 18. Compostella, F., L. Franchini, G. De Libero, G. Palmisano, F. Ronchetti, andimportance in APCs that express CD1a and in the setting of in- L. Panza. 2002. CD1a-binding glycosphingolipids stimulating human autoreative T-cells: synthesis of a family of sulfatides differing in the acyl chain moiety.creased sulfatide availability. Upregulation of the production of Tetrahedron 58: 8703–8708.sulfatide and other glycosphingolipids in activated DCs has already 19. Gallati, H., I. Pracht, J. Schmidt, P. Haring, and G. Garotta. 1987. A simple, ¨been shown to play a role in CD1-mediated immunity (12). The high rapid and large capacity ELISA for biologically active native and recombinant human IFN g. J. Biol. Regul. Homeost. Agents 1: 109–118.levels of expression of CD1a, long a marker of LCs, on myeloid 20. Diez-Roux, G., and A. Ballabio. 2005. Sulfatases and human disease. Annu. Rev.DCs point to its importance in self- and foreign lipid Ag pre- Genomics Hum. Genet. 6: 355–379.sentation. Further, because so many pathways of internalization of 21. van der Wel, N. N., M. Sugita, D. M. Fluitsma, X. Cao, G. Schreibelt, M. B. Brenner, and P. J. Peters. 2003. CD1 and major histocompatibility com-exogenous Ags traverse the early endocytic system, the role of plex II molecules follow a different course during dendritic cell maturation. Mol.CD1a as the principal lipid Ag-presenting molecule in this location Biol. Cell 14: 3378–3388.highlights its distinctive role as the main sentry at this entry point. 22. Cao, X., M. Sugita, N. Van Der Wel, J. Lai, R. A. Rogers, P. J. Peters, and 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: 4770–4777.Acknowledgments 23. Schlesinger, L. S., T. M. Kaufman, S. Iyer, S. R. Hull, and L. K. Marchiando.We thank Dr. Duarte Barral for his expert advice. 1996. Differences in mannose receptor-mediated uptake of lipoarabinomannan from virulent and attenuated strains of Mycobacterium tuberculosis by human macrophages. J. Immunol. 157: 4568–4575.Disclosures 24. Delamarre, L., M. 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