Early Recycling Compartment Trafficking of CD1a Is Essential for Its Intersection and Presentation of Lipid Antigens This information is current as Manuela Cernadas, Marco Cavallari, Gerald Watts, Lucia of September 28, 2021. 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 References This article cites 33 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/184/3/1235.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Early Recycling Compartment Trafficking of CD1a Is Essential for Its Intersection and Presentation of Lipid Antigens Manuela Cernadas,*,1 Marco Cavallari,†,1 Gerald Watts,‡ Lucia Mori,† Gennaro De Libero,†,2 and Michael B. Brenner‡,2 A major step in understanding differences in the nature of Ag presentation was the realization that MHC class I samples peptides transported to the endoplasmic reticulum from the cytosol, whereas MHC class II samples peptides from lysosomes. In contrast to MHC class I and II molecules that present protein Ags, CD1 molecules present lipid Ags for recognition by specific T cells. Each of the five members of the CD1 family (CD1a–e) localizes to a distinct subcompartment of endosomes. Accordingly, it has been widely assumed that the distinct trafficking of CD1 isoforms must also have evolved to enable them to sample lipid Ags that traffic via Downloaded from different routes. Among the CD1 isoforms, CD1a is unusual because it does not have a tyrosine-based cytoplasmic sorting motif and uniquely localizes to the early endocytic recycling compartment. This led us to predict that CD1a might have evolved to focus on lipids that localize to early endocytic/recycling compartments. Strikingly, we found that the glycolipid Ag sulfatide also localized 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 tail chimeras, that retain the same Ag-binding capacity as CD1a but traffic based on the cytoplasmic tail of CD1b to lysosomes, failed http://www.jimmunol.org/ to present sulfatide efficiently. Thus, the intracellular trafficking route of CD1a is essential for efficient presentation of lipid Ags that traffic through the early endocytic and recycling pathways. The Journal of Immunology, 2010, 184: 1235–1241. 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 T 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 protein have been shown to present both exogenous microbial Ags as well as AP-3 through interactions with the cytoplasmic tyrosine-based by guest on September 28, 2021 endogenous 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 myeloid APCs and have been implicated in adaptive immunity against mi- dendritic cell (DC) subsets (2). CD1a is distinct among the CD1 crobial lipids. In contrast, CD1d-restricted NK T cells are innate- isoforms in that it does not contain a tyrosine-based cytoplasmic like 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 or trafficking 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 molecules † Boston, MA 02115; and Experimental Immunology, Department of Biomedicine, have also been shown to localize in Birbeck granules, which are University Hospital Basel, Basel, Switzerland intracellular compartments unique to LCs that have been demon- 1M.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 November cluding didehydroxymycobactin, a lipopeptidic Ag isolated from 17, 2009. Mycobacterium tuberculosis, and sulfatide (7, 8). Sulfatide (39- This work was supported by grants from the National Institutes of Health (AI 028973 to M.B.B.), the Swiss National Science Foundation (3100AO-109918), and Roche sulfated b1-D-galactosylceramide) is an endogenous glycolipid Research Foundation (to G.D.L.). highly expressed in neuronal cells, kidney, and pancreas (9–11), and Address 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 including Medical School, Smith Building, Room 552, 1 Jimmy Fund Way, Boston, MA 02115. E-mail address: [email protected] 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 the endocytic recycling compartment; LAM, lipoarabinomannan; LAMP-1, lysosome- ability to stimulate CD1a-restricted T cells over a 3 d period. In associated 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 same Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 time period. These findings suggest that CD1a has a unique ability www.jimmunol.org/cgi/doi/10.4049/jimmunol.0804140 1236 CD1a TRAFFICKING IS ESSENTIAL FOR LIPID ANTIGEN PRESENTATION to 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. extensive colocalization of the Ag-presenting molecule with the Ag. Transient transfections were performed with FuGENE 6 (Roche, Basel, 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 DCs lipids that are primarily or, in the case of sulfatide, almost exclusively, were generated as previously described (15). Briefly, CD14+ monocytes localized 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-Paque shown to be critical for the sustained ability of CD1a molecules to PLUS (GE Healthcare, Buckinghamshire, U.K.) and monocytes positively stimulate 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- has significant implications for