Presenting fats with SAPsMitchell Kronenberg

CD1 presents lipids to unique subsets of T cells, but how are these lipids loaded onto CD1? A series of three papershas shed light on the molecules that facilitate this process.

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126 VOLUME 5 NUMBER 2 FEBRUARY 2004 NATURE IMMUNOLOGY

An effective antigen-presenting functionrequires the surveillance of different

intracellular compartments. To achieve this,antigen-presenting cells orchestrate theintracellular trafficking of both antigens and antigen-presenting molecules. The peptidepresentation pathways for the major histo-compatibility complex (MHC)-encodedclass I and class II molecules have been par-tially elucidated. However, much less isknown about the intracellular pathwaysleading to lipid presentation by the CD1family of molecules. As lipids tend to insertinto the bilayer of membranes, an unre-solved issue is the means by which lipids areremoved from membranes for loading ofCD1 molecules. Two reports in this issue ofNature Immunology, by Kang and Cresswelland Winau et al., as well as a recent paper byZhou et al. in Science1–3, have revealed thatthe sphingolipid activator proteins (SAPs)are chief participants in the loading of CD1,and they provide important insight intotheir mechanism of action.

CD1-reactive T lymphocytes includepathogen-specific cells with diverse T cellantigen receptors (TCRs) and apparentlyself-reactive regulatory T cells. HumanCD1a, CD1b and CD1c present variouslipid-containing antigens from the myco-bacterial cell wall to lymphocytes withdiverse TCRs4. They also can present brain-derived lipids to autoreactive T cells. Afourth human molecule is CD1d; itsorthologs constitute the only CD1 proteinin mice. Mouse CD1d is recognized by cellswith an invariant Vα14 TCR rearrangementand expression of natural killer (NK) recep-tors (Vα14i NKT cells). These cells havebeen reported to influence the maintenanceof tolerance, the regulation of autoimmunediseases, and the responses to microbialpathogens and tumors4. Vα14i NKT cellsshow self-reactivity for CD1d when it pre-sents a still-uncharacterized antigenderived from an endosomal compartment.

Their responses are greatly enhanced, how-ever, when CD1d presents the glycolipid α-galactosyl ceramide (α-GalCer), originallyisolated from a marine sponge4. Humanshave a homologous population thatexpresses an invariant Vα24 rearrangementwith identical specificity for α-GalCer pre-sented by CD1d.

Several important ideas have emergedfrom previous studies of CD1-mediatedantigen presentation5 (Fig. 1). First, CD1molecules probably are loaded with phos-phatidylinositol-containing compounds inthe endoplasmic reticulum5. With a fewexceptions, these are not antigenic, but theycan be exchanged for antigenic lipids either

on the cell surface or in endosomes,depending on the structure of the antigen.Antigens with shorter lipid tails are loadedon the cell surface, whereas those withlonger tails require loading in late endo-somes5, perhaps because the acidic pH therefacilitates antigen extraction from mem-branes or is required for conformationalchanges in CD1. Additionally, antigens withmore than one carbohydrate may requirecarbohydrate processing in lysosomes6.Second, the different human CD1 isoformssurvey different endosomal compartments,with CD1a located in sorting and earlyendosomes and CD1b preferentially local-ized to late endosomes and lysosomes5.

Mitchell Kronenberg is at the La Jolla Institute for

Allergy and Immunology, 10355 Science Center

Drive, San Diego, California 92121, USA.

e-mail: mitch@liai.org

Figure 1 Five steps in the traffic and antigen loading of human CD1b and mouse CD1d molecules. (1) Synthesis and assembly in the endoplasmic reticulum (ER), loading of phosphatidylinositol(PI)-related lipids and rapid arrival at the cell surface via the trans-Golgi network (TGN). (2)Internalization, mediated by AP-2 for CD1b, and multiple rounds of recycling through endosomes.(3) Transport to lysosomes or lysosome-like antigen-processing compartments via AP-3. ‘?’indicates there may be a subpopulation of newly synthesized CD1 molecules that traffic directly tolysosomes. (4) Action of SAPs to make lipid antigens available for CD1 loading in lysosomes,including antigen removal from the lipid bilayer and facilitated exchange of lipids bound to CD1molecules. (5) Return of CD1 to the cell surface from lysosomes. Parts of this pathway are likelyto be shared by human CD1c and CD1d.

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Third, a tyrosine-containing motif found inthe cytoplasmic tail of all CD1 molecules(other than CD1a) is important for theirendosomal localization. This motif binds toadaptors of the AP family, which can medi-ate sorting to endosomes5.

Mouse CD1d and human CD1b appearrapidly on the cell surface after synthesisand undergo several rounds of internaliza-tion and recycling7,8, with internalizationof CD1b mediated by AP-2 (ref. 8). CD1band mouse CD1d eventually localize to lateendosomes and lysosomes, which is essen-tial for their presentation of certain anti-gens. Their movement to lysosomes ismediated by another adaptor, AP-3, whichis required not only for antigen presenta-tion9–11 but also for the development ofVα14i NKT cells10,11, probably because of afunction in the generation or CD1d loadingof the ligands that select these cells. Despitesimilarity in their ectodomains, the cyto-plasmic tails of mouse and human CD1dare different, and human CD1d does notinteract with AP-3 (ref. 9).

Those previous findings left open theimportant question of the factors that makelipid antigens available for CD1 loadinginside late endosomes. The SAPs were goodcandidates, because it was already knownthat they reside in lysosomes and they workat the lipid-aqueous interface to make gly-cosphingolipids in the membrane availablefor degradation by water-soluble enzymes.The four saposins, SAP-A, SAP-B, SAP-Cand SAP-D, are highly homologous and arederived from the lysosomal cleavage of asingle precursor, prosaposin12. A fifth SAP,the GM2 activator protein, has an entirelydifferent structure and was originally iden-tified because it facilitates the degradationof ganglioside GM2. Despite sequence simi-larity between the four saposins, they maynot act in a similar way12. SAPs can bind tothe hydrophilic head group of glycolipids,acting to disrupt the bilayer and to freeindividual glycolipids, an activity that char-acterizes SAP-B, the GM2 activator, andperhaps others. Although SAP-C can berecruited to membranes, it has beenreported to act mainly by protein-proteininteractions, causing the allosteric activa-tion of the enzyme that degrades glucosyl-ceramides12.

The new studies focus on antigen presen-tation by human CD1b to T cells reactivewith microbial antigens3, and by human2

and mouse CD1d1 to NKT cells with an

invariant Vα rearrangement. In experimentswith cell lines from patients or gene-tar-geted mice deficient for the prosaposin pre-cursor, it was found that antigenpresentation is decreased in the absence ofprosaposin and that this is not because of ageneral disruption of lysosomal function1–3.Lipid antigen presentation could be restoredby providing SAPs1–3. The magnitude oftheir influence depended on the antigen andthe antigen-presenting molecule. The effectof SAP-C on the presentation by CD1b ofthree structurally different mycobacterialantigens was notable3. The effects of pros-aposin deficiency on the CD1d autoreactiv-ity of Vαi NKT cells were mixed, with astrong effect in mice1 but no effect whenhuman NKT cells were tested2. This couldreflect the different factors controlling thetrafficking of mouse and human CD1d5.There was only a marginal effect of saposinson the presentation of α-GalCer by eithermouse or human CD1d1,2, although thepresentation of disaccharide analogs thatrequire carbohydrate antigen processingwas prosaposin dependent1. When humanCD1d molecules in lysosomal membraneswere analyzed, however, α-GalCer presenta-tion also was saposin dependent2. Thisobservation is consistent with the fact thatα-GalCer can be loaded into CD1d on thecell surface, which would minimize anyeffect of the saposins, as well as in late endo-somes and lysosomes, whereas the disaccha-ride requires late endosomes6.

In mice lacking prosaposin, Vα14i NKTcells were not detected1, indicating a functionfor saposin in the generation of the naturalligand(s) bound to CD1d responsible forNKT cell positive selection. This result pro-vides further evidence for an endosomalrequirement for the processing and/or load-ing of this elusive ligand.

Saposins colocalized with CD1b andmouse CD1d in late endosomes, and theywere immunoprecipitated with CD1 mole-cules1,3. Moreover, Zhou et al. used an ele-gant in vitro system to demonstrate thatsaposins and the GM2 activator protein canexchange the lipids bound to mouse CD1d1.An unexpected feature of these interactionsis their high degree of specificity. HumanCD1b seems to work exclusively with SAP-C, which promotes its presentation of dif-ferent types of antigens3. For mouse CD1d,however, the association with SAP-A or theGM2 activator protein depended on the gly-colipid antigen bound to CD1d1. Because

the lipid tails are buried in the CD1dgroove, this indicates that the SAPs recog-nize the exposed hydrophilic head groupsof the bound antigens, perhaps togetherwith CD1. This result led to the proposalthat not only do the SAPs have a detergentfunction that aids lipid presentation byremoval of antigens from membranes butthey also may serve an editing function bybinding to and removing antigens that areweakly bound to CD1, with different SAPsresponsible for binding to different CD1-bound antigens 1.

A broad analogy can be made betweenthe functions of the SAPs for CD1 and thefunctions of TAP (transporter associatedwith antigen processing) and tapasin forMHC class I. In both cases, these moleculesmake the antigen available for loading, theyinteract with the antigen-presenting mole-cule and they have a ‘quality-control’ orediting function. It is still early, but so far,all the molecules known to be involved inCD1 trafficking, antigen loading or antigenprocessing participate in general cellularpathways and therefore are not CD1 dedi-cated. This characteristic stands in contrastto the invariant chain and HLA-DM forMHC class II, and tapasin and TAP forMHC class I, which are more specialized forantigen-presenting function. Perhaps thisrelative simplicity reflects different selectivepressures on the CD1 system, or its earlierorigin in phylogeny, with a more primitivecharacteristic. This apparent simplicity maybe due to the incomplete state of ourknowledge, however, as it is likely that addi-tional accessory proteins, yet to be identi-fied, are important for the uptake,transport and loading of diverse lipid anti-gens in early as well as late endosomes.

1. Zhou, D. et al. Science (22 December 2003) doi:10.1126/science.1092009.

2. Kang, S.-J. & Cresswell, P. Nat. Immunol. 5,175–181 (2004).

3. Winau, F. et al. Nat. Immunol. 5, 161–174 (2004).4. Vincent, M.S., Gumperz, J.E. & Brenner, M.B. Nat.

Immunol. 4, 517–523 (2003).5. Moody, D.B. & Porcelli, S.A. Nat. Rev. Immunol. 3,

11–22 (2003).6. Prigozy, T.I. et al. Science 291, 664–667 (2001).7. Jayawardena-Wolf, J., Benlagha, K., Chiu, Y.H.,

Mehr, R. & Bendelac, A. Immunity 15, 897–908(2001).

8. Briken, V., Jackman, R.M., Dasgupta, S., Hoening, S.& Porcelli, S.A. EMBO J. 21, 825–834 (2002).

9. Sugita, M. et al. Immunity 16, 697–706 (2002).10. Elewaut, D. et al. J. Exp. Med. 198, 1133–1146

(2003).11. Cernadas, M. et al. J. Immunol. 171, 4149–4155

(2003).12. Schuette, C.G., Pierstorff, B., Huettler, S. &

Sandhoff, K. Glycobiology 11, 81R–90R (2001).

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