TAP genes and immunity

TAP genes and immunity James McCluskey
, Jamie Rossjohn and Anthony W Purcell The transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette transporter family that specializes in delivering cytosolic peptides to class I molecules in the endoplasmic reticulum. The TAP is a major target of genetic alteration in tumours and disruption by viral inhibitors. In some species, TAP genes have co-evolved with MHC class I molecules to deliver peptides that are customised for particular alleles. In humans, MHC class I polymorphism determines the level of tapasin-mediated association with TAP and subsequent peptide optimisation within the peptide-loading complex (PLC). MHC class I molecules that still load peptides without complexing to the TAP might be more resistant to viral interference of the PLC and less sensitive to competition for TAP by other class I allotypes. Addresses Department of Microbiology and Immunology, The University of Melbourne, Victoria, Australia
e-mail: [email protected]

loops between TM segments 4–6, involving both TAP1 and TAP2 [1]. The unrelated ER protein tapasin can be considered a third subunit of TAP that is co-opted to stabilize TAP expression and to optimise the peptide-loading of MHC class I molecules in the ER. Tapasin is a crucial broker in creating the multimeric peptide-loading complex (PLC) comprising the TAP heterodimer that is bridged by tapasin to empty MHC class I–b2-microglobulin complexes associated with the chaperone calreticulin and the thio-oxidoreductase ERp57 [3]. Thus, TAP function is best considered in the context of the multimolecular interactions that take place during assembly and function of the PLC, where polymorphism in any of the components can potentially impact upon peptide loading. Here we review the salient features of TAP biology and immunogenetics with particular emphasis on recent developments.

Current Opinion in Immunology 2004, 16:651–659

TAP polymorphism and peptide selection

This review comes from a themed issue on Immunogenetics Edited by Chester Alper

The TAP loci are linked and lie adjacent to the genes encoding the two low molecular weight polypeptide (LMP) immunoproteasome subunits, LMP2 and LMP7 in the class II region of the MHC, see Figure 2. Tapasin is also encoded in the MHC near HLA-DP. This observation suggests the co-evolution of the linked genes controlling the creation of peptide antigens in the cytoplasm (immunoproteasomes LMP2 and 7), the capture and transportation of these peptides into the ER (TAP1 and TAP2) and then their presentation to T cells (MHC class I molecules). One consequence of the linkage of these genes might be their coordinated regulation by cytokines [4,5], which might benefit from open chromatin or shared promoter elements, such as in the intergenic region between TAP1 and LMP2 (see Figure 2; [6,7]). Another explanation for MHC linkage of the TAP loci could involve the selection of favourable combinations of TAP alleles with alleles of the immunoproteasome to customise peptide specificities for polymorphic MHC class I molecules. However, specific patterns of linkage disequilibrium that fit this design have not been observed in humans despite apparent linkage disequilibrium between HLA-A and tapasin [8]. Instead, human TAP (hTAP) selects peptides that are generally well suited to the binding preferences of polymorphic HLA class I molecules, suggesting the co-evolution of these genes independent of their linkage [9].

Available online 19th August 2004 0952-7915/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2004.07.016 Abbreviations ER endoplasmic reticulum HCMV human cytomegalovirus hTAP human TAP LMP low molecular weight polypeptide MHC major histocompatibility complex PLC peptide-loading complex rTAP rat TAP TAP transporter associated with antigen processing TM transmembrane

Introduction The transporter associated with antigen processing (TAP) is a protein that delivers cytosolic peptides to the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The TAP heterodimer comprises TAP1 and TAP2 proteins, members of the ATP-binding cassette (ABC) family of transporter molecules, which contain multiple transmembrane (TM) spanning segments and a cytoplasmic nucleotide-binding domain (Figure 1a; [1,2]). Peptides bind to the cytosolic www.sciencedirect.com

Nonetheless, TAP polymorphism in the rat, but not human [9] or mouse [9,10], does affect peptide selection Current Opinion in Immunology 2004, 16:651–659

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Figure 1

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1 MASSRCPAPR GCRCLPGASL AWL.GTVLLL LADWVLLRTA S R 1 .......... ......MAAH VWLAAA.LLL LVDWLLLRPM .......... ......MAAH AWPTAALLLL LVDWLLLRPV S HS 100 AAALGLALPG LALFRELISW GAPGSADSTR LLHWGSHPTA Mm 76 VAALSLALPG LALFRELAAW GTLREGDSAG LLYWNSRPDA

LPGIFSLLVP .EVPLLRVWV VGLSRWAILG LGVRGVLGVT .......AGA HGWLAALQPL 75 LPGIFSLLVP .EVPLLRVWA VGLSRWAILG LGVRGVLGVT .......AGA RGWLAALQPL 76

Rn 77 VAALGLALPG LASFRKLSAW GALREGDNAG A Hs 200 GEMAIPFFTG RLTDWILQDG SADTFTRNLT Mm 176 GEMAIPFFTG RITDWILQDK TVPSFTRNIW Rn 177 GEMAIPFFTG RITDWILQDK TAPSFARNMW

FVVSYAAALP AAALWHKLGS LWVPGGQGGS GNPVRRLLGC LGSETRRLSL FLVLVVLSSL 199 FAISYVAALP AAALWHKLGS LWAPSGNRDA GDMLCRMLGF LGPKKRRLYL VLVLLILSCL 175 W LLHWNSRLDA FVLSYVAALP AAALWHKLGG FWAPSGHKGA GDMLCRMLGF LDSKKGRLHL VLVLLILSCL 176 S LMSILTIASA VLEFVGDGIY NNTMGHVHSH LQGEVFGAVL RQETEFFQQN QTGNIMSRVT EDTSTLSDSL 299 LMSILTIAST ALEFASDGIY NITMGHMHGR VHREVFRAVL RQETGFFLKN PAGSITSRVT EDTANVCESI 275 H TM2 LMCILTVAST ALEFAGDGIY NITMGHMHSR VHGEVFRAVL HQETGFFLKN PTGSITSRVT EDTSNVCESI 276

Hs 300 SENLSLFLWY LVRGLCLLGI MLWGSVSLTM VTLITLPLLF LLPKKVGKWY QLLEVQVRES LAKSSQVAIE ALSAMPTVRS FANEEGEAQK FREKLQEIKT 399 V V Mm 276 SGTLSLLLWY LGRALCLLVF MFWGSPYLTL VTLINLPLLF LLPKKLGKVH QSLAVKVQES LAKSTQVALE ALSAMPTVRS FANEEGEAQK FRQKLEEMKT 375 D TM3 TM4 Rn 277 SDKLNLFLWY LGRGLCLLAF MIWGSFYLTV VTLLSLPLLF LLPRRLGKVY QSLAVKVQES LAKSTQVALE ALSAMPTVRS FANEEGEAQK FRQKLEEMKP 376 Hs 400 LNQKEAVAYA VNSWTTSISG MLLKVGILYI GGQLVTSGAV SSGNLVTFVL YQMQFTQAVE VLLSIYPRVQ KAVGSSEKIF EYLDRTPRCP PSGLLTPLHL 499 C L Mm 376 LNKKEALAYV AEVWTTSVSG MLLKVGILYL GGQLVIRGAV SSGNLVSFVL YQLQFTQAVQ VLLSLYPSMQ KAVGSSEKIF EYLDRTPCSP LSGSLAPSNM 475 T H TM5 TM6 Rn 377 LNKKEALAYV TEVWTMSVSG MLLKVGILYL GGQLVVRGAV SSGNLVSFVL YQLQFTRAVE VLLSIYPSMQ KSVGASEKIF EYLDRTPCSP LSGSLAPLNM 476 Hs 500 EGLVQFQDVS FAYPNRPDVL VLQGLTFTLR PGEVTALVGP NGSGKSTVAA LLQNLYQPTG GQLLLDGKPL PQYEHRYLHR I Mm 476 KGLVEFQDVS FAYPNQPKVQ VLQGLTFTLH PGTVTALVGP NGSGKSTVAA LLQNLYQPTG GQLLLDGQCL VQYDHHYLHT R WA Rn 477 KGLVKFQDVS FAYPNHPNVQ VLQGLTFTLY PGKVTALVGP NGSGKSTVAA LLQNLYQPTG GKVLLDGELL VQYDHHYLHT S Hs 600 AYGLTQKPTM EEITAAAVKS GAHSFISGLP QGYDTEVDEA GSQLSGGQRQ AVALARALIR KPCVLILDDA TSALDANSQL G Q Q C D WB Mm 576 AYGLNRTPTM EEITAVAVES GAHDFISGFP QGYDTEVGET GNQLSGGQRQ AVALARALIR KPLLLILDDA TSALDAGNQL Rn 577 AYGLTRTPTM EEITAVAMES GAHDFISGFP QGYDTEVGET GNQLSGGQRQ AVALARALIR KPRLLILDDA TSALDAGNQL Hs 700 TQHLSLVEQA DHILFLEGGA IREGGTHQQL MEKKGCYWAM VQAPADAPE Mm 676 TQQLSLAEQA HHILFLREGS VGEQGTHLQL MKRGGCYRAM VEALAAPAD Rn 677 TQQLSLAERA HHILFLKEGS VCEQGTHLQL MERGGCYRSM VEALAAPSD T K

QVAAVGQEPQ VFGRSLQENI 599 QVAAVGQEPL LFGRSFRENI 575

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MRLPDLRPWT SLLLVDAALL WLLQGPLGTL LPQGLPGLWL EGTLRLGGLW GLLKLRGLLG FVGTLLLPLC LATPLTVSLR ALVAGASRAP PARVASAPWS 100 MALSYLRPWV SLLLADMALL GLLQGSLGNL LPQGLPGLWI EGTLRLGVLW GLLKVGRLLG LVGTLLPLLC LATPLFFSLR ALVGGTASTS VVRVASASWG G T E MALSYPRPWA SLLLVDLALL GLLQRSLGTL LPPGLPGLWL EGTLRVGVLW GLLKVGGLLR LVGTFLPLLC LTTPLFFSLR ALVGSTMSTS VLRVASASWG H S L N V

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WLLVGYGAAG LSWSLWAVLS PPGAQEKEQD QVNNKVLMWR LLKLSRPDLP LLVAAFFFLV LAVLGETLIP HYSGRVIDIL GGDFDPHAFA SAIFFMCLFS 200 WLLAGYGAAA LSWAVWAVLS PAGVQEKEPG QENRTLLMKR LLKLSRPDLP FLIAAFFFLV VAVWGETLIP RYSGRVIDIL GGDFDPDAFA SAIFFMCLFS V TM1 TM2 WLLAGYVAAA LSLAVWAVLS PAGAQEKEPG QENNRALMIR LLRLSKPDLP FLIVAFIFLA MAVWGETLIP HYSGRVIDIL GGDFDPDAFA SAIFFMCLFS D G V W MF

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FGSSLSAGCR GGCFTYTMSR INLRIREQLF SSLLRQDLGF VGSSFSAGCR GGSFLFTMSR INLRIREQLF SSLLRQDLGF VGSSLSAGCR GGSFLFTMSR INLRIREQLF SSLLRQDLAF AE PFTIAAEKVY NTRHQEVLRE IQDAVARAGQ VVREAVGGLQ

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PLTIAAEKVY NPRHQAVLKE IQDVVAKAGQ A DGELTQGSLL SFMIYQESVG SYVQTLVYIY AGEVTRGGLL SFLLYQEEVG QYVRNLVYMY S TM6 AGEVTRGGLL SFLLYQEEVG HHVRNLVYMY Q LVGPNGSGKS TVAALLQNLY QPTGGQVLLD S LVGPNGSGKS TVAALLQNLY QPTGGQLLLD LVGPNGSGKS TVAALLQNLY QPTGGKVLLD QL WA GEKGSQLAAG QKQRLAIARA LVRDPRVLIL

Hs 401 Mm Rn Hs 501 Mm Rn Hs 601 Mm Rn

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VVREAVGGLQ GDMLSNVGAA GDMLSNVGAA GDMLSNVGAA EKPISQYEHC GEPLTEYDHH GEPLVQYDHH

FQETKTGELN SRLSSDTTLM SNWLPLNANV LLRSLVKVVG FQETKTGELN SRLSSDTSLM SRWLPFNANI LLRSLVKVVG FQETKTGELN SRLSSDTSLM SRWLPFNANI LLRSLVKVVG Q SL TM3 TVRSFGAEEH EVCRYKEALE QCRQLYWRRD LERALYLLVR T I TVRSFGAEEQ EVSRYKEALE RCRQLWWRRD LEKDVYLVIR H TVRSFGAEEQ EVSRYKEALE RCRQLWWRRD LEKELYLVIR FR S Q EKVFSYMDRQ PNLPSPGTLA PTTLQGVVKF QDVSFAYPNR EKVFSYLDRR PNLPQPGILA PPWLEGRVEF QDVSFSYPRR K EKVFSYLDRR PNLPKPGTLA PPRVEGRVEF QDVSFSYPSR N L YLHSQVVSVG QEPVLFSGSV RNNIAYGLQS CEDDKVMAAA T V YLHRQVVLVG QEPVLFSGSV KDNIAYGLRD CEDAQVMAAA YLHRQVVLVG QEPVLFSGSV KDNIAYGLRD CEDAQVMAAA

LYGFMLSISP RLTLLSLLHM 300 LYFFMLQVSP RLTFLSLLDL LYYFMLQVSP RLTFLSLLDL RVLHLGVQML MLSCGLQQMQ 400 RVMALGMQVL ILNCGVQQIL

TM5

RVMALGMQVL ILNCGVQQIL V PDRPVLKGLT FTLRPGEVTA 500 PEKPVLQGLT FTLHPGTVTA PEKPVLQGLT FTLHPGKVTA QAAHADDFIQ EMEHGIYTDV 600 QAACADDFIG EMTNGINTEI QAACADDFIG EMTNGINTEI

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DEATSALDVQ CEQALQDWNS RGDRTVLVIA HRLQTVQRAH QILVLQEGKL QKLAQLQEGQ DLYSRLVQQR LMD C A GEKGGQLAVG QKQRLAIARA LVRNPRVLIL DEATSALDAQ CEQALQNWRS QGDRTMLVIA HRLHTVQNAD QVLVLKQGRL VEHDQLRDGQ DVYAHLVQQR LEA GEKGSQLAVG QKQRLAIARA LVRNPRVLIL DEATSALDAE CEQALQTWRS QEDRTMLVIA HRLHTVQNAD QVLVLKQGQL VEHDQLRDEQ DVYAHLVQQR LEA R

C

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Current Opinion in Immunology 2004, 16:651–659

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TAP genes and immunity McCluskey et al. 653

and translocation to the ER. Initially called the MHC class I modification locus (cim), the rat TAP2 locus is essentially dimorphic with alleles differing by up to 25 amino acids; for example, TAPu versus TAPa, Figure 1c. The TAP2A (TAPa) allele supplies peptides suitable for presentation to RT1-A class I molecules of the RT1-Aa and RT1-Au haplotypes. By contrast, TAP2B (TAPu) supplies peptides that preferentially bind RT1-Au and are unsuitable for RT1-Aa molecules, thereby slowing their assembly and transport [11]. Human TAP [9,12] and rat TAPa (rTAPa; [11,13]) translocate peptides with hydrophobic or basic carboxyl termini that suit binding to most MHC class I molecules, including RT1-Aa, which prefers carboxyl terminal arginines. By contrast, mouse TAP (mTAP) and rTAPu translocate peptides with hydrophobic carboxyl termini that preferentially bind to mouse MHC class I molecules and RT1-Au [13]. The crystal structures of two RT1-A allotypes (RT1-Aa and RT1-Ac, the latter of which behaves in a similar way to the RT1-A1u allotype) that differentially associate with TAP2A and TAP2B alleles show how the chemical properties of the F-pockets of the antigen-binding cleft match those of the corresponding carboxyl termini of the peptides [14]. Functional polymorphism in rTAP has an even greater impact on the surface expression of other rat class I molecules, such as RT1-A10 and RT1-A2, than it does on RT1-A1a, leading to the initial misclassification of these class allotypes as low/non-expressors [15]. In Xenopus, two distinct alleles of the TAP1 and TAP2 loci are thought to have diverged from each other 60 million–100 million years ago [16]. These lineages were present as defined haplotypes of LMP7, TAP1 and TAP2 at different frequencies for X. laevis and X. tropicalis, suggesting that these ‘class I region’ biallelic lineages are maintained in the population by selection [16]. It will be of interest to determine whether ancient TAP

genes, such as those found in the lamprey [17], are also polymorphic.

Human TAP polymorphism and disease There are six hTAP1 and four hTAP2 alleles formally recognized but other rare alleles have been described in the literature [18–20]. Polymorphism in hTAP genes generally results in differences in only one or two amino acids, and these are scattered throughout the protein (Figure 1b,c). Of 15 possible TAP1 and TAP2 haplotypes present in a panel of genotyped cell lines, 11 were actually observed [18], consistent with other reports of little or no linkage disequilibrium between alleles at the TAP1 and TAP 2 loci [21–24]. Similarly, there is no apparent linkage disequilibrium between the alleles at the LMP2 and LMP7 loci, or between these and the adjacent TAP1 and TAP2 loci [25], except perhaps in some restricted populations such as Brazilian Amerindians [26], Mexican Mezistos and Seri Indians [27,28]. Indeed, there is evidence for a recombinational hot spot in the intron 2 of the TAP2 locus [24,29], an observation apparently at odds with the linkage disequilibrium between HLA-DP and HLA-DR/DQ that operates across this site in some haplotypes [23,30–32]. Presumably the low levels of TAP and LMP polymorphism, and the lack of measurable functional differences among human TAP alleles [9,12], allows the selection of certain DR-DQ-DP haplotypes in the face of genetic variation at the intervening TAP/ LMP loci [23]. Accordingly, there is little evidence in humans that preferred combinations of TAP1 and TAP2 alleles are selected for functional reasons, although some linkage disequilibrium with MHC class II alleles is reported [18]. The location of TAP polymorphisms does not rule out functional differences in peptide selection or transport, but this has not yet been documented for any hTAP alleles (Figure 1; [9,12]). This might explain why TAP polymorphisms are either weakly associated with

(Figure 1 Legend) A topological model of human and rodent TAP molecules. (a) Human TAP consists of a heterodimer of TAP1 (748 amino acids) and TAP2 (703 amino acids). Human TAP1 is predicted to have a core six-transmembrane-spanning domain, up to four amino-terminal TM helices [94] and a carboxy-terminal nucleotide-binding domain, the structure of which has recently been determined [2]. TAP2 is also predicted to have a core six-transmembrane-spanning domain and three amino-terminal TM helices in addition to the carboxy-terminal nucleotide-binding domain. Naturally occurring allelic polymorphism is evident in both TAP1 and TAP2, and is highlighted on the topological model by red stars. The large red star at the carboxyl terminus of TAP2 denotes a deletion of 18 residues in TAP2*0101. The naturally occurring amino acid polymorphisms span the entire length of the molecule, and in the human do not appear to contribute to substrate specificity or functionality of the TAP molecules. Some of the salient structural and functional regions of the molecule are colour coded as in Figure 1b,c (transmembrane regions, cyan; nucleotide-binding domains, magenta; and, peptide-binding domains, green). Model adapted from [1,94]. Alignments of human and rodent TAP1 (b) and TAP2 (c) are illustrated, in which the salient structural features of the molecules are highlighted on the basis of sequence alignments and theoretical and biochemical analysis of the human TAP sequences [58,94]. Of note is the highly conserved nature of the carboxy-terminal nucleotide-binding domain, including strict sequence conservation of the functional regions of the nucleotide-binding domain including the Walker A and B (WA and WB respectively), the switch (Sw) domain and the C, D and Q loops. These regions act in concert to fix ATP, control hydrolysis and communicate this process to the transmembrane domain. Other than the extreme amino-terminal transmembrane helix of hTAP, rTAP appears to adhere very well to this topological model, as anticipated from their high sequence homology (77–76% sequence identity for TAP1 and 73% sequence identity for TAP2 between mouse, human and rat species). Naturally occurring allelic polymorphisms in all three species are highlighted by amino acid residues in red, with alternative residues listed underneath the consensus sequence. These are mainly dimorphisms at given amino acid positions. Polymorphisms in human hTAP and mTAP do not appear to alter substrate specificity. In the rTAP, however, two clustered polymorphism regions at amino acids 217–218 and 262–266 distinguish the rTAP2a and TAP2u, and alter the carboxy-terminal preference of translocated peptides (for an overview, see [1,94]). These polymorphisms occur in the loop between TM2 and TM3 of the core domain of TAP2, a region proximal to the peptide-binding domain, and of the 25 amino acid differences between these two alleles, the two clusters of dimorphic residues seem to critically influence the ability of these alleles to translocate peptides terminating in an arginine residue that is best suited to RT1a molecules. www.sciencedirect.com

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Figure 2

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Current Opinion in Immunology

A map of the class II region of the human major histocompatibility complex on the short arm of chromosome 6. HLA class II loci and genes involved in Ag presentation are shown in orange. Recombination ‘hot spots’ are shown as red bars, and the more fixed chromosomal regions, sometimes known as frozen blocks, are shown as blue bars (after [29]). The DQB pseudogene that forms a boundary on one hot spot is shown in black. Genes near the tapasin locus are awaiting functional characterisation and are shown in green. The enlarged section depicts the TAP/LMP loci showing the exon-intron structure and transcriptional direction of each gene (not to scale). TAP1 and LMP2 share a bi-directional promoter. A recombination hot spot is localised to intron 2 of the TAP2 locus [24]. The map of the MHC is adapted from http://www.path.cam.ac.uk/~mhc/ map/cl2BIG.jpg. The gene organisation and genomic structure of the TAP/LMP complex was constructed from the ensembl genome browser publicly available through the Wellcome Trust Sanger Centre website at http://www.ensembl.org/.

immune disorders [33–36] or not at all [37–40]. Assigning a role for TAP polymorphism in disease susceptibility will require carefully powered studies with stratification of the data to control for linkage disequilibrium with other genes in the MHC class II region. It is nonetheless tantalizing that the factors that maintain natural TAP polymorphisms in humans remain unexplained given the localization of some polymorphisms to functional regions of the molecule (Figure 1; [20]). Inherited TAP deficiency is rare and manifests as the downregulation of HLA class I molecules or Type 1 bare lymphocyte syndrome [41]. Deficiency of either TAP1 or TAP2 genes typically manifests as recurrent bacterial infections and necrotising granulomatous skin lesions early in life [41]. These lesions may reflect exaggerated T-helper-1-like immunity related to activated natural killer and gd T cells [41], a possibility supported by the skewed repertoire of killer-cell inhibitory receptor (KIR) expression observed in TAP-deficient patients [42]. Tapasin is also polymorphic, with two major structural alleles that differ by a single residue at amino acid position 240 (Arg!Thr) [43]. These alleles are almost equally distributed in the UK population [44] and are also Current Opinion in Immunology 2004, 16:651–659

found in the Japanese [45], where linkage disequilibrium with MHC class II alleles has allowed the definition of common haplotypes. There was no linkage disequilibrium with alleles at any of the MHC class I loci in the UK population [44]. There is no evidence for functional differences in tapasin alleles. The replacement of all 19 cysteines in TAP1 and TAP2 results in a functional TAP with respect to ATP-dependent peptide transport, class I peptide loading and inhibition by ICP47 of herpes simplex virus, indicating that these cysteines are unlikely to be critical for these functions [46]. Interestingly, two of the cysteines are sites of natural polymorphisms (Figure 1).

TAP and the PLC as targets of viral subversion The TAP, and associated components of the PLC, are not only essential for direct antigen presentation to CD8+ T cells, but are also required for the cross-presentation of exogenous antigen delivered to the cytoplasm via phagosomes [47,48,49,50,51]. Cross-presentation might be particularly important for generating immunity to microbes that do not infect dendritic cells, as these are the antigen-presenting cells that prime immune responses [52]. It is not surprising, therefore, that viral genes frequently interfere with components of the PLC www.sciencedirect.com

TAP genes and immunity McCluskey et al. 655

to impair antigen presentation. The large dsDNA herpes viruses are particularly adept at immune evasion through subversion of antigen presentation [1,53,54]. Impaired TAP expression can be achieved transcriptionally or by specific interference of TAP function by viral proteins [55]. For example, during lytic pseudo rabies virus infection, the product of the virion host shut-off (vhs) gene, UL41, induces degradation of cellular mRNAs, including those encoding MHC class I and TAP, although other mechanisms also contribute to class I downregulation during infection [56]. The herpes simplex virus protein ICP47 blocks TAP function by binding the cytosolic face of the TAP complex [57], and the human cytomegalovirus (HCMV) US6 protein interacts with TAP inside the lumen of the ER [58]. Both viral inhibitors prevent peptide translocation by binding different ends of the transmembrane segment core of TAP forming the translocation channel [59], consistent with the data that ICP47 binds stably to the peptide-binding domain of TAP [60]. TAP blockade is also mediated by an early protein of equine [61] and bovine [62] herpes virus-1, suggesting that this is probably a general feature of herpes virus-1. The immune evasion protein US3 from HCMV directly binds tapasin and inhibits peptide loading, thereby preventing the optimization of the peptide repertoire presented by class I molecules [63]. The loss of classical MHC class I expression after viral interference of antigen presentation should theoretically alert NK cells to these class I ‘low’ cells; however, TAP blockade by HCMV glycoprotein US6 does not affect HLA-E expression, which normally prevents infected targets from undergoing NK cell lysis [64]. The murine gamma-herpesvirus-68 MK3 protein inhibits CD8+ T-cell recognition of peptide antigen by directing the degradation of MHC class I heavy chains, tapasin and TAP [54]. Biochemical evidence suggests that TAP1 is the primary binding partner for MK3 protein in the peptide-loading complex [54]. Viral subversion of TAP-mediated peptide delivery has also revealed that TAP-independent pathways of viral antigen presentation might operate to maintain some level of immune control under these conditions [65]. Thus, the evolutionary ‘arm wrestle’ between the adaptive immune system and the ubiquitous herpes family of viruses generally leads to persistent virus infection through the concerted evasion of host immunity coupled with inherent mechanisms for viral latency.

HLA class I polymorphism, TAP association and susceptibility to viral evasion The structural sites involved in tapasin-mediated crosslinking of MHC class I molecules and the TAP heterowww.sciencedirect.com

dimer are now being characterised. A nine-amino-acid region in the immunoglobulin-like domain of tapasin strongly influences formation of the PLC [66], and mutations clustered between amino acids 122 and 136 in the MHC class I heavy chain a2 domain and position 222 in the a3 domain, resulted in the loss of MHC class I interaction with tapasin [67]. The amino-terminal domains of TAP1 and TAP2 are essential for the recruitment of tapasin and the assembly of the PLC [59,68]. Other interactions within the PLC include the intermediate disulphide bond, which forms between ERp57 and tapasin [69], and the interactions between the aminolinked glycans at Asn86 of the human MHC class I heavy chain and calreticulin [3]. Within the known structurally important sites controlling PLC interactions, only the MHC class I molecules harbour significant genetic polymorphism. HLA class I polymorphism can significantly alter the tapasinmediated interaction with TAP, thus modulating the incorporation of particular class I allotypes into the PLC. For example, most class I allotypes, such as HLA-B*4402, HLA-B8 [70] and B*1510 [71], associate strongly with TAP and are highly dependent upon tapasin expression for effective antigen presentation and cell surface expression [70]. However, other class I molecules, such as HLA-B*2705 [70,72], HLA-B*1501, B*1518 [71] and RT1.Au [73], can load peptides without incorporation into the PLC, even though some of these allotypes are normally incorporated in the presence of tapasin [70,71,74]. The structural features of class I molecules that confer the capacity for peptide loading without TAP assembly appear to focus on the F-pocket of the antigen-binding cleft involving residues 116 [71,72,74,75] and 114 [76]. For example, HLA-B*4402 (Asp116) is highly dependent upon incorporation into the PLC for peptide optimisation and antigen presentation [70,77], whereas HLA-B*4405 (Tyr116) is not detectable in the PLC despite competent antigen presentation under physiological conditions [78,79] and in the absence of tapasin [72]. The biological significance of MHC polymorphisms that affect association with TAP through incorporation into the PLC might reside in resistance to viral subversion of antigen presentation [53,54,63,70,79]. Thus, class I molecules that can load with peptides in the absence of TAP association (or incorporation into the PLC) are less sensitive to viral inhibitors of the PLC, such as the HCMV US3, a tapasin inhibitor that only affects class I alleles that are dependent on tapasin for peptide loading and surface expression [63]. The ability of certain MHC class I allotypes to load antigen without associating with TAP might also be important under conditions of limiting peptide supply when competition for the TAP excludes some MHC class I molecules from the PLC [11,79]. The mechanism by which MHC class I molecules load in Current Opinion in Immunology 2004, 16:651–659

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the absence of incorporation into the PLC is unclear. Tapasin might still play a role in optimising the peptide repertoire under these circumstances [11,79] and heat shock proteins could protect short-lived ER peptides facilitating PLC-independent antigen loading. In addition, some class I molecules, such as HLA-A2, can bind signal sequence peptides that are imported into the ER in a TAP-independent manner [80]. However, those MHC class I molecules that can capture a repertoire of ‘normal’ peptides without associating with the PLC might also have intrinsic structural features in the F-pocket of their antigen-binding cleft, which engender rapid peptide binding in the absence of chaperone assistance [79].

TAP genes and malignancy Mutation of the PLC genes, particularly TAP mutations, in tumours can significantly impair antigen presentation, generally leading to truncation of the protein and loss of expression. TAP gene defects are common in cervical carcinoma (>50%; [81]), where they correlate with progression of the disease [82]. TAP loss is also common in melanoma [83–85], as is tapasin loss [86]. Cervical carcinoma and melanoma are malignancies under some degree of immune surveillance, suggesting immune selection of class I deficient cells through CTL activity on class I-positive tumour cells [87]. By contrast, TAP gene defects are not common in renal carcinoma lesions [88], even though downregulation of TAP and tapasin expression was found in more than half of these lesions [89]. Nor is TAP expression altered in hepatocellular carcinomas [90], suggesting selective pressure for TAP loss only occurs in certain tumours. TAP gene lesions can be structural [83], or regulatory, such as the single-nucleotide deletion that leads to rapid degradation of TAP1 mRNA in a melanoma cell line [85]. Coordinated loss of all the components of the PLC and other gene products involved in antigen presentation occurs in retinoblastoma [91], colorectal carcinoma [92] and in a mouse model of metastatic fibrosarcoma [93].

The TAP gene is frequently mutated or lost in tumours, and TAP proteins are substrates for viral inhibitors of antigen presentation. In the rat, TAP polymorphism has co-evolved with MHC class I genes to custom deliver peptides suited to particular MHC class I molecules, but functional specialisation of TAP alleles does not appear to be the case in humans or in mice. In humans, polymorphism in MHC class I determines their level of TAP association and subsequent tapasinmediated peptide optimisation in the PLC. Some MHC class I molecules still load peptides and present antigen in the absence of TAP association, and these may be more resistant to viral subversion of the PLC, or to competition for TAP by other class I allotypes. These concepts highlight a subtle layer of polymorphism in MHC genes that influences TAP interaction, the peptide loading pathway and the host adaptation to viral evasion mechanisms.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:  of special interest  of outstanding interest 1.

Bauer D, Tampe R: Herpes viral proteins blocking the transporter associated with antigen processing TAP–from genes to function and structure. Curr Top Microbiol Immunol 2002, 269:87-99.

2.

Gaudet R, Wiley DC: Structure of the ABC ATPase domain of human TAP1, the transporter associated with antigen processing. EMBO J 2001, 20:4964-4972.

3.

AWilliams A, Peh CA, Elliott T: The cell biology of MHC class I antigen presentation. Tissue Antigens 2002, 59:3-17.

4.

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5.

Brucet M, Marques L, Sebastian C, Lloberas J, Celada A: Regulation of murine Tap1 and Lmp2 genes in macrophages by interferon gamma is mediated by STAT1 and IRF-1. Genes Immun 2004, 5:26-35.

6.

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7.

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8.

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Obst R, Armandola EA, Nijenhuis M, Momburg F, Hammerling GJ: TAP polymorphism does not influence transport of peptide variants in mice and humans. Eur J Immunol 1995, 25:2170-2176.

Conclusions The TAP heterodimer is the gateway to efficient peptide loading by MHC class I molecules. Polymorphisms in the hTAP genes have not yet been reconciled with any functional variation, and the role (if any) of TAP genes in disease susceptibility is not clear. The functional biology of TAP transport of peptides, however, is being unravelled, and the importance of the PLC for peptideloading of most, but not all, MHC class I molecules is evident. Tapasin plays a crucial role in augmenting expression and function of TAP by formation of the PLC and optimisation of MHC class I peptide cargo. Tapasin can therefore be considered a third subunit of TAP that stabilizes TAP while forming a molecular bridge with MHC class I molecules. Current Opinion in Immunology 2004, 16:651–659

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82. Fowler NL, Frazer IH: Mutations in TAP genes are common in cervical carcinomas. Gynecol Oncol 2004, 92:914-921.

93. Garcia-Lora A, Martinez M, Algarra I, Gaforio JJ, Garrido F: MHC class I-deficient metastatic tumor variants immunoselected by T lymphocytes originate from the coordinated downregulation of APM components. Int J Cancer 2003, 106:521-527.

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