Peptide selection by class I molecules of the major histocompatibility complex

REVIEW

Peptide selection by class I molecules of the major histocompatibility complex

Tim Elliott*, Michael Smith+, Paul Driscoll+ and Andrew McMichael* * Molecular Immunology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.

+Department of Biochemistry, University of Oxford, Oxford, UK.

Class I molecules of the major histocompatibility complex (MHC) bind peptides derived from cytoplasmic proteins. Comparison of over 100 such peptides reveals

the importance of the carboxy-terminal residue in selective binding. Recent evidence implicates the proteases and transporters of the processing pathway in

providing peptides with the correct residues at the carboxyl terminus.

Introduction

Cellular immunity mediated by cytotoxic T lymphocytes is an essential part of the specific immune response to virus infections. By killing cells infected with viruses or other intracellular parasites, cytotoxic T cells eliminate, or at least control, infections. In 1974, Zinkernagel and Doherty 111 made the remarkable observation that class I molecules of the major histocompatibility complexes (MHC) are critically involved in this process. The class I MHC glycoproteins have a high degree of genetic polymorphism, and 2ink:ernagel and Doherty showed that virus specific cytotoxic T cells, when tested on infected cells from different inbred strains of mice, only lysed these targets if they shared their MHC class I alleles 121. It was subsequently shown that MHC-restricted cytotoxic T cells recognize peptides derived from cytoplasmic viral proteins 1541, implying that MHC molecules act as receptors for viral peptides, presenting them at the cell surface in a suitable form for recognition by cytotoxic T cells. Different class I molecules present different peptides [3,51.

Dramatic support for this view came from the crystallographic structure of the MHC class I molecule HLA-A2. This showed that the two membrane-distal domains of the MHC class I molecule (al and a21 are arranged into an unique ‘superdomain’, with an antigen-binding site formed between two c1 helices and a floor of a f3 sheet 16, 71. Many of the residues that are polymorphic line this site. Electron dense material was observed within the groove, and Bjorkman et al. suggested that this might be bound peptide antigen. Similar density was also shown to be present in the crystal structures of HLA-Aw68.1 and HLA B2705, where its definition was better and it coqld be clearly identified as peptide 1891. More recently,:,a. number of reconstituted class I-peptide com$&es have been crystallized, directly locating bound peptides to this groove 110-121.

We have learnt a great deal about the nature of peptides bound by class I molecules from a series of bold experiments pioneered by Rotzschke et al. and van Bleek and Nathenson 113,141, in which peptides were eluted from purified class I MHC molecules and then sequenced 113-173. The results showed that the source of class I-binding peptides is diverse, including normal cellular proteins as well as exogenous viral proteins. It has been estimated that the class I molecules on a single cell can bind over 1000 different peptides 1171, and if any one foreign peptide binds above a threshold of about 100 molecules per cell it can stimu- late a cytotoxic T-cell response 1181. In a virus-infected cell there is a balance between processing of the foreign proteins to peptide epitopes, and the generation of new virus particles. If virus-specific cytotoxic T cells caq lyse infected cells before new virus particles are released, the virus infection can be controlled 12,191. ‘2 As more and more peptides that bind different class I molecules are being defined, we are beginning to understand the rules that govern peptide-MHC interactions. In addition, our understanding of the processes by which peptides are generated in the cytoplasm and then reach the class I molecules in the lumen of the endoplasmic reticulum (ER) is rapidly increasing, with the identification of some of the molecules involved. In addition, it is becoming apparent that these steps also play a role in selection of the peptides that will bind to the MHC class I molecules. In this review we survey the peptides that are known to bind to several different class I molecules in humans and mice. This offers clues as to how peptides bind, and how events that preceed binding, collectively known as antigen processing, could contribute to the selection of viral epitopes for presentation at the cell surface. An understanding of the rules that govern this selection should simplify the search for peptide epitopes, which is important for the study of cytotoxic T cell responses and for the development of vaccines.

Correspondence to: ‘Tim Elliott.

854 0 Current Biology 1993, Vol 3 No 12

MMC class I-binding REVIEW 855

to MHC class I molecules comprise a highly polymorphic, 45kD heavy chain and an invariant, 12kD light chain, known as BZmicroglobu’lin. The heavy chain has three extracellular domains, each of around 90 amino acids. The amino ..terminal domlains, crl and 0.2, comprise an eight stranded B-sheet with two anti-parallel cl-helices bordering the peptide binding groove; this structure is supported by immunoglobulin-like a3 and fl2-microglobulin domains 1201. The T-cell receptor of the cytotoxic T cell recognizes peptides bound to this structure.

A striking feature of the peptides that bind to class I molecules on cells is that they are short, most commonly nine amino acids in length 113,14,211. Peptides bound to a given class I allelic product show common amino acids at particular positions in the sequence, described as anchor residues, which make specific contacts with the class I molecule t&9,13-171. At higher resolution, it can be seen that the anchor side chains bind to pockets within the antigen-binding groove 121,221. The peptides that have been fully characterized as virus (or other pathogen) epitopes for cytotoxic T cells, or that have been eluted and sequenced from a variety of

Fig. 1. The peptide-binding groove of MHC class I molecules, showing: (a) the relative position of the six specificity pockets (A-F) that line the groove; (b) a longitudinal section through the class I-peptide complex to illustrate the van der Waals contacts; (c) the conserved hydrogen bonds made between (conserved residues lining the A pocket and the amino terminus of bound peptide; (d) the conserved bonds between the carboxyi termilnus of bound peptide and residues lining the F pocket. (a) is based on the HLA A2 structure, with the solvent- accessible surface shown in green for residues contributing to the peptide-binding groove. (b) is based on the structure of HLA 82705, with peptide RRIKAIILK modelI,& into the peptide-binding site; water molecules (x) fill the space between the floor of the groove and the peptide. (c) and (d) are based on ‘theHLA B2705 structure containing the bound peptide RRIKAIILK. Conserved class I residues involved in hydrogen bonding are shown in yellow. Hydrogen bonds are shown as white dashed lines; at the amino terminus (c) a hyrodgen bond network is formed with a water molec:ule. Note that residue W147 in (d) makes a conserved bond with the penultimate carbonyl group of bound peptide, and does not form aart of the F pocket. (This figure is based on data from Saper et al. [22] and Madden et al. [S].)

856 Current Biology 1993, Vol 3 No 12

Class I molecule/ epitope

,_ sauce References !

H-2D= 25 c N M

I

Flu NP Ad5 ElA SVCO-T SV40-T HPV E7 LCMVGP2 LCMVNP LCMVGPl

366-374 ASNENMDAM SGPSNTPPEI

207-215 AINNYAQKL 223-,231 CKGVNKFYL

47-51 RAHYNIVTF SGVENFGGZL FQPQNGCFI KAVYNFATC

[13,711 [721 [731 [731 (741

H-2Kb 25 C F L Y

Ovalbumin vsv NP SV NP Insulin

H-2Ra

BLA Cw3 FLU NP LM l'lysin P815 TumAg Mu self PY csp PB csp MCMVpp89

H-2Xk

2%2;4 SIINFEKL [751 RGYVYQGL [141

324.-332 FAPGNYPAL [761 31.-39 CE~LVEAL [771

170-179 RYLKNGKE'TL 1781 147-155 TYQRTRALV' 113,791

91-99 GYKDGNEYI 1801 14-22 KYQAVTTTL 1811

Eluted SYFPEITHI [Zll 277-285 SWPSAEQI 1781 249-257 SYLPSAEKI [781 167-176 ~PHFMPTNL [821

2 C E I D

Flu NP 50.-58 SDYEGRLI [831 Flu HA 259.-266 FEANGNLI [831 Flu HA2 lo--18 IEGGWTGMI [831 Flu NSl 152,-160 EEGAIVGEI [841

H-2L' 2 C P L

MC.WJ pp89 168-176 YPHFMPTNL [821 LCMV NP 118-126 RPQASGWM [851 Al10 wewtide Mouse-L?A

ELuted LSP FPFDL 1861 Eluted XPLEANYQXF t29j

Bovine endo Eluted APQPGMENFK t291 Me-transferase Eluted QPQRGQENF [291 P.rich wrotein Eluted XP OGPREO 1291 Chicken-class1 Eluted XPQPNLYQL i29j Superox.dismut.Eluted XP AXAYPY i291

Mouse DMD Eluted Mouse kinase,Eluted

HLA A2

Flu M 56-68 HTLVlTax 1.1-19 HCMV g% 619-628 HBV Ncp 18-27 HIV-1 pal 476-484 HIV-1 gag 77-85

Eluted Eluted Eluted Eluted Eluted Eluted

HLA B35

XPNVNIHNF 1291 XPQKAGGFLM i291

2 C V

L L GILGFVFTL SLFGYPVYV IAGNSAYEYV FLPSDFFPSV ILKEPVHGV SLYNTVATL SiPSGGiGV LLDVPTAAV GiVPFiVSV SLLPAIVEL YLLPAIUHI TLWVDPYEV

2 c P Y

[30,31,871 1881 [89J [901 [911 1921 ii71 [171 Cl71 [171 [171 [171

PF cp26 368-375 KPKDELDY [381 PF cp29 368-375 KSKDELDY [381 PF Is8 1850-1857 KPNDKSLY 1381 HIV-l nef 74-81 VPLRPMTY 1701 HIV-2 gag 245-253 NPVPVGNIY t CMVpp65 123-131 IPSINVHHY 1931

HLA B53 2 C P F

W

PF ls6 1786-1794 KPIVQYDNF 1381 PF cp6 300-308 .MPNDPNRNV* [381 PF sh6 77-85 MPLETQLAI* (381 HIV-2 gag 173-181 TPYDINQML [941 HLA a3 Eluted YPAEITLTW [381 Proteas.C3 Eluted DPSGAYFAW 6 CKSh 1 Eluted EPEPHILLF §

I HLA B8 35 c KK I

Flu NP 386-394 ELRSRYWAI 1261 EBV EBNA3 339-347 FLRC~FAYGI [951 HIV gag p17 24-32 GGKKKYKLK [261 aI+ PO1 185-193 DPKVKOWPL 1261 HIV gag p24 331-339 DCKTILKAL [261 HIV gag p24 261-269: GEIYKRWII I261 HIV gp41 586-593 YLKDQ QLL [921

H&A B27

Flu NP 383-391 HIV gag 265-274 EBV EBNA3C 258-266 HIV gpl20 314-322 Histone H3 Eluted Hsp 89a Eluted

2 C R K

R

SRYWAIRTR t961 XRWIILGLI;IK [9?1 RRIYDLIEL [981 GRAFVTIGK [151 RRYQKSTEL [151 RRIKEIVKK [151

human and murine class I molecules, are compiled in Table 1, As shown in Table 1, each peptide has at least two anchor residues, one of which is always at the carboxyl terminus. The other anchor residue is most commonly the second amino acid of the peptide (PZ), but can sometimes be the third fP3) or fifth (P5), depending on the-class I allele. These observations correlate well with th$ X-ray crystallographic structures of ,the class I molec$$ HLA AZ, HLA A68.1, HLA B2705 and H-Z Kb, which show six ‘specificity determining pockets’, labelled A to F, lining the peptide-binding groove (Fig. la) l&-1.2,22]. While pockets A and F are conserved in structure, pockets B-E are polymorphic.

Peptides bound in the class I groove adopt an extended conformation (Fig, lb), with their amino and carboxyl termini fitted into the A and F pockets, forming hydrogen bonds with conserved residues at each end: Y7, Y59, Y159 and Y171 for the amino terminus (Fig. 1~);

T143 and Y84 for the carboxyl terminus (Fig. Id). In addition, residue W147 makes a conserved hydrogen bond with the penultimate peptide carbonyl group (Fig. lb). The conservation of the A and F pockets in all class I molecules sequenced implies that all peptides are orientated the same way. Occasionally a small variation is found, for instance the murine non-classical MHC glycoprotein, H-2M3, has phenylalanine at

MHC class l-binding pep-tides REVIEW 857

Hsp 89b Eluted RRVKEWKK 1151 I

HLA A3.1 23 C Ef2 Eluted RRWLPAGDA 1151 IF K Helicase Eluted RRSKEITVR i15i Ribosome Eluted GRIDKPILK [151 Ribosome L28 Eluted FRYNGLIHR [I51

Eluted KRFEGLTQR [151 ..' Elut.ed RRFTRPEHX 1151 Eluted RRISGVDRY [15j Elut.ed ARLFGIRAK [151

HLA B’l

HLA DP signal Elut:ed

HLA A2 signal Elut:ed CD20 Eluted Topo-iso II Eluted HLA Blsignal Elut:ed

Elutled Elul:ed Eluted Eluted Elut:ed Elutzed Eluted

Histone Hl Elut:ed Elut:ed

HLA A68

Flu NP 91-99 HBV NC 141-151

123 C APR L

V

APRTVALTA [27] APRTVALTAL [27] APRTLVLLL 1271 RPRSNIVLL i27j SPRYIFTML [27] AMPRTVL [271 APRiPiTGi [27] APRASPPSi 1271 APRiiiiii i27j APAPTVAVi [271 MPRGVVVYi [27] RPSGPGPEi 1271 RVMAPRAii 1271 ASRERSGVSL [271 APFlAFiPiPV [27]

2 C T R V

KTGGPIYKR [lo] STLPETTWRR 1991

L Y

HIV-1 nef 73-82 QVPLRPMTYK [lo01 Eluted KXFKMILRK [28] Eluted KLFKNILYK [281

YLXVRXAXN 1281 KLHKORAKS 1281

Eluted Eluted Eluted Eluted Eluted Eluted Eluted Eluted Eluted

HLA Al1

EBV EBNA4 HIV gag HIV nef P53

HLA A31 HBV cAg

HLA B62 HIV gag p17

HLA Al MAGE-1

HLA B14

325-333 AIFQSSMTK iiOlj 14-82 PLRPMITYK [loll

343-351 ELNEALELK [loll

142-152

21-29

SLFKiWTK [28j KXFVKXLXY [281 SLFNTHLXK [281 TLANDXWP 1281 GIFAXXXVKA i28j TXFVKXLXY [281 SLFDHILXKH [28]

2 C I K L

IVTDFSVIK r1011

WLPETTWR [991

RLRPGGKKKY si

EADPTGHSY [102]

Rib 60s Hsp B

Elut:ed DVFRDPALK [24j Elut:ed TVFDAKRLIGR [241

HIV g~41 589-597 ERYLKDQQL [921 I HIV gag 305-313 RAEQASQEV [701

Eluted EVAPPEYHR 1241 Eluted AVAAVAARR [241 Eluted EVILIDPFHK [241

HLA A24 HIV g~4 584-591 YLKDQQLL [1031

Only peptide epitopes that have been optimized and titred for length are included. We have ommitted many epitopes where this has not been done, even though they may contain a near-perfect motif. Only epitopes have been assigned a position in the amino-acid sequences. For eluted peptides the source is given if known. In some instances of eluted peptides, certain residues are unknown; these are designated as X. Where mass spectroscopy has been used to determine sequence, it is not possible to distinguish between leucine and isoleucine, so these residues are given as 2’. Ai&ve the peptide sequences are shown the dominant anchor residues as defined by the peptide sequences shown. Motifs are not shown where there are only one or two peptides known. Peptides that were shown to bind to HLA B53 or B35 but were not defined as epitopes for CTL are marked *. (Note that peptide sequences longer than nonamers have been condensed using a smaller font to keep the anchor residues aligned.)

1

t S Rowland-lones, oersonal communication. * J Cairin and MBA‘Oldstone, personal communication. S AVS Hill and D Hunt, personal communication. ¶ P Johnson, personal communication.

Key Flu, influenza A virus; Ad5, adenovirus-5; SV, Sendai virus; HPV, human papilloma virus; VSV, vesicular stomatitis virus; LM, Listeria monocytogenes; PY, Plasmodium yoleii; PB, Plasodium berghei; PF, Plasmodium fakiparum; MCMV, murine cytomegalovirus; LCMV, lymphocytic choriomeningitis virus; HTLV, human T cell leukaemia virus; HCMV, human cytomegalovirus; HBV, hepatitis B virus; HIV, human immunodeficiency virus; EBV, Epstein-Barr virus.

position 171 and as a result only binds peptides with formylated amino termini [231.

The hydrogen bonding between the amino and car- boxy1 termini of bound peptides and conserved residues in the A and F pockets of class I molecules explains why most tig,htly bound peptides are nine amino-acids long, but is also consistent with the observation that some cl%?s I alleles can bind to peptides shorter or longer thar@e usual nonamer. The first indi- cation of this cam& &om the structure of H-2 Kb co-crystallized with octamer and nonamer peptides, which showed the octamer fully extended and the nonamer bulging slightly [11,121. Subsequently, the

refined structure of Aw68.1 showed that the amino- and carboxy-terminal amino acids of bound peptide mix- tures could be traced, but the electron density map of the central peptide residues was indistinct (constrasting with the clear resolution of the peptide density in the HLA B27 structure> suggesting that peptides longer than nine amino acids bulge from the binding site between anchor residues [241.

At the ‘left-hand’ end of the groove of HLA A2, Aw68.1 and B2705, the side chain of peptide residue P2 enters the deep B pocket formed by class I residues 9, 24, 45, 66, 67 and 70 [8,9,221. The functional specificity conferred by the B pocket has recently been nicely


illustrated by a pocket transplant experiment 1251. Residues 9, 24, 45, 66, 67 and 70 from HLA A2 were transferred to B2705 by site-directed mu~genesis. The hybrid molecule now bound to and presented peptides only wherr they had leucine at P2 (as for HLA A2), rather than arginine which is required for binding to native HLA B27. ~though P2 is an anchor residue for most class I molecules analysed so far, in some class I molecules a phenylalanine or tyrosine at MHC position 67 probably blocks the B pocket, limiting its availability as a specificity-deternlining pocket 1261. Similarly, an aromatic residue at position 45 could contribute to the occlusion of the B pocket observed in the Kb crystal structure (Y45) 112,121. Class I molecules like this either use other pockets (true, for example, of II-2 Kb, Db, Kk, and BS), or may often require proline, which has a compact side-chain at P2 (true of HIA B35, B7, B51 and B53; see Table I).

The side chain of the carboxy-terminal peptide residue, which is always an anchor, points directly into the bottom of the F pocket, above the polymorphic class I residue 116, When the amino acid residues of eluted peptides and well-characterized epitopes (Table 1) are compared, some striking features are seen. First, the amino acids found at the carboxyl termini (P-C residues) are greatly restricted: isoleucine, leucine, valine, arginine, lysine, tyrosine or phenylalanine are found in more than 95% of epitopes. This restriction also holds true of ind.ividual sequenced peptides eluted from HI.A A2, B27 and B7 [15-17,271, although there are a few exceptions in peptides eluted from HLA A3.1 and H-2Ld 128,291; it may also be obscured in the sequences of pooled eluted peptides because of the length hetero- geneity. It is quite likely then, that the conservation of the P-C residue is closely related to the nature of the pocket into which the carboxy-terminal side chain fits (Fig. 1).

Class I residues 116 and 77 appear to play key roles in determining the nature of the amino acid side chains that will fit into the F pocket. Table 2 shows that all class I molecules that prefer residue P-C to have a positively charged side chain have D77 and Dllb, whereas two out of three alleles that prefer Y at position P-C have S77 and SlI6, though Y can also fit into D77/DlI6 and NT~/DII~ pockets. Eight out of ten class I molecules that bind peptides with hydrophobic P-C residues have F or Y at 116. From the available crystal structures, it can be seen that class I residue 116 defines the floor of the F pocket; in the B27 and Aw68.I structures, residue 116 makes contact with the P-C side chain of bound peptides, although this does not appear to be the case for ~116 in the HIA A2 and II-2 Kb structures. Thus, there is a correlation between the three common amino acids at class I residue II6 - Y,

carboxy-terminal anchor residues Y, respectively. There are some

Iy in peptides where the fit else- where is particularly good, but in most cases this ‘carboxy-end rule’ is observed. Figure 2 shows how the carboxy-termina1 peptide side chain fits into the F

pocket in I-U B27, HLA A2 and H-2Kb, together with a predicted fit for HLA B35.

In the case of class I molecules preferring P-C residues with hydrophobic side chains, the F-pocket interactions show an additional feature. Structures have ‘been solved for two class I molecules of this type (H-2Kb and HLA A2), and it can be seen that residue ~116 occupies a different position in each [11,12,221. The H-2 Kb structure was solved as a complex with single bound peptides, each with L at P-C, whereas the HLA A2 structure represents the average structure of many different HLA A2-peptide complexes. In the latter case, the electron density for ~116 was indistinct; indeed, ~116 was placed in a different orientation in the refigled A2 strut- ture than in the original 1987 model [6&?1. This suggests that either Yll6 is relatively mobile in the HLA A2 molecule, or that each HLA A2 molecule can adopt more than one confo~ation depending on the nature of its bound peptide ligand. In contrast to the motif described for H-2Kb, with L or M at P9 1211, pooled sequencing of peptides eluted from HLA A2 shows a preference for V at P9 [Zll, although we know that peptides with L at P9 can bind with high affinity 130,311. Thus, we suggest that the original structure of HLA A2 represents an average of a set of complexes with peptides that most commonly have V at P9, but some have L at P9 and the two cases require ~116 to be in a different position. This could account for the difficulty in allocating an unambiguous orientation to this amino acid in the HLA A2 crystal structures.

Madden et al. 1321 have recently solved the crystal structures of five HLA A2-peptide complexes. Four of these complexes contain peptides with V at P9 and, in three of these, Yll6 is orientated in the same. way as in the

Tqbl& 2. Correlation between peptide carboxy-terminal residue and class I residue 116.

Class 1 molecule

> 2

Peptide carboxy-terminal

F-pocket residue

residue 77 81 95 116

Db

$

Cd A2 88 67 B14” A24*

f362* 63.5 B53

B2705 Al? A33* A3.1 A68 A31* Al

M/I I/L L

L L/V l/L

L/V L N A L Y

z : : i: NAI S

Data derived from Table 1. *Class I molecules in which the carboxy-terminal anchor residue has been assigned only ~rov~siona~~y.

C class I-binding pep&ides REVIEW 8.55

Fig. 2. Accommodation of the carboxy-terminal side chain by the F pockets of (a) HLA B27, (b) HLA 835, (c) H-2Kb and (d) HLA A2. In each case, the view shown is from a position in the peptide-binding groove of the class I molecule, looking towards the carboxy-terminal end of the peptide. The residues (81, 84, 95, 147, 116, 124 and 146) that comprise the F pocket are shown in pink; the two carboxy-terminal residues of the bound pleptide are shown in brown. In each case, the side chain of the penultimate peptide residue (P8) is directed upwards out of the peptide binding groove towards the T-cell receptor. The last residue (P9) points down into the F pocket. The dotted surface represents the molec:ular surface (calculated with the peptide removed) of the groove of the MHC molecule in the vicinity of the F pocket. The HLA B27 and H-2Kb graphics are derived from the refined X-ray structures of MHC-peptide complexes. The H-2Kb structure was obtained using a preparation of molecules containing a single peptide [l 11. In the case of HLA B27, the peptide electron density has been interpreted in terms of ,a single peptide structure [91. For HLA A2, we used molecular modelling to build the peptide into the groove of the HLA A2 coordinates [24] in such a way as to maintain all the appropriate hydrogen-bonding interactions at the amino- and carboxy- termini of the peptide. For HLA 635, we modelled both positions of the peptide and the side chains of the polymorphic residues surrounding the F pocket. It can be seen that there is an overall correspondence between the size of the F-pocket represented by the molecular surface of the MHC molecule and the size of the side chain, leading to a maximum of non-bonded contacts in each case. Coordinates of HLA B27 were obtained from Dean Mladden; coordinates of HLA A2 and H-2Kb are from the Brookhaven Data Bank; the pictures were made using the program QUANTA (Molecular Simulations Inc).

averaged structure (pointing towards D77). In the fifth, with L at ~9, the orientation of Yll6 is shifted towards the amino-terminal residue of the bound peptide, as in the H-2 K%eptide &mplex. Thus, although the corre-

heavy chain could dictate the position of the sidechain of residue 116 in the fully assembled class I molecule. Alternatively, the folding of the peptide binding site could be influenced by (as yet unknown) co-factors,

lation is not absolute; these experiments clearly demon- and subtle differences in the way that these co-factors strate that the relative orientation of Yll6 in HLA A2 interact with newly synthesized class I heavy chains could depend on the nature of the bound peptide. could affect the repertoire of peptides bound by Early interactions between peptide and nascent class I a single class I molecule. We would expect these


differences to be apparent at the cell surface, giving a phenotype in which a single class I molecule is capable of binding to different subsets of peptides, depending on the cellular context in which it is expressed. Two such phenotypes have been described [33,341 although there is no evidence that this explanation applies (this is discussed further in a later section in the context of peptide transporters). At a practical level, these observations on the carboxyl terminus and its relationship to the F pocket should be useful in refining cytotoxic T cell epitopes, or even in predicting them from the primary sequence of antigenic proteins. The complete absence at peptide position P-C of residues E,D,N,G,H and S, and very rare occurrence of T, P, Q and C, should be particularly useful in epitope definition.

The forces that bind the peptide include, therefore, a highly conserved pattern of hydrogen bonding at the amino and carboxyl termini, hydrogen bonds with the backbone and the finely tuned non-bonding interactions with the anchor residue side-chains in the specificity-determining pockets. The relative importance of these interactions has been investigated by studying the binding of peptides in which either the amino or car- boxy1 termini were blocked, or where the anchor residues were changed to alanine Ml. All modifications led to a substantial reduction in binding capacity: changing the carboxy-terminal anchor decreased binding by about loo-fold; changing the second anchor by about 500-fold; blocking the carboxyl terminus by amidation led to a loo-fold reduction in binding; and acetylation of the amino terminus decreased binding by almost four orders of magnitude. In addition, positions outside the principal anchor positions may be important for determining allele peptide-binding specificity. This has been illustrated by the finding that binding of peptides to class I molecules can be impaired when non-anchor peptide residues are altered M-371.

Peptide binding and iassembly of class I molecules

The nature of the peptides that are selected for presentation at the cell surface, including naturally processed viral epitopes, has been elucidated by determining the pooled sequences of class I-bound peptides. The sequences of almost all viral epitopes that have been identified conform to the rules inferred from these pooled sequences. However, there is ample evidence that for any given viral protein, only a few of the total number of class l-binding peptides that could theoreti- cally be generated are actually selected for use as epitopes (for example, see 1381). Furthermore, there are examples where identical class I molecules in different settings present different peptide epitopes 134,391. Thus, the chemistry of the peptide binding is not the whole story of epitope sel5Ftior-i.

The evidence to da&indicates that binding of peptides to class I molecules’i~‘highly selective. Indeed, peptide binding is closely linked to the assembly of class I molecules, that is to say, peptides and &-2 microglobulin bind cooperatively to class I heavv chains 140.411.

Peptide binding to heavy chain-p-2 microglobulin heterodimers stabilizes the interaction between the two class I chains [40,42-441. The most stable complexes are formed with naturally processed epitopes; peptides that contain the same core sequence but that are ,extended at either the amino or carboxyl termini bind less tightly 1421. It has been suggested that stable peptide binding is associated with a conformational change in the heavy chain-p-2 microglobulin heterodimer 145-471. Only naturally processed epitopes are able to form quasi-stable intermediates with class I heavy chains in the absence of p-2 microglobulin 1411. In doing so, they induce the heavy chains to adopt the conformation they have in native class I molecules. This reaction could be at the heart of epitope selection, and it is particularly interesting that it does not require the presence of the heavy- chain a3 domain [351, and- that peptides may induce the conformational change by lowering the activation energy required for it to happen 1431. Given that this reaction requires peptides to be reduced to the correct

(a) Peptide 20 frequency 18 (No.164)

16 -

14 -

Observed at N-l

El Expected

12 -

IO -

8 6

4

2

0 ACDEFGH IKLMNPQRSTVWY

1 (b) Peptide 20 1

: 2 Amino acid

16 -

14 -

12 -

10 -

8-

6

0 ACDEFGH

Observed at C

cl Expected

K LMNPQRSTVWY

Amino acid

1

Fig. 3. Frequency of amino acids at (a) the position preceding the amino terminus (N-l 1, and (b) the carboxy-terminal position C, in peptides that bind to class I MHC molecules. A comparison is made between the actual frequencies koloured bars) and the expected frequencies fgrey bars) based on the natural abundance of each amino acid 1691. (Data for 64 wotides from Table 1 .I

MHC class l-binding peptides REVIEW 861

length, it may greatly limit peptide selection from a pool of potentially binding peptides.

As explained above, one general observation made concerning the nature of peptides bound to class I molecules is that the side chain of the carboxy-terminal amino acid is invariably an anchor residue and the chemical nature of this side chain appears to be limited to L, I, V, R, K, Y or F. The extent of this restriction is even more striking whlen the relative frequency of amino acids immediately before the amino terminus is compared to the relative frequency of amino acids at the carboxyl terminus (Fig. 3). There are a number of possible explanations for this observation. The very existence of a specificity-determining pocket for accom- modating the side chain of the carboxy-terminal amino acid almost certainly places the greatest constraint on the nature of the residue at the carboxyl terminus. The high frequency of hydrophobic and basic amino acids at this position could also reflect specificities of the proteolytic enzymes involved in generating peptides or of the transporters responsible for importing the peptides from the cytosol to the ER.

These possibilities are addressed by an experiment that directly investigates the involvement of the carboxy- terminal amino acid in binding of peptides to class I

molecules, Matsumura et aE. 1481 synthesized a random mixture of nonomeric peptides in which each of the 20 amino acids was equally represented at each peptide position. These were then offered to empty H-2 Kb molecules in vitro, and the pooled sequence of all bound peptides was determined. Unlike the pooled sequence of peptides eluted from H-2 Kb molecules that had been purified directly from the cell surface, in the case of the peptides that bound H-2 Kb molecules in vitro no predominant carboxy-terminal residue was identified, although Y/F was predominant at P5. These results suggest that the requirement for a particular side-chain at the carboxyl terminus of a class I-binding peptide is not dictated solely by the nature of the class I binding groove, and imply that processes upstream of class I binding could contribute to the observed in vivo binding specificity. However, it will be necessary to ensure that the peptides with alternative carboxyl terminal residues bind to class I with affinities sufficient for the generation of stable complexes at the cell surface, before ruling out the F pocket as the main determinator of selection at the carboxyl terminus.

There is growing evidence to suggest that the peptide transporters encoded by the TAP genes (reviewed in 1491) show some degree of substrate specificity. Recent experiments by Neefjes et al. [501 demonstrate a degree

Fig. 4. Assembly of class I molecules with peptides, showing alternative ways in which the class I-peptide complexes may be formed. Cytosolic proteases may generate optimal peptides (shown in red) or, more likely, longer peptides extended at the amino terminus, with the potential epitope at the carboxyl terminus. Both may be transported into the lumen of the endoplasmic reticulum. The longer peptides may be shortened at the amino terminus by peptidases to generate the optimum epitope which then binds to newly synthesized class I molecules. The optimal epitope‘may initiate folding of the free heavy chain (pathway 1) which then binds to b-2 microglobulin to give the stable class I-peptide complex ttid&s tranlocated to the cell surface. Alternatively, the optimal peptide may bind to the folded but empty heavy chain-b-2 microglobulin complex (pathway 2) to give the same stable class I-peptide complex. Longer peptides containing the epitope may be able to bind to the empty heavy chain-p-2 microglobulin complex and may then be shortened by peptidases while bound to the MHC molecule (pathway 3), as suggested by Rotzschke and Falk [62].

862 Current Biology 1993, ‘V/o1 3 No 12

of sequence specificity in the transport process, by showing that a decameric peptide ending in threonine is not transported intlo the ER of permeablized cells; peptides that were identical apart from having histidine or glutamate at the carboxyl terminus were efficiently transported. These experiments imply that the absence of class I-binding peptides with acidic carboxyl termini is not due to their inability to be transported into the ER. Similarly, Shepherd et al. [511 have shown that peptides of varying length and sequence compete with different efficiencies for transport of an index peptide, though no particular size or sequence specificity was apparent. Androlewicz et al. 1521, however, using a similar assay, claim that the optimal substrates for the TAP transporters are S-10 amino acids long, similar to the optimal length for class I binding. Although the degree of selectivity exerted at the level of peptide transport is uncertain, there is one good example of genetic polymorphism. of the peptide transporter affect- ing the nature of epitopes bound to class I molecules. The rat class I molecule, RTla, presents different peptide epitopes according to the transporter phenotype of the animal i.331. Expression of either of the two transporter allotypes l’eads to selective binding by RTla of different sets of peptides with different carboxy- terminal residues. As a consequence, the same class I molecule can present mutually exclusive sets of epitopes to T cells, depending on the TAP2 allele expressed.

The best studied agent of protein degradation in the cytosol is the multi-subunit complex known as the proteasome 1531. There has been much speculation about the role of the proteasome in generating peptides for binding to class I molecules, supported by several indi- rect observations 154,551 and the discoveries that two of the proteasome subunits (LMP 2 and 7) are encoded by genes in the MHC and are IFN-y inducible 155-581. The role of LMP 2 and 7 in antigen processing has been

called into question by the observation that normal class I expression and antigen presentation can be restored in mutant cell lines lacking both LMP and TAP genes by the reintroduction of the TAP genes alone 158,591. Nevertheless, several recent reports suggest that proteasome subunits LMP 2 and 7 do play a role in the regulation of the specifity of the cleavages that generated class I-binding peptides. Of the three ATP-independent proteolytic specificities of the proteasome, the activity of those cleaving after hydrophobic and basic residues is increased after IFN-y stimulation 160,611, whereas the third, cleaving after acidic residues, is depressed [6ll. The modulation of these activities appears to be dependent on the presence of LMP 2 and 7. The provocative implication is that the unwanted specificity of the proteasome - no F pocket has yet been shown to accept an acidic residue - is sup- pressed by LMP 2 and 7. Furthermore, by reducing the proteolytic specificities, epitopes containing acidic residues will be spared degradation.

Whether proteasomes are partially or wholly responsible for the proteolysis of intracellular proteins that generates class I-binding peptides awaits further investigation, but the fact that fewer amino acids are represented at the ~9 position of bound peptides than at the P-l position suggests that different sets of enzymes could be involved in the generation of the amino and carboxyl termini, with known proteasome specificities being evident at P9 but not P-l. Indeed, the amino terminus of peptide epitopes could be optimized by amino-peptidases after their TAP-dependent transport into the ER. Optimal peptides might then interact with newly synthesized class I heavy chains, initiating their folding and assembly with p-2 microglobulin. Alternatively the amino terminus might be trimmed after binding of a precursor peptide to the class;1 molecule as first proposed by Rotschke and Falk 162l.‘However, for an enzyme to gain access to the

influenza AP

Al B27

HIV gag

Pl7 Q24 r

PI,5 I ,

B62 657 B62

A33 B35 862 4 1993 Current Biology

Fig. 5. Epitope maps for class I-presented peptides in the viral proteins influenza virus NP, HIV gag and HIV pal, showing how the epitopes are clustered. The epitope sequences and positions are given in Table 1 and 1701.

MHC class l-binding pepiides REVIEW 863

N-l-P1 peptide bond, disruption of the Pl-A and P2-B pocket interactions would probably be required, result- ing in rapid dissociation of the bound peptide. These possibilities are illustrated in figure 4.

Epitope clustering

One final point strikes us. As more and more epitopes for different alleles are identified from a given viral protein, it appears that they are not randomly distrib- uted but clustered, sometimes with overlapping epitopes for different class I molecules (Fig. 5). This is particularly noticeable fior influenza NP, HIV gag and HIV nef proteins, in which large numbers of cytotoxic T-cell epitopes have been mapped. This could be fortu- itous, or it could be a reflection of the fact that the distribution of all 20 amino acids in proteins is not uniform with, for example hydrophobic amino acids clustering in regions of a protein where they are required for the maintainance of a stable tertiary structure. Thus, motifs that rely heavily on hydrophobic anchor residues may be expected to cluster in these regions. Similarly, DNA-binding proteins, which seem to be particularly frequent as dominant antigens recognized by cytotoxic T c’ells, may contain stretches of basic amino acids; HLA B8 and B2705 bind peptides with positively charged anchors, and there are two instances where B8- and B2705-presented epitopes overlap. Such overlapping of epitopes could mean that different class I molecules in the ER compete for the same precursor peptide. One might expect, therefore, epitope selection by one type of class I molecule would be influenced by the presence of other types of class I molecule, in some cases at least. Preliminary evidence suggests that this is the case [631. This has interesting implications for HLA-disease associations, which if dependent on presentati’on of a particular (self) peptide might be influenced by the full set of class I molecules expressed by an individual (and thus his/her complete HLA type).

In addition to the non-random distribution of amino acids in proteins, the tertiary structure of an antigenic protein could also influence the location of epitopes. If we assume that a potential antigenic protein must be unfolded prior to partial hydrolysis, the exact pathway of this unfolding could contribute substantially to the availability of epitopes. This may account for the finding [641 that the generation of a T-cell epitope was sensitive to its position in a recombinant protein antigen. The overall rate of intracellular degradation varied among the different chimeric proteins, perhaps reflecting different unfolding pathways. An alternative explanation for the result (the one favoured by the authors) is that the differences in primary sequence flanking the inserted epitope affect recognition by proteolytic enzymes. Similarly, Cerundolo et al. [651 found that an influenza NP, elpitope presented by HLA A68 was not presented when cells were infected with the 1934 as opposed to the,,1968 virus even though a synthetic peptide corresponding to residues 91-99 was presented equally well to the same cytotoxic T cells. Small differences in the flanking sequences between the

forms of NP encoded by the two viruses could have been responsible. However, Chimini et al. [661 have placed a cytotoxic T-cell epitope in different regions of the HLA Cw3 molecule with little effect on its presentation, atid Hahn et al. [671 have done the same with influenza haemagglutinin with the same result. The effect of the flanking sequences on processing can now be addressed by transfecting target cells with minigenes encoding an epitope embedded in a carrier protein, with the flanking sequences varied by mutagenesis 1681.

Conclusions

Despite recent advances, the relative contributions to epitope selection of class I-binding chemistry and upstream processes, such as protein unfolding, hydrolysis, and transport, remain uncertain. It seems likely that the processing pathway could play a major role in selecting the regions of the antigen that are presented, and thus explain why many good binding peptides are not natural epitopes. If proteasomes prove to be responsible for the cleavage that generates the carboxyl termini of class I-presented peptides, then, with the notable exception of those that are negatively charged, the peptides will find suitable binding pockets available in the common class I molecules. What happens at the amino terminus remains a mystery, but this should be soluble by studying reconstituted systems in vitro. That the transporter can impose some selection has been demonstrated in the rat, and further examples in other species are being sought. However, once peptides are in the ER, the class I molecule must be the primary selector, although the possibilities that proteolysis con- tinues in the ER, and that class I molecules compete to bind overlapping peptides, are intriguing. Acknowledgements We are grateful to Dean Madden and Fred Goldberg for helpful discussions and access to unpublished data. We also thank Sarah Rowland-Jones, Adrian Hill, Paul Johnson and Vie Engelhard for permision to include unpublished peptides.

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Received: 17 August 1993; revised 22 October 1993.

Peptide selection by class I molecules of the major histocompatibility complex

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