Critical residue combinations dictate peptide presentation by MHC class II molecules

Peptides, Vol. 15, No. 4, pp. 583-590, 1994 Copyright© 1994ElsevierScienceLtd Printedin the USA.All rightsreserved 0196-9781/94 $6.00+ .00

Pergamon 0196-9781(94)E0009-T

Critical Residue Combinations Dictate Peptide Presentation by MHC Class II Molecules J E A N - F R A N ( ~ O I S H E R N A N D E Z , .1 F R A N C O I S C R E T I N , ~ 2 S U Z A N N E L O M B A R D - P L A T E T , t J E A N - P A U L SALVI,* N A D I A W A L C H S H O F E R , * D E N I S G E R L I E R , t J O E L L E PARIS* AND CHANTAL RABOURDIN-COMBEt 3

*Laboratoire de Chimie ThOrapeutique, Facult~ de Pharmacie, UniversitO Lyon L 8, avenue Rockefeller, France and ~'Immunobiologie MolOculaire, UMR 49, CNRS-ENS Lyon, 69364 Lyon Cedex 07, France Received 20 October 1993 HERNANDEZ, J.-F., F. CRETIN, S. LOMBARD-PLATET, J.-P. SALVI,N. WALCHSHOFER,D. GERLIER, J. PARIS AND C. RABOURDIN-COMBE. Criticalresiduecombinations dictatepeptidepresentation by MHC class H molecules. PEPTIDES 15(4) 583-590, 1994.--Peptides encompassingthe core hen egg lysozyme HEL(52-61) peptide elongated or not and substituted or not with natural and unnatural amino acids were used to find a peptide motif for binding to the major histocompatibility complex (MHC) class II 1-Ak. Using a T-cell recognition functional assay, nine out of 10 positions were found to be somehow involved in the I-Ak binding, and six out of 10 residues were involved in T-cell recognition. The deleterious effect of single substitutions could be rescued by changing peptide length and/or sequence. Thus, efficient binding to MHC class I1 molecules requires not only few anchoringresiduescorrectlyinterspaced, but a complex, nonrandom combination of residueswith appropriate orientation of the peptide backbone and some crucial side chains. Antigen peptide MHC class I1 TcR recognition Amino acid substitutions Nonnatural amino acids

Peptidebinding motif Hen egg lysozyme Structure-activity relationship

ABBREVIATIONS Amino acids--mAba, meta-aminobenzoic acid; pAba, paraaminobenzoic acid; Aib, a-aminoisobutyric acid; D-AIa; D-alanine; Ava, 5-aminovaleric acid; D-Leu, D-leucine; Nva, norvaline, Sar, sarcosine. Others--tBoc, tert-butyloxycarbonyl; BOP, benzotriazole-1-yl-oxy-tris(dimethylaminophosphonium hexatluorophosphate); DCM, dichloromethane; DIC, diisopropylcarbodiimide; DMF, dimethylformamide, DMSO, dimethylsulfoxyde; Fmoc, 9-fluorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; Mtr, 4-methoxy-2,3,6-trimethylbenzenesulfonyl; RP-HPLC, reverse phase-high performance liquid chromatography; TFA, trifluoroacetic acid. PEPTIDES have recently been highlighted by the crucial role they have in the regulation of the immune response. Two set of molecules ensure the specific recognition of a molecule by the immune system. The molecule recognized is called an antigen. The antibodies used by B lymphocytes as antigen-specific cell surface receptors bind directly to the antigen. The T-cell receptor (TcR) expressed at the surface of T lymphocytes cannot bind to the antigen. Instead, it binds to a bimolecular complex made of major histocompatibility complex (MHC) molecules and pep-

tides derived from proteolytic degradation of the antigen. These peptide-MHC complexes are expressed at the cell surface of antigen-presenting cells (APC). Recent studies have revealed that peptide binding to MHC molecules occurs and participates in the maturation of MHC molecules [see (16) for review]. The main characteristics of the MHC molecules are their high polymorphism localized in the peptide binding groove domains. As a result, each MHC molecule can bind to its own set of peptides. Two structural and functional subsets of MHC molecules, class I and class II, coexist. MHC class I molecules are made of one polymorphic heavy chain and the monomorphic /32-microglobulin. They are expressed in most tissue and, complexed with a peptide, they activate CD8 ÷ T cells with cytolytic function. MHC class II molecules are made of two polymorphic cz and/3 chains. They are expressed only on a few specialized cells (macrophages, dendritic and B cells) and, complexed with a peptide, they stimulate CD4 + T cells. CD4 ÷ T cells play a major role in the initiation, amplification, and control of both humoral and cellular immune responses. Generation of antigen-derived peptides able to associate with MHC class II molecules requires the endosomal degradation of the antigen into peptides fitted to accommodate the peptide binding groove of MHC class II molecules [see (1 6) for review].

Present address: LEM, Institut de Biologie Structurale, 41 Avenue des Martyrs, 38027 Grenoble Cedex 1, France. 2 Present address: Laboratoire d'Immunochimie, DBMS/ICH, INSERM U238, CENG 85X, 38041 Grenoble Cedex, France. 3 Requests for reprints should be addressed to Chantal Rabourdin-Combe, CNRS-ENS Lyon, UMR 49, 46 Allre d'Italie, 69364 Lyon Cedex 07, France.

583

584

HERNANDEZ ET AL.

Delineating the biochemical constraints of peptide association with MHC class II molecules and identifying the side chains of the peptide residues involved in the recognition by the TcR is crucial for our understanding of how the T-cell repertoire is shaped by MHC-restricted thymus education, and for prediction of MHC class II-restricted T-cell epitopes useful for developing artificial vaccine and for generating antagonist peptides able to control autoimmune reactions. Indeed, such an approach has proven to be quite successful in predicting the peptides able to associate with MHC class I molecules after identification of allelespecific peptide motif for efficient binding (11,14,30). Identification of naturally processed peptides eluted from several MHC class II molecules has revealed that, in contrast to peptides eluted from MHC class I molecules, their length is longer and may vary from 12-24 residues (13,18,19,36,37). This is in agreement with the crystallographic structure of MHC class II molecules (8). The peptide binding cleft of MHC class II molecules, though very similar to that of MHC class I molecules as hypothesized earlier (9), is opened at both ends, thus allowing the binding of a peptide with a length exceeding that of the cleft. In addition, the naturally processed peptides eluted from class II molecules do not exhibit a clear allele-specific binding motif (13,18,19,36,37). We attempted to uncover the rules governing the association of peptides with the mouse MHC class II I-Ak molecule. The IA k binding and T-cell stimulation abilities of synthetic hen egg lysozyme (HEL) peptides encompassing the minimal HEL(5261) peptide core, substituted or not with natural and unnatural amino acids, were tested using a T-cell functional assay with two closely related HEL(52-6 l)-specific I-Ak-restricted 2A 11 and 3A9 T-cell hybridomas. METHOD

Synthetic Peptides and Peptide Analogues The peptides and peptide analogues were synthesized using Fmoc-substituted amino acids. Fmoc amino acids including Asn, Gln, Ser(O-tBu), Thr(O-tBu), Tyr(O-tBu), Lys(e-Boc), Arg(NgMtr), Asp(O-tBu), and Glu(O-tBu) were purchased from Bachem, Bubendorf, Switzerland, and Neosystem, Strasbourg, France. The Fmoc derivatives of Ava, mAba, and pAba (Aldrich, St Quentin Fallavier, France) were prepared by using Fmoc-Nhydroxysuccinimide (31). Solid-phase synthesis of the peptides on p-alkoxybenzyl alcohol resin (33,38) was done manually to facilitate a rapid synthesis and to ensure a complete coupling/deblocking for each cycle. Monitoring was done using the Kaiser test (22). Removal of the Fmoc group was effected by a 20% solution (v/v) of piperidine in DMF for 4 + 8 min. Resin washing was accomplished by repeated application of DMF, methanol, and DCM. Loading of first residue on resin was performed as described by Lu et al. (26). Couplings were mediated by DIC in either DCM, DMF, or mixtures thereof, depending upon the solubility of the particular Fmoc amino acids, and the mixture BOP/HOBt (1/1) was used for difficult couplings. Fmoc-Asn and -Gln were incorporated into the peptide with unprotected side chains in the presence of two equivalents of HOBt in DMSO-DMF or DMSODCM. The peptides were released from the peptide resins by treatment at 37°C for 7 h with 20 ml/g of a freshly prepared mixture of TFA, thioanisole, H20, and DCM (45/10/1/44). The peptides were precipitated from the cleavage solution and washed three times by the addition of tbutyl methyl ether (100 ml/g). The peptides were then dissolved in a 0.1% TFA solution (100 ml/g) and the pH adjusted to 6 with 1 M NaOH. The resin was separated by filtration and the filtrate was lyophilized. The crude

peptides were purified by semipreparative RP-HPLC (33,34). The purified peptides were characterized by analytical RP-HPLC, amino acid analysis, and fast atom bombardment-mass spectrometry using the facilities of the Service Central d'Analyse du C.N.R.S., Solaize, France. For use in the T-cell stimulation assay, peptides were first solubilized in either acid or basic conditions or in DMSO to obtain optimal concentrations and were next neutralized before addition to medium culture. The highest concentration of each peptide that could be used in the bioassay was limited by the peptide solubility and/or the cell toxicity of the preparation.

T-Cell Stimulation Assay Anti-HEL I-Ak-restricted 2A11 and 3A9 T-cell hybridoma recognizing the minimal HEL(52-61) peptide determinant were kindly provided by P. A. Allen (4). Specific antigen stimulation of the T-cell hybridomas was performed by cocultivating 105 hybridoma cells and 3 × 10 4 H-2 k CH27 B cells in the presence or absence of serial dilutions of peptides in a final volume of 200 ul. After incubation for 20 h at 37°C in 96-well microplates, IL-2 production in supernatants was measured in a biological assay using the IL-2-dependent CTL-L2 cell line (10). The growth of CTL-L2 cells was evaluated using the MTT assay (17). The results were expressed as stimulating peptide concentration (S.P.C.) where S.P.C. was the graphically determined peptide concentration giving 50% of the maximal T-cell stimulation. For competition experiments, serial dilutions of the competing peptide were mixed with 5 uM of HEL(52-61) before addition to CH27 and 3A9 cells. The results were expressed as the competing peptide concentration (C.P.C.) where C.P.C. was the graphically determined peptide concentration inhibiting 50% of the 3A9 T-cell stimulation induced by the 5 ~zM of HEL(5261 ). All the experiments were repeated several times with similar results. For substituted peptides found to be unable to compete for presentation of HEL(52-61) to 3A9 T cells, the highest peptide concentration that could be tested was usually limited by the peptide solubility. RESULTS

Extension of the HEL(52-61) Peptide to Its N- or C-End Results in Enhanced Binding to I-A k and~or Recognition by T Cells The extension of the HEL(52-61) sequence at its C-end by adding the HEL[Trp62-Trp63] results in the enhancement of presentation to both 2AI 1 and 3A9 T cells, because to induce 50% of the maximal T-cell stimulation sevenfold and 20-fold less HEL(52-63) peptide was required, respectively (compare S.P.C. ofpeptide 1 and 28, Table 1). The addition ofHEL[Aspas-Gly49SerS°-Thr5~] sequence at the N-end of HEL(52-61) also strongly enhanced the presentation to 3A9 T cells (100-fold enhancement, compare S.P.C. of peptide 32 with that of peptide 1, Table 1). Further elongation with HEL[Trp 62] residue at the C-end results in a similar stimulation efficiency (peptide 34, Table 1). In contrast to the C-end elongation, N-end elongation resulted in a peptide with a 20-30-fold reduced stimulating activity of 2A 11 T cells when compared to HEL(52-61) peptide (peptides 32 and 34, Table 1), thus confirming that 2A11 T cells preferentially recognized a shorter HEL peptide as initially observed by Allen et al. (3). It should be stressed that the S.P.C. values observed for the stimulation by HEL(52-61) peptide of 3A9 and 2AI 1 T cells were very similar to those previously reported by Allen et al. (3).

CRITICAL RESIDUE COMBINATIONS DICTATE PEPTIDE PRESENTATION

A Poly-Ala Peptide Containing the Three Putative I-A k Anchoring Residues Cannot Compete for Presentation of HEL(52-61) by I-A k Allen et al. (2) have previously proposed that, within the sequence of HEL(52-61), only three residues (Asp52, Ile58, and Arg 61) were acting as anchoring residues for binding to I-Ak. We then first attempted to determine whether the HEL(53-57, 59-60) residues are acting only as spacer residues to maintain the three postulated I-Ak anchoring residues at the appropriate distance. As previously reported for defining human MHC class II anchoring residues (20), a poly-Ala 10-mer peptide, includingthe three putative anchoring residues, was tested for its ability to compete for presentation of HEL(52-61) to 3A9 T cells. As expected, this peptide was not recognized by either T cells, but was also unable to compete with the parental peptide, indicating that it cannot bind to I-Ak molecules (peptide 24, Table 1). These data clearly indicated that Asp52, Ile58, and Arg61 residues, if maybe necessary, were not sufficient for HEL(52-61) binding to I-Ak.

Substituting Two Residues With an Amino Acid Analogue of Similar Length Resulted in the Impairment of HEL(52-61) Binding to I-A k Molecules In an attempt to determine if at least the HEL(54-55) residues were acting only as spacer residues, as postulated by Allen et al. (2), amino acid analogues with carbon backbone of similar length to that of the peptidic backbone of the dipeptide Gly54-Ile55were used. Substituted HEL(52-61 ) peptide with Ava 54-55,pAba 54-55, and mAba54-55(peptides 25, 26, and 27, respectively) were poorly or not recognized by 2A 1 1 and 3A9 T cells and were unable to compete with HEL(52-6 l) peptide for 3A9 T-cell stimulation (Table 1), indicating a very poor I-Ak binding ability of these peptide analogues. These data were a further indication that the I-Ak binding ability of HEL(52-61) peptide may not simply rely on few anchoring residues.

Single Residue Substitutions at Positions 53, 54, 55, 56, 58, 59, and 60 Can Strongly Impair HEL(52-61) Binding to I-A k Molecules This led us to reassess the role of every residue in HEL(5261) peptide presentation by I-Ak molecule by testing the effect of single substitutions with various natural and unnatural amino acids. The Tyr53 to Pro 53 (peptide 2), GIy54 to D-AIa54 (peptide 3), lie55 to D-Leu 55 (peptide 5), Leu 56 to either Aib56, D-Leu56, Phe 56, Nva 56, Thr 56, or D-AIa56 (peptides 6, 7, 8, 9, 10, and 14 respectively), lie5s to Phe 5s (peptide 20), Asn59to D-Ala59(peptide 21), and Ser6° to D-Ala6° (peptide 23) single substitutions resulted in peptides poorly (peptides 3, 5, 7, 9, 20, 21, 23) or not (peptides 2, 6, 8, 10, 14) recognized by 2A1 1 or 3A9 T cells and unable (or very poorly able) to compete with HEL(52-61) peptide for stimulation of 3A9 T cell (Table 1). Allen et al. (2) have previously reported that HEL(52-[Phe56]-61) peptide can compete with the parental peptide for presentation to T cells, but the inhibition was limited and not dose dependent, suggesting that this compound may not be a full inhibitor as indicated by our data. It should be stressed, however, that for a given position only some residues are forbidden for efficient I-Ak presentation to T cells. For example, Gly54 to Sats4 and the lie5s to Nva 5s substitutions hardly affected the ability to stimulate the 2A 1 1 T cells (peptide 4 and 17). Likewise, Ser6° to Ala6° substitution (peptide 22) did not strongly affect the binding onto I-Ak because the peptide was still very efficiently recognized by 2A l 1 T cells. This latter result showed a strong discrepancy with a previously published work (2), but in this previous report, the HEL(52-

585

[Ala6°]-61) peptide structure was not verified by fast atom bombardment-mass spectrometry. Taken together, our data indicate that all deleterious single substitutions primarily affect the binding ability of the HEL(52-61) peptide onto I-Ak molecules.

The I-A k Binding Ability of an HEL(52-61) Peptide With a Deleterious Single Substitution Can Be Rescued by Substituting One or Several Other Residues or by Extending Its Length The Ile58 to Nva 58 substitution in addition to the deleterious Leu 56to D-Ala56substitution was found to partly rescue the ability of the substituted HEL(52-61) peptide to bind to I-Ak and to be recognized, albeit moderately, by the two T-cell hybridomas (peptide 15 to be compared with peptide 14, Table l). Similarly, whereas the Leu 56 to Thr 56 change resulted in abrogation of binding to I-Ak (peptide 10), simultaneous additional residue substitution Asp 52 to T h rs2, Tyr 53 to Asn53, and Ile55 to Va155 restored the ability to bind to I-Ak molecule, as shown by strong ability to compete with HEL(52-6 l) peptide for recognition by 3A9 T cells (compare peptide l0 and 13). However, the restoring effect on I-Ak binding is limited to some residue combinations because, for example, a single Tyr 53to Asn 53and a Gln 57to Ala57 additional substitution could not rescue the detrimental effect of the Leu 56 to Thr 56 substitution (compare peptides 1 1 and 12 with 13). The extension ofHEL peptides with two more residues HEL[Trp62-Trp63] to their C-end was also able to restore some partial or even full I-Ak binding capacity, as shown by the ability of peptides with substitutions with Leu 56 to Thr 56, lle58 to Leu 58, or lle58 to Nva 58 to inhibit HEL(52-61) presentation to 3A9 T cells and/or to stimulate 2AI 1 and 3A9 T cells (peptide 29, 30 and 31 respectively, to be compared with peptide 10, 19, 17). Likewise, the Ile58 to Nva58-substituted HEL, when extended to its N-end by adding the HEL(48-51 ) sequence, recovered a good ability to stimulate the 3A9 T cells (peptide 33 to be compared with peptide 17); this stimulation efficiency was maximalized when the peptide was also extended at its C-end with HEL[Trp 62] (peptide 35). The apparent conflicting results obtained in this last experiment when studying the ability to stimulate 2A11 T cells were due to the fact that elongation ofHEL(52-61 ) peptide at its N-end strongly diminished its potency to stimulate 2A 11 T cells [(20) and see above].

Single Residue Substitutions at a Few Positions Directly Affect HEL(52-61) Recognition by Either 2All or 3A9 T Cells The comparison of the ability of I-Ak binder-substituted peptides to be recognized by 2A 11 and 3A9 T cells gave some information on residues likely to be involved in contacting the TcR of these two related but independent T-cell lines. On one hand, the Gly54 to Sat 54 change (peptide 4) induced a limited eightfold reduction in the recognition by 2A 1 1 cells but completely abolished recognition by 3A9 T cells (more than 400fold reduction). Similarly, the Ile58 to Nva 58 or to Leu 58 change moderately affected the recognition by 2 A l l cells (6-40-fold reduction) (peptide 17 and 19), whereas recognition by 3A9 T cells was abolished (more than 400-fold reduction). However, lle58 may also be involved in 2A1 1 T-cell recognition because the change to Phe 58 abrogated 2A 11 T-cell stimulation, although the substituted peptide was still able to compete with HEL(5261) for recognition by 3A9 T cells with a C.P.C. similar to that of HEL(52-[Leu58]-61), which was well recognized by 2Al 1 T cells (peptide 20 and 19, respectively). On the other hand, the Gin 57 to Glu 57 change (peptide 16) strongly decreased the recognition by 2A l 1 T cells (200-fold reduction) without affecting the recognition by 3A9 T cells (l.3-fold reduction). These data

586

H E R N A N D E Z ET AL.

TABLE 1 ABILITY OF NATURAL AND SUBSTITUTED HEL PEPTIDES TO STIMULATE 2AII AND 3A9 T CELL HYBRIDOMA OR TO COMPETE FOR THE PRESENTATION OF HEL (52-61) PEPTIDE BY I-Ak MOLECULES TO 3A9 T CELLS No. HEL

2All S.P.C.*

~ptide ~quence

3A9 S.P.C.

(HEL52-61) C.P.C.t

48 -49 -50 -51 -52 -53 -54 -55 -56 -57 -58 -59 -60 -61 -62 -63 Asp-Gly-Ser-Thr-Asp-Tyr-Gly-Ile-Leu-Gln-Ile-Asn-Ser-Arg-Trp-Trp

1 2 3 4 5 6 7 8 9 10 11

--

Pro

--

--

DAIa

--

--

Sar DLeu Aib DLeu Phe Nva Thr

--

Asn

--

--

Thr

Asn

--

Val

12 13

5 nM n.s.$ 150 uM 40 nM 700 #M n.s. 150 #M n.s. 1 gM n.s. n.s.

Thr Thr

Ala

Thr

14

DA1 a

15 16 17 18 19 20 21 22 23 24

oAla

Nva

Glu

Nva Val Leu Phe pal a Ala -DAla - --

Ala

Ala

Ala

Ala

Ala

-- A l a

Ala

--

n.c.§ n.c. n.c. n.c. n.c. n.c. n.c. n.c. n.c.

n.s.

n.s.

n.c.

n.s.

n.s.

I #M

n.s.

n.c.

n.s.

--

600 nM n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s.

8 #M 1 izM 30 nM 150 nM 200 nM n.s. 4 #M 150 nM 90 jzM n.s.

250 tsM 800 nM n.s. 6 uM n.s. n.s. n.s. 30~M n.s. n.s.

3 #M 80/~M 100 ~zM n.c. n.c. n.c.

25

Ava

90 # M

n.s.

n.c.

26

pAba

90 # M

n.s.

n.c.

27

mAba

90 # M

n.s.

n.c.

28 29

Thr

0.7 n M

30 n M

n.s.

n.s.

9 #M

30

Leu

4 nM

n.s.

3 ~M

31

Nva

2 nM

40 n M

32 33 34 35

Nva Nva

150 nM 10 ~M 100 nM 300 nM

6 500 6 6

nM nM nM nM

* Stimulating peptide concentration, see the Method section. t Competing peptide concentration, see the Method section. $ Nonstimulating at a concentration of at least: 60 ~M (peptide 4, 8, 11, 14, 29, 30); 100 ~M (peptide 2, 9, 12, 17, 21, 23, 24, 25, 26, 27); or 250 t~M (peptide 3, 5, 6, 7, 10, 13, 19, 20). § Noncompeting at a concentration of at least: 60 ~M (peptide 8, 11, 14); 100 #M (peptide 2, 9, 12, 21, 23, 24, 25, 26, 27); or 250 uM (peptide 3, 5,6,7, 10).

would suggest that Gly 54 and Ile 5s H E L residues may directly contact the 3A9 TcR and that Gln 57 and Ile 5s residues may contact the 2A 1 1 TcR. Finally, the single Leu 56 to Thr 56 change in HEL(52-63) completely abrogates the stimulation o f both 2A11 and 3A9 without inhibiting its ability to bind to I-A k molecule (peptide 29, C.P.C. o f 9/~M), confirming that Leu 56 is involved in the recognition o f the peptide by the TcR o f both T-cell hybridomas, in agreement with previous reports (2,3). DISCUSSION Various synthetic peptides encompassing the HEL(52-61) core sequence substituted at different positions with natural and

unnatural a m i n o acids were used to determine the role o f every residue involved in the binding to I-A k M H C class II molecule and in the recognition by HEL(52-61)-specific T cells. F r o m the inability o f at least one substituted peptide to c o m p e t e for the presentation o f HEL(52-61 ) peptide to 3A9 T cells, the Tyr 53, Gly 54, Ile 55, Leu 56, Ile 5s, Asn 59, and Ser 6° residues were found to be involved s o m e h o w in the binding o f this peptide to I-A k molecule. This result was not expected, because in the original work o f Allen et al. (2), only Asp 52, Ile 58, and Arg 61 were ascribed as I-A k binding residues. Furthermore, the Trp 62 a n d / o r T I p 63 residues are also likely to be involved in the I-Ak binding o f HEL(5263) because this 12-mer peptide exhibited an increased affinity

CRITICAL RESIDUE COMBINATIONS DICTATE PEPTIDE PRESENTATION

towards I-Ak. Both T-cell hybridomas required less amount of this peptide than that of its HEL(52-61) 10-mer counterpart. For example, when a Leu 56 to Thr 56 deleterious residue substitution was made, the longest peptide was still able to bind to IA k. Likewise, Ser5° and/or Thr 51 residues can also be involved in HEL(50-61) binding to I-Ak molecules because HEL(50[Phe56]-61)-substituted 12-mer peptide does bind to I-Ak molecule, as previously reported by Babbitt et al. (5), whereas its HEL(52-[Phe56]-61) 10-mer counterpart does not compete with HEL(52-61 ) binding and presentation to 3A9 T cell (see peptide 8, Table l). Finally, when studying the ability of HEL peptides to strongly associate with I-Ak molecules to render them SDS stable in the cold, Thr 5j and Trl062were found to act as stabilizing residues (5). Thus, as summarized in Table 2, within the HEL(50-63) sequence all the residues but one (Gln57) could be demonstrated to be somehow involved in the binding of this peptide to the IA k molecule. However, it should be stressed that, at position Gln 57, the effect of only two substitutions [Glu57, this study, and Ala57, (2)] has been explored. Similar data were obtained by Kurata and Berzofsky (23) when studying the interaction between a sperm whale myoglobin peptide with I-Ed molecules. These authors interpreted their findings by proposing that a given peptide may bind in more than one way to the same MHC class II molecule. Recently, Boehncke et al. (7) have described some dominant negative effects of amino acid side chain substitutions on peptide binding to I-Ed molecules. However, our data show that the loss of binding ability of HEL(52-61) peptide to I-Ak molecule due to a single residue substitution can be rescued either by substituting one or several others residues, or by increasing the length of the peptide. We then propose that efficient peptide binding to I-Ak molecule does not simply rely on the presence of few invariable dominant anchor residues within the peptide sequence, as suggested by the description of few putative MHC class II allelespecific peptide binding motifs ( 12,18,20,29). Rather, only some residue combinations will result in a peptide structure, allowing

587

a favorable conformation of the peptide backbone and the appropriate degree of freedom of some critical residue side chains for efficient anchoring in the groove of the MHC class II molecule. For any position within a peptide sequence, a forbidden residue substitution may be found, thus excluding any residue to act as a pure spacer residue. This may explain why the search of MHC class II allele-specific peptidic motifs for binding to MHC class II molecules has been so poorly rewarded (13,15,19,29,36). Interestingly, a recent reappraisal of the role of the peptide residues other than the main anchor ones in the binding to H-2Kb mutants has led to a very similar hypothesis on how a peptide can bind to MHC class I molecules (36). The data obtained by substituting at position 53, 54, 55, 56, 59, and 60 with amino acid analogues known to introduce strong local conformational restrictions fit to our proposal. Peptides 2 (Pro53), 3 (D-AlaS4), 5 (D-Leu55), 6 (Aib56), 7 (D-keu56), 14 (DAlaS6), 21 (D-AlaS9), and 23 (D-Ala6°) were much less or not at all efficient in stimulating 2 A l l T cells (800- to 140,000-fold less for compounds 3, 5, 7, 21, 23; no stimulation for 2, 6 and 14) and not efficient in stimulating 3A9 T cells. These results must be correlated to a decrease in I-Ak binding because they did not compete with HEL(52-61) peptide for stimulation of 3A9 T cells. Again, this shows that the previously so-called spacer and T residues (2) are also important for binding to I-Ak. As the corresponding L-Ala-monosubstituted analogues were able to stimulate the 2A11 T cells or to compete with HEL(52-61) (2), it is unlikely that the replaced side chains make crucial contact with some I-Ak residues. Instead, these results might be due to indirect effects, either conformational, disturbing the local orientation of the peptide backbone, or steric. The side chains of D-Ala, D-Leu, and Aib (Aib possesses two side chains) [Fig. l(b)] having a different orientation compared to the natural L-amino acids might make deleterious contacts with some I-Ak residues. These could not be accommodated without a reorientation of the peptide within the binding groove, and such an orientation would not be possible without loss of binding except if it is balanced by another re-

TABLE 2 H E L R E S I D U E S I N V O L V E D ( D I R E C T L Y O R I N D I R E C T L Y ) I N B I N D I N G O F HEL(50-63) P E P T I D E T O I-A k M O L E C U L E A N D R E S I D U E S D I R E C T L Y O R I N D I R E C T L Y I N V O L V E D IN R E C O G N I T I O N BY 2 A l l A N D 3A9 H Y B R I D O M A T CELLS

Residues Involved

In binding to I-Ak

50 -51 -52 -53 -54 -55 -56 -57 -58 -59 -60 -61 -62 -63 Ser - Thr-Asp- Tyr- Gly - I i e - Leu- Gin- I i e - A s n - Ser- Arg- Trp - Trp

(C) (C)

C

C

In recognition by 2AI 1 T cell

C

C

In recognition by 3A9 T cell

C

-

C

C

C

-

C

-

C

C

C

C

C

-

C

C

C

C (C) (C)

C critical residue; (C) critical residue only inferred from data presented in this paper or from the literature.

References

aa 50/51, inferred from (23-25) aa 52, (21) aa 53, 54, 55, 56, this paper aa 58, (21) & this paper aa 59, 60, this paper aa 61, (21) aa 62/63, inferred from (24) & this paper aa 52, (20, 23) aa 53, (20) aa 56, (20, 21) & this paper aa 57, (21) & this paper aa 58, this paper aa 52, (20, 23) aa 54, this paper aa 56, (20) & this paper aa 58, this paper

588

HERNANDEZ ET AL.

(OC) ~ql0

H2N

pAI~.

mAba

Ava

b)

H2N-~c/COOH J ~ H CH3 Ah

H2N'~c/COOH J ~ H3C H DAla

H2N.~cf COOH I ~ H3C CH3 bib

H3~ HN-~cH2.,,'COOH Sat

c) H2N--~H--COOH [3~H2

H2N--~H--COOH ~H--CH3

H2N--~H--COOH ~H2

CH3

CH~

CH3

Leu

lie

Nva

H2N--~H--COOH [3~H--CH3

Val

FIG. 1. Structure of some amino acids used in this study. (a) Amino acids substitute for a dipeptide: atoms equivalent to the dipeptide backbone atoms are indicated [a, C(O), N(H)]. pAba possesses a structure similar to a cis peptide bond; mAba is one atom shorter than a dipeptide backbone; Ava is also called carba(Gly-Gly)(CO-NH replaced by CH2CH2). (b) Structure of alanine derivatives and of sarcosine (N-methylGly). (c) Structure of close residues, Leu, Ile, Nva, and Val. orientation conferred by a substitution in another position (for example, Ile58 to Nva 58 in addition to Leu 56 to D-AlaS6). Replacement of the Tyr53 residue by a proline (peptide 2) abolished I-Ak binding, As the side chain of Tyr 53 is considered to point toward the TcR (2,3,5), this result might be explained by the Nsubstitution of proline, conferring local constraint of the backbone as well as steric hindrance and impossibility of hydrogen bonding. This would not permit the crucial adjacent I-Ak binding Asp52 to be correctly positioned. The same reasoning can be made for the peptides 25, 26, and 27, where the residues Gly54 and Ile55 were replaced by a pseudodipeptide (Fig. 1). mAba and pAba bring conformational constraint in this region of the peptide; on the contrary, Ava is more flexible than a dipeptide. They all lack the lie side chain and, more importantly, a peptide bond, thus lacking the possibility of hydrogen bonding at this level. These characteristics might explain that these compounds bind very poorly to 1-Ak [they are very poorly recognized by 2 A l l T cells and do not compete for stimulation of 3A9 T cells with HEL(52-61) peptide]. Altogether, these results suggest that the backbone of these residues makes close contact with residues of the I-Ak binding groove. Such an assumption is supported by the recent determination of the crystalline structure of the human MHC class II HLA-DRI (8,9). The backbone of the bound peptides was shown to contact conserved hydrogen bonding residues along the a helices. In HLA-DR1, one of these conserved residues, Ash62, seemed to hydrogen bond with the antigenic peptide by its 3,-amide group. Interestingly, it seems to correspond to Asn66 of I-Ak, which is considered to be an antigen contact residue because its replacement by an alanine abolished the presentation of HEL(46-61) (32). This would suggest that the lack of a hydrogen bond is sufficient to loosen binding to I-Ak. There is also evidence that some peptide side chains are likely to truly contact the I-Ak molecule and thus contribute to the binding of HEL(52-61) peptide. For example, substitutions of Leu 56 with amino acids not able to introduce, per se, an important conformational change of the peptide, such as Phe 56(peptide 8), Nva 56 (9), and Thr 56 (10), resulted in peptides poorly or not

stimulators of the two T cells. Only peptide 9 kept some stimulatory activity for 2A 11 T cells (200 times less well than the unsubstituted peptide). Its Nva 56 residue has a side chain structure very close to that of Leu with the same length but lacking the 3,-methyl group [Fig. l(c)]. This indicates that this methyl makes a crucial contact and confirms the great specificity already observed at this position (2). The lack of recognition by T cells primarily correlates with the inability to bind to I-Ak because none of these compounds was able to compete with HEL(52-61). A similar reasoning can be made for Ile5s previously considered as I-Ak binder because Ala58 substitution abrogates the binding (2). Although the Nva 58-, Va158-, Leu 58-, and Phe58-substituted peptides (peptide 17, 18, 19, and 20, respectively) were still able to bind to I-Ak, their competing activity decreases when the size of the side chain increases (Nva, Val < Leu < Phe). Accordingly, the enhancing effect on binding to I-Ak of elongating Leu 56to Thr 56 or to Phe 56, and lle58 to Leu 58 substituted peptides to 12-mers at the N- or C-terminus (see above) would be to give more freedom to the peptide to choose a different orientation, releasing deleterious contacts of Thr 56, Phe 56, and Leu 58 with I-Ak molecule. This reorientation would be destabilizing for shorter analogues. To map the peptide residues directly involved in the recognition of the HEL(52-61)-I-Ak complexes by the TcR, two independent l-Ak-restricted T-cell hybridomas recognizing the same minimal HEL(52-61) core peptide (3) but expressing different TcR (21 ) were used. The quantitative analysis of the effect of some residue substitutions, which, per se, does not grossly affect the peptide's ability to bind to I-Ak molecule, allows the identification of Leu 56, Gln 57. and lle5s as critical residues for recognition by 2A 11 T cells, and Gly54, Leu56, and Ile58 as critical residues for recognition by 3A9 T cells. As summarized in Table 2, some of these residues have been previously identified as well as two others (Asp52 and Tyr 53) (2,3,5). Thus, three residues would be "seen" by both TcR, interspaced with one residue "seen" only by one of the TcR (Table 2). In addition, the side chain of the lle58 residue would be "seen" differently by the two TcR because they can accommodate different side chain substitutions. In summary, within the HEL(52-61), six out of 10 residues are critical for recognition by at least one TcR. All those six residues may have their side chain pointing out of the I-Ak peptide binding cleft to have a chance to contact at least one TcR. But, as pointed out above, five out of six of these residues are also influencing the peptide binding ability to I-Ak molecule. These apparent conflicting data could be reconciled by considering that some of these five residues might only indirectly play a role in the binding to I-Ak by allowing some other critical residues having their peptide backbone and/or their side chains available for efficient anchoring in the peptide binding cleft of the MHC class 1I molecule, as proposed above. Alternatively, but not exclusively, some of these side chains may not directly contact the TcR but may allow a specific local configuration of other surrounding residues, enabling their side chains (and/or their peptide backbone?) to functionally interact with the TcR. The Leu 56 and Ile58 are examples of residues likely to be only indirectly involved in the recognition by a TcR. The Leu 56 residue has been shown to be essential for T-cell recognition (2,3), but as discussed above, we favor that it is a true I-Ak contact residue. In this case, changing Leu 56 to Thr 56 (peptide 29) or to Phe 56 (3) in long 12-mer peptides that can be accommodated by I-Ak due to the length effect could force the peptide to reorient within the peptide binding groove, abolishing recognition by the TcR. The lle5s residue also appears to be important for T cell

CRITICAL RESIDUE COMBINATIONS DICTATE PEPTIDE PRESENTATION stimulation because compounds 17 (NvaS8), 19 (Leu58), and 20 (Phe 58) did not stimulate the 3A9 T cells and peptide 20 was not recognized by 2A 11 T cells. The 3A9 T cells are more sensitive to a change in position 58 than 2A11 T cells. A modulation of the recognition was observed with peptides 17 (Nva 58) and 18 (ValS8), which were only 10 times less stimulatory than the parent peptide. Nva and Val are structurally close to lie, the former being as long but lacking the/3-methyl of Iie, and the latter being one methylene shorter but possessing this ¢3-methyl [Fig. l(c)]. Our results suggest that this ~-methyl is crucial for recognition by the 3A9 T cells, indicating a very precise interaction. Moving this methyl from ~- to y-position, as in Leu [Fig. 1(c)], abolished the stimulation. Although much less pronounced, a similar modulation was observed with the 2A 11 T cells but in an opposite way, as peptide 17 is fivefold more active than peptide 18. We favor that Ile 5s is a true I-Ak contact residue (see above) and then that the observed effect on T-cell recognition is indirect. Indeed, Leu 58 (peptide 19) and Phe 5s (peptide 20) substitutions resulted in peptides with comparable low competing activity whereas peptide 20 is more than 1000 times less stimulating for 2A11 T cells than peptide 19. If we consider that Ile~s makes a very precise contact with I-Ak, its change would result in a different orientation of the TcR contact residues not recognized anymore. Elongating the peptide towards its C-terminus attenuated the effect of substituting Ile 5s. Compound 30 (Leu sS) could still not stimulate 3A9 T cells, but was competing 26 times better than its shorter counterpart 19. This increase in I-Ak binding correlates with the 50 times better activity on 2A 11 T cells. Surprisingly, the NvaSS-substituted peptide 31 was shown to stimulate the 3A9 T cells at a level similar to that of its unsubstituted counterpart (peptide 28). The N-lengthened peptide HEL(48-[NvaSS]-61 (peptide 33) was also able to stimulate the 3A9 T cells but 83 times less efficiently than the parent compound 32 (a similar effect was observed for 2A11 T cells), confirming the influence of this residue. Only the presence of at least TIp 62 was able to compensate the adverse effect of Nva 58, as HEL(48-[Nva58]-62) is as active as its parent 34. Again, we can postulate that the addition of TIp 62 would modulate the reorientation of the peptide due to the Nva 58 substitution, thus restoring appropriate orientation of some crucial T-cell contact residues. The Gly 54 residue could be considered as likely contacting a TcR. The compound 4, where Gly 54 was replaced by a special residue, sarcosine, or N-methyl glycine [Fig. 1(b)], stimulated the 2A 1 1 T cells still efficiently (only eightfold less well than the parent peptide), indicating that it binds to I-A k. However, it did not stimulate 3A9 T cells, suggesting that this residue is important for recognition by the 3A9 TcR. As has been discussed above for binding to I-Ak, several reasons can be proposed. The presence of a methyl group on this peptide bond would introduce conformational restriction, resulting in an orientation of the TcR contact residues not recognized by the 3A9 T cells. Alternatively, this methyl group would have a steric effect by making a deleterious contact with a TcR residue, or it would suppress an important hydrogen bond between the NH ofglycine and a TcR residue, making Gly 54 an essential TcR contact residue. It is unlikely that the methyl group makes a contact with an I-A k residue, resulting in a reorientation of the peptide in the binding groove because, in this case, a worse effect on presentation to 2AI 1 T cells would be expected as Gly 54 is adjacent to the crucial Tyr 53 residue [(20) and see above].

589

Among the natural HEL peptides used in this study, three of them [HEL(52-63), HEL(48-61), and HEL(48-62)] have been described to be naturally processed peptides (28). Those peptides differ dramatically in their ability to stimulate 2A 1 l and 3A9 T cells, HEL(52-63) being the best stimulator for the former and HEL(48-61) or HEL(48-62) being the best stimulator for the latter. This raises the possibility that the two independent T-cell clones that give rise to these two Tcell hybridomas may have been originally stimulated only by their respective best stimulatory HEL peptides. Indeed, it is striking that when I-A k and invariant chain transfected L cells were used as APC to present HEL to 2A11 and to 3A9 T cells, only the latter can be efficiently stimulated, whereas when using CH27 B cells as APC, both hybridomas are efficiently stimulated [(35,36); Gerlier, unpublished data]. Because HEL processing by L cells is not optimal, this result can reflect the production of only the most abundant longer HEL peptides (28) or a specific proteolytic defect necessary to generate the shorter peptide. Two I-Ak-restricted T-cell hybridomas recognizing the HEL(35-45) core peptide also have been reported to differ in their ability to recognize peptides of different lengths (26). Therefore, the generation of several related complexes formed from a given MHC class II allele combined to the same related antigen peptide differing only by their length should increase the T-cell repertoire responding to the same region of an antigen. In conclusion, by testing the effect of various residue substitutions and peptide elongation on the ability of a single antigen peptide to bind to a given MHC class II molecule and to be recognized by antigen-specific T cells, most of the antigen peptide residues were found to be somehow involved in the binding to MHC class II molecule and more than half of them to be somehow involved in a T-cell recognition process. This strongly suggests that binding of antigen peptide to MHC class II molecules does not solely rely on few dominant anchor residues, but rather on critical residue combinations, resulting in a favorable conformation of the peptide backbone and in an appropriate orientation of the side chain of some critical residues. Complementary studies where the effect of residue substitutions will be tested using a naturally processed peptide such as the HEL(52-63) are required to ascertain the results obtained with the short HEL(52-61) peptide. Crystallographic data of complexes made from a given MHC class II molecules combined with the various substituted peptides will also allow determining which (and how) antigen peptide residue(s) truely contact(s) the MHC class II and which can contact a TcR when the peptide-MHC class II complex is stable. However, only the determination of the kinetics of peptide binding with purified empty MHC class II molecule in physicochemical conditions closed to the physiological situation, namely, just after invariant chain dissociation in an acidic pH, will give the final clue on the most favorable peptide conformation for binding to MHC class II molecules.

ACKNOWLEDGEMENTS The authors thank M. C. Trescol-Birmont, I. Fugier-Vivier, K. Brlorizky, G. Varior-Krishnan, C. Gimenez, and D. Naniche for helpful discussions, and L. Ettouati for synthesizing the peptide 15. This work was supported in part by grants from INSERM (C.R.-C. 920611), ARC (C.R.-C. No. 6108), E.D.I.M. (C.R.-C. and J.P.), and Rrgion Rh6neAlpes (J.P.).

590

H E R N A N D E Z ET AL.

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