A R P S Q R H G. *Acl-1 1[4Y: Ac A S. Y R P S Q R H G. Ac A A Q K R P A A A AAA. Ac3.5.6. AcA A Q A R P A A A AAA. QAc3.5.6{4Y]:. Ac A A Q Y R P A A A A A.
Proc. Natl. Acad. Sci. USA Vol. 91, pp. 767-771, January 1994 Immunology
Minimum structural requirements for peptide presentation by major histocompatibility complex class II molecules: Implications in induction of autoimmunity ANAND M. GAUTAM*t, CHRISTOPHER B. LOCK*, DAWN E. SMILEKt, CECELIA I. PEARSON*, LAWRENCE STEINMAN§, AND HUGH 0. MCDEVITT*¶ *Departments of Microbiology and Immunology, §Neurology and Neurological Sciences and Medicine, 94305; and tImmuLogic Pharmaceutical Company, Palo Alto, CA
IStanford University School of Medicine, Stanford, CA
Contributed by Hugh 0. McDevitt, August 26, 1993
ABSTRACT The precise mechanisms of failure of immunological tolerance to self proteins are not known. Major histocompatibility complex (MHC) susceptibility alleles, the target peptides, and T cells with anti-self reactivity must be present to cause autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a murine model of a human autoimmune disease, multiple sclerosis. In EAE, residues 1-11 of myelin basic protein (MBP) are the dominant diseaseinducing determinants in PL/J and (PL/J x SJL/J)Fl mice. Here we report that a six-residue peptide (five of them native) of MBP can induce EAE. Using peptide analogues of the MBP-(1-11) peptide, we demonstrate that only four native MBP residues are required to stimulate MBP-specific T cells. Therefore, this study demonstrates lower minimum structural requirements for effective antigen presentation by MHC class H molecules. Many viral and bacterial proteins share short runs of amino acid similarity with host self proteins, a phenomenon known as molecular mimicry. Since a six-residue peptide can sensitize MBP-specific T cells to cause EAE, these results derme a minimum sequence identity for molecular mimicry in autoimmunity.
recognition in vivo, using EAE as a measure of activation. The data presented in this communication show that a short peptide of six amino acids with a core of only five native Acl-11 amino acids induces EAE. The data imply that TCR recognition of a peptide-MHC class II complex is specific, and it may involve only a few residues of a peptide in a distinct conformation. It is this conformation of peptide and MHC class II molecules that may generate the rigid specificity for TCR recognition. The presence of similar amino acid sequences in environmental pathogens (e.g., viral) and host proteins is more common than originally supposed (7-9). Since a short sequence of amino acids with limited number of native MBP residues can cause EAE, a cross-reactive mode of T-cell stimulation may be one of the mechanisms in breakdown of self-tolerance in autoimmune diseases.
MATERIALS AND METHODS Mice. PL/J and (PL/J x SJL/J)F1 mice (8-12 weeks of age) were obtained from The Jackson Laboratory. Peptides. Peptides were synthesized by standard fluoren9-ylmethoxycarbonyl (Fmoc) chemistry using the Applied Biosystems 431A peptide synthesizer. Peptides were analyzed by HPLC and purified if necessary, and structures were confirmed by amino acid analysis and mass spectrometry. D amino acids and a-aminobutyric acid were bought from Bachem and Calbiochem. Ovalbumin amino acids 322-339 [Ova-(322-339) peptide] was biotinylated by a short-chain N-hydroxysuccinimide biotin (NHS-biotin) from Pierce as described previously (10). T-Cell Hybridoma Assay. The T-cell hybridoma 1934.4 was established from the Acl-11-specific T-cell clone PJR-25 (5). Activation of 1934.4 was assessed by measuring interleukin 2 production, using [3H]thymidine incorporation by the interleukin-2-dependent cell line HT-2 as described previously (10). Cell Surface Binding Assay. A total of 105 cells expressing AauA3 were incubated either with biotinylated Ova-(322339) peptide [Bio-Ova-(322-339); 15 ,uM] alone or with competitor peptides (see legends for concentrations) and BioOva-(322-339) (15 ,uM) together for 18-20 hr. After washing in cold phosphate-buffered saline/0.05% sodium azide/1% bovine serum albumin, cells were incubated (30 min at 4°C) with streptavidin Texas red (Pierce) and analyzed by flow cytometry as described (6, 9). To measure the relative amount of streptavidin Texas red bound, the mean fluores-
The recognition of peptides associated with major histocompatibility complex (MHC) class II molecules by T cells is a key event for mounting an effective immune response. MHC class II molecules bind peptides and present them to helper T cells (1-3). The acetylated myelin basic protein (MBP) peptide Acl-11 binds to the MHC class II molecule AauAp3u and induces experimental autoimmune encephalomyelitis (EAE) in PL/J and (PL/J x SJL/J)F1 mice (4). EAE is considered a useful model for the human demyelinating disease multiple sclerosis. We have previously identified key residues in Acl-11 for binding to AaUAI3U and for recognition by the T-cell receptor (TCR) (5). Recently, we have shown that a polyalanine peptide with only five native MBP residues is able to induce EAE in (PL/J x SJL/J)F1 mice (6). However, further analysis also showed that an 11-amino acid peptide, consisting mainly of alanines with only four native Acl-11 residues (Ac3.5.6; see Table 1), was able to induce T-cell hybridoma proliferation (6). These findings led us to propose that most side chains in MBP Acl-11 are essentially irrelevant for Aa"Af3pu binding, T-cell stimulation, and disease induction. Taking an approach of introducing either D amino acids or unnatural amino acids in place of L amino acids in MBP Acl-11 analogues, we now show that T cells (at least in this case) recognize only a short stretch of six or seven amino acids. More importantly, this stretch contains only four native MBP Acl-11 residues. We have also tested T-cell
Abbreviations: EAE, experimental autoimmune encephalomyelitis; MHC, major histocompatibility complex; MBP, myelin basic protein; TCR, T-cell receptor; Bio-Ova-(322-339), biotinylated ovalbumin peptide (residues 322-339). tPresent address: The John Curtin School of Medical Research, Human Genetics Group, The Australian National University, Canberra, ACT, Australia 2601.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
767
768
Proc. Natl. Acad. Sci. USA 91 (1994)
Immunology: Gautam et al.
Table 1. Summary of alanine-substituted peptides and their ability to stimulate the T-cell hybridoma 1934.4 and to bind to cell surface AauA,3U molecules Binding to T-celi stimulation EAE Acd-11 peptides with alanine substitutions AauAIBu Acl-11 +++ Ac A Z Q + B G &9 Q B L -!Ac3.4.5.6 + Ac A A Q2 B P A A A A A +++ ++++ Ac3.5.6.10 Ac A A Q A B E A A A AA ++++ + Ac3.5.6 ++++ Ac A A Q A B E A A AAA ++++Ac3.5 +++Ac A A Q A B A A A AAA NT A A AAA ++++ Ac5.6 Ac A A A A B NT Ac3.6 Ac A A Q A A A A A A A + Results are from ref. 6. Amino acids are written in the single-letter code. Underlined letters indicate native Acd-11 residues. NT, not tested.
cence was determined for at least 5000 propidium iodidenegative cells. Specific binding is expressed as percent inhibition of the control binding of Bio-Ova-(322-339) in the absence of competitors as before (6, 10).
A
3
Ac3.5.6:
Ala
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EAE Induction. EAE was induced as described (4, 6) by injecting MBP Acl-1l peptide emulsified in complete Freund's adjuvant subcutaneously at the base of the tail in a total volume of 0.1 ml. Two hundred nanograms of pertussis
Ala
Ala
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-
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D.Ala D.Ala
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Ac3.5.6 01 01.2 07 08.9.10.11 01 0 01. OVA 323-339
FIG. 1. T-celi response is abolished by introducing D amino acids from the amino but not the carboxyl terminus. (A) Amino acid sequence of peptide Ac3.5.6 is shown in the top row. Subsequent sequences show D-alanine (D.Ala) substitutions. (B) T-celi hybridoma 1934.4 stimulation. Change to D-Ala at positions 2 and 7 diminishes response to Ac3.5.6. (C) Cell surface binding assay: Inhibition of binding by D-Alacontaining peptides. Competitors in this experiment were used at 400 ,uM with Bio-Ova-(322-339) at 15 MM. Mean fluorescence intensity background and signal by BioOva-(322-339) were 1.2 and 8.4, respectively. Competition by peptides at a single dose is shown for brevity. All peptides have been tested (at various doses) at least three times in this assay with a similar pattern of inhibition.
encephalitogenic T cells, then a short peptide with a limited identity to MBP might induce EAE. Peptide analogues of Ac3.5.6 in which L-alanines were replaced with D-alanines at the amino and carboxyl termini in various combinations were synthesized (Fig. 1A). Replacement of an L amino acid with a D amino acid has a profound effect on peptide conformation by affecting the position of adjacent residues (11). These peptides were analyzed for T-cell hybridoma activation and MHC class II binding. Only peptides in which D-alanines were introduced at the carboxy terminus (not at the amino terminus) stimulated the T-cell hybridoma and bound to AaUAW (Fig. 1 B and C; compare peptides D8.9.10.11 and D1.2). Substitution with D-alanine at the seventh residue abolished binding and T-cell recognition (Fig. 1 B and C). Since most substitutions with D amino acids permitted stimulation of the Acl-li-specific hybridoma (though reduced in the case of the Di peptide), these results suggest that the TCR recognizes only six residues in MBP Acl-11 bound to AaUA3. To determine more precisely what Acl-11-specific T cells recognize in a peptide bound to AaUAW, we also synthesized peptides in which all of the alanines in Ac3.5.6 were substi-
toxin (JRH Biosciences, Woodland, CA) was injected intravenously at the time of immunization and again 48 hr later. Mice were examined daily for 30-40 days and were scored as follows: 1, limp tail; 2, partial hind-limb paralysis; 3, complete hind limb paralysis; 4, hind- and forelimb paralysis; and 5, moribund. The data are presented as cumulative incidence, calculated as total number of mice that showed signs of EAE at any point during the experiment.
RESULTS AND DISCUSSION Table 1 summarizes our previous results (6). Since peptides Ac3.5.6 (AcAAQARPAAAAA) and Ac3.4.5.6 (AcAAQKRPAAAAA) (see Table 1; bold letters show native MBP residues) were able to stimulate an Acl-11-specific hybridoma in vitro, and since peptide Ac3.4.5.6 was also able to induce EAE, we have chosen these peptides to further study T-cell recognition and mechanisms which may be involved in the initiation of an autoimmune disorder. Sequence identity between viral and bacterial proteins and mammalian proteins usually involves short stretches, 4-6 amino acids (6). If molecular mimicry (7-9) were to play a role in sensitizing D A *Acl-11 5 Ac3.4.5.6
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769
Proc. Natl. Acad. Sci. USA 91 (1994)
Immunology: Gautam et al.
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Ac3.5.6[4Y.6aa]
FIG. 2. T-cell stimulation and cell surface binding by peptides 6 to 11 amino acids in length. (A) Peptides with Lys (native residue) at position 4. 8aa, 7aa, and 6aa indicate peptides with eight, seven, and six amino acids, respectively. (B and C) In vitro stimulation of the Acl-il-specific T-cell hybridoma 1934.4 and cell surface competition assay, respectively, as described in the legend of Fig. 1. (D-F) Peptides with Ala at position 4 (Lys to Ala substitution), their ability to stimulate the Acl-il-specific hybridoma, and cell surface binding as assessed by competition with Bio-Ova-(322-339) (15 ,IM). (G-I) Peptides with Tyr at position 4 (Lys to Tyr substitution), their ability to stimulate the Acl-il-specific hybridoma, and cell surface binding, respectively. Mean fluorescence intensity backgrounds ranged from 1.0 to 1.2, and the signals given by biotinylated peptide were between 31.2 and 48.4. Large boxes in A, D, and G highlight the core region of the peptides, while the narrow boxes in the middle of the large boxes highlight the Lys, Ala, and Tyr residues in the core.
770
Immunology: Gautam et al.
tuted with an unnatural amino acid, a-aminobutyric acid (Abu). A peptide containing Abu residues at all positions except Gln, Arg, and Pro at positions 3, 5, and 6 bound AauAWu and stimulated the Acl-li-specific hybridoma (data not shown). Together these results demonstrate that the Acl-11-specific TCR recognizes a distinct but very short conformation generated by just a few of the native MBP residues bound to AaUA,W. The experiments above indicate that only a short core of native amino acids in a six- or seven-amino acid peptide segment is required to stimulate Acl-11-specific T cells. Therefore, we synthesized peptides of various lengths, 6-11 amino acids. Peptides were synthesized in which position 4 was Ala, Tyr, or Lys (the native residue) as shown in Fig. 2 A, D, and G. Tyr was chosen because it has been shown that this residue enhances peptide binding to AaUAJ3u beyond that observed for Ala at position 4 (12). Fig. 2 shows the T-cell hybridoma responses and cell surface AaUA3u binding when peptides with Lys (Fig. 2 A-C), Ala (Fig. 2 D-F), and Tyr (Fig. 2 G-I) at position 4 were used. As expected, peptides containing Lys at position 4 (4K peptides) (Fig. 2A) required high concentrations of peptides to stimulate the T-cell hybridoma (Fig. 2B). 4K peptides with fewer than eight amino acids marginally stimulated T cells. When tested for binding to cell surface AaUApu by their ability to compete with Bio-Ova-(322-339), these peptides competed very poorly (Fig. 2C). In contrast, when the peptides were synthesized with Ala at position 4 (4A peptides) (Fig. 2D), the T-cell hybridoma was stimulated by a peptide with only seven residues (Fig. 2E). The 4A peptides were also able to compete with Bio-Ova-(322-339), providing the length of the peptide was at least seven amino acids (Fig. 2F). The stimulation of the T-cell hybridoma was further enhanced by peptides with Tyr at position four (4Y peptides) (Fig. 2G). In this instance, even a six-amino acid peptide with Tyr at position 4 was able to stimulate the T-cell hybridoma, albeit at higher concentrations (Fig. 2H). This peptide also competed with Bio-Ova(322-339) for the cell surface binding of AauA,f& (Fig. 2I). To relate these findings to induction of autoimmunity, we next asked whether it is possible to induce AauA,3u-restricted T-cell responses in vivo with short peptides. Although peptides with Lys (a native residue) or Ala at position 4 stimulate the same T-cell clones, only peptides with Lys at position 4 induce EAE (5). Therefore, peptides with Lys at position four were .tested for their ability to induce EAE in (PL/J x SJL/J)Fl mice. Surprisingly, peptides seven and six amino acids in length were able to induce EAE in at least 30-40% and 15-20% of mice, respectively (Fig. 3). Despite the delay in onset of EAE in these mice, the clinical scores were identical to those obtained with 11-amino acid peptides. It is also interesting that the short peptides barely stimulated the T-cell hybridoma in vitro as shown in Fig. 2B. Therefore, the delayed induction of EAE in mice immunized with short peptides may indicate a slow sensitization of encephalitogenic T cells to threshold numbers in vivo. Prior to these results, 2,4-dinitrophenyl (DNP) heptapeptides containing L-lysine and L-alanine were the shortest peptides reported to be immunogenic (13). MHC class II molecules bind and present peptides to T cells of the helper subset (14-16). For MBP Acl-11 as a model peptide, we have now defined the minimum length requirements for effective peptide presentation by these molecules. These results have interesting implications for peptide presentation in the induction of autoimmunity. Molecular mimicry has been implicated as a mechanism for the induction of autoimmune disease (7-9). Viruses such as measles virus, Epstein-Barr virus, and hepatitis B v'irus have been shown to sensitize T lymphocytes to MBP in some subjects (17, 18). MBP and influenza virus share sequence homology (7). MBP is constitutively expressed in the central
Proc. Natl. Acad. Sci. USA 91 (1994)
w
Ac-1 1
w
60
-
Ac3.4.5.6[8aa]
a) C, -0
c
._
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-
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3
0
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Ac-1 1: Ac3.4.5.6: Ac3.4.5.6[8aa]:
Ac3.4.5.6[7aa]: Ac3.4.5.6[6aal:
25
30
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5
after immunization
Ala Ser Gln Ala Ala Gln Ala Ala Gln Ala Ala Gin Ala Ala Gln
4
Lys Lys Lys Lys Lys
5
6
Gin Arg Pro Arg Pro Ala Arg Pro Ala Ala
Arg Pro Arg
Arg
Aia
Gly la
Ala
Pro
FIG. 3. A six-amino acid peptide induces EAE. Groups of 8-10 mice were immunized for induction of EAE with Acl-11, Ac3.4.5.6, Ac3.4.5.6[8aa], Ac3.4.5.6[7aa], or Ac3.4.5.6[6aa]. Mice were monitored for up to 45 days for clinical disease. Data represent total number of mice pooled from at least two separate experiments. Mice with EAE from each group showed severe clinical signs with at least hind leg paralysis at some stage of the disease development.
nervous system and not in the thymus. T cells reactive to MBP epitopes presumably escape thymic clonal deletion and may remain in the periphery in a resting state. Under certain conditions (e.g., viral infection), these T cells may become activated and cause disease. Since a six-amino acid peptide with only five native residues can induce EAE, it is possible that a pathogen with limited homology to self proteins (i.e., five residues identical) may be able to sensitize autoreactive T cells. Earlier results indicated that several residues (e.g., Table 2. Sequence similarities between MBP-(1-6) and other proteins Source Sequence A S QKRP Native MBP-(1-6) A A QKRP EAE-inducing sequence A A QRRP Shallot virus X DNA sequence A A QRRP Saimirine herpes virus T L QKRP Hepatitis B virus open reading frame 1 N A QKRP Measles virus (strain Ip-3-Ca) M V QKRP Influenza A/swine/Tennessee/26/77 (HlNl) R P QKRP Epstein-Barr virus nuclear antigen 1 I C QKRP Japanese encephalitis virus Q D QKRP Herpes simplex virus 1 K R A A S T I A L A V
D P X A S E W A C H E
QKRP QKRP QKRP QRRP QKRP QKRP QKRP HKRP QKRP QKRP QKRP
Coxsackie virus B4 Mouse hepatitis virus Salmonella typhimurium
Thiobacillus ferroxidans F1Fo-ATPase Bacillus subtilis operon regulatory protein Escherichia coli plasmid ColB2 finO gene E. coli AroM protein Bracket fungus A-a Z 3 protein Yeast (Saccharomyces cerevisiae) LEU2 gene Yeast vacuolar PRCI gene Candida albicans repeat element Results of a protein data base search. Similar sequences from viruses, bacteria, and fungi are shown. A standard search in GenBank was conducted, using six amino acids on AXQKRP (X at position 2 represents any amino acid) and the FASTA method of Pearson and Lipman (19).
Immunology: Gautam et al. amidonitrobenzyl-Tyr-Gly) can be added to Acl-1l without diminishing AauAu binding or T-cell stimulation (5). Thus identity at only four residues might be sufficient. In a protein data base search, we found a surprisingly high incidence of QKRP or QRRP in various viral, bacterial, and fungal protein sequences (Table 2). We speculate that autoimmunity may develop when a fQreign agent (such as a virus) expresses proteins with relatively short sequence identity with host self proteins. A recent report shows that transgenic mice expressing MBP-(1-11)-specific TCRs develop EAE spontaneously in a dirty environment (20). These results provide further support for the involvement of environmental pathogens in induction of autoimmune diseases. Since there are many pathogens in the environment carrying short sequences similar to self proteins, cross-reactive induction of autoimmunity becomes an even more important consideration. Finally, in this communication we have determined minimum structural requirements for a peptide presentation and T-celi recognition by MHC class II molecules. We thank Prof. Gordan Ada for useful comments. This work was supported by an award from the National Institute of Allergy and Infectious Diseases to H.O.M. A.M.G. was funded by the National Multiple Sclerosis Society. C.B.L. was supported by the Juvenile Diabetes Foundation. 1. Babbitt, B. P., Allen, P. M., Matsueda, G., Haber, E. & Unanue, E. R. (1985) Nature (London) 317, 359-362. 2. Buus, S., Sette, A., Colon, S. M., Jenis, D. M. & Grey, H. M. (1986) Cell 47, 1071-1077. 3. Davis, M. M. & Bjorkman, P. J. (1988) Nature (London) 334, 395-398. 4. Zamvil, S. S., Mitchell, D. J., Lee, N. E., Moore, A. C.,
Proc. Natl. Acad. Sci. USA 91 (1994)
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9. 10. 11. 12.
13. 14.
15. 16. 17.
18. 19.
20.
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Kitamura, K., Steinman, L. & Rothbard, J. B. (1986) Nature (London) 324, 258-260. Wraith, D. C., Smilek, D. E., Mitchell, D. J., Steinman, L. & McDevitt, H. 0. (1989) Cell 59, 247-254. Gautam, A. M., Pearson, C. I., Smilek, D. E., Steinman, L. & McDevitt, H. 0. (1992) J. Exp. Med. 176, 605-609. Fujinami, R. S. & Oldstone, M. B. A. (1985) Science 230, 1043-1045. Jahnke, U., Fisher, E. H. & Alvord, E. C. J. (1985) Science 229, 282-284. Oldstone, M. B. A. (1987) Cell 50, 819-820. Gautam, A. M., Pearson, C. I., Sinha, A. A., Smilek, D. E., Steinman, L. & McDevitt, H. 0. (1992) J. Immunol. 148, 3049-3054. Degrado, W. F. (1988) Adv. Protein Chem. 39, 51-124. Wraith, D. C., Bruun, B. & Fairchild, P. J. (1992) J. Immunol. 149, 3765-3771. Levin, H. A., Levine, H. & Schlossman, S. F. (1970) J. Immunol. 104, 1377-1383. Brown, J. H., Jardetzky, T. S., Gorga, J. C., Stern, L. J., Urban, R. G., Strominger, J. L. & Wiley, D. C. (1993) Nature (London) 364, 33-39. Rudensky, A. Y., Preston-Hurlburt, P., Hong, S. C., Barlow, A. & Janeway, C. A. J. (1991) Nature (London) 353, 622-627. Jorgensen, J. L., Esser, U., Fazekas de St. Groth, B., Reay, P. A. & Davis, M. M. (1992) Nature (London) 355, 224230. Johnson, R. T., Griffin, D. E., Hirsch, R. L., Wolinsky, J. S., Roedenbeck, S., Lindo De Soriano, I. & Vaisbery, A. (1984) N. Engl. J. Med. 310, 137-141. Waksman, B. (1985) Nature (London) 318, 104-105. Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448. Goverman, J., Woods, A., Larson, L., Weiner, L. P., Hood, L. & Zaller, D. M. (1993) Cell 72, 551-560.
