Activation of autoreactive T cells is a crucial event in the pathogenesis of autoimmune diseases. Cross-reactivity between microbial and self Ags (molecular mimicry) is one hypothesis that could explain the activation of autoreactive T cells. We have systematically examined this hypothesis in experimental autoimmune encephalomyelitis using mice bearing exclusively myelin basic protein (MBP)-specific T cells (designated T+ α). A peptide substitution analysis was performed in which each residue of the MBPAc1–11 peptide was exchanged by all 20 naturally occurring amino acids. This allowed the definition of the motif (supertope) that is recognized by the MBPAc1–11-specific T cells. The supertope was used to screen protein databases (SwissProt and TREMBL). By the search, 832 peptides of microbial origin were identified and synthesized. Of these, 61 peptides induced proliferation of the MBPAc1–11-specific transgenic T cells in vitro. Thus, the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only two native MBP-residues were sufficient to activate MBPAc1–11-specific T cells in vitro, and experimental autoimmune encephalomyelitis could be induced by immunizing mice with a mimic peptide with only four native MBP residues.

Experimental autoimmune encephalitis (EAE)3 is an acute inflammatory demyelinating disease of the CNS. EAE is mediated by CD4+ cells that are specific for CNS Ags such as myelin basic protein, proteolipid protein, or myelin oligodendrocyte glycoprotein (reviewed in Ref. 1). The disease can be induced in experimental animals by immunization with CNS Ags in CFA followed by the i.v. injection of pertussis toxin (PT) or by adoptive transfer of activated CD4+ cells from diseased animals into syngeneic recipients (reviewed in 1). EAE is among the best characterized T cell-mediated autoimmune diseases and serves as an animal model for multiple sclerosis (MS). A crucial step in the pathogenesis of autoimmune diseases such as MS or EAE is the activation of autoreactive T cells. Abundant clinical (2), epidemiological (3), and experimental evidence (4, 5, 6, 7) link MS and other autoimmune diseases (reviewed in Refs. 8, 9) with infectious diseases, suggesting autoimmunity as a potential sequel of infection. Furthermore, in some transgenic animal models, EAE develops spontaneously only if the animals are kept in conventional facilities but not in germfree animals, indicating a pathogenic role for the presence of microbes (10, 11). Several mechanisms could lead from infection to autoimmunity, including the release of normally sequestered autoantigens through direct tissue damage (12) and the induction of proinflammatory cytokines or costimulatory molecules by microbial products such as LPS and lipoproteins (13, 14), toxins (15, 16), or CpG-rich oligonucleotides (17). The local inflammation induced by such factors facilitates the nonspecific recruitment of T cells including those specific for autoantigens expressed at the site of inflammation. Virally encoded superantigens have recently been implicated in the pathogenesis of MS (18). One attractive hypothesis is based on the concept that sequence similarity between microbial and self Ags (“molecular mimicry”) could activate autoreactive lymphocytes, thus enabling such cross-reactive lymphocytes to cause autoimmune damage in the host (19, 20). Supporting this hypothesis, several authors have reported on cross-reactive T cells that could recognize both a microbial peptide and a highly homologous self peptide (4, 21, 22). In several instances, autoimmunity was elicited by immunization with the microbial peptide (23, 24) albeit at much reduced incidence and severity (25) or only at significantly higher Ag doses (26) as compared with the self Ag.

More recently, it was demonstrated that individual TCRs could recognize different peptide/MHC complexes that do not show strong sequence homology (5, 6, 27, 28, 29, 30, 31, 32). Structural analyses have demonstrated that the antigenic peptide contributes little to the TCR-peptide-MHC interface. Furthermore, this interface shows a poor shape complementarity that could accommodate a wide range of different peptides, thus providing a structural base for the degenerate recognition of peptide-MHC complexes by individual TCRs (33). Consequently, two groups have demonstrated cross-reactivity for MBP-specific T cell clones from MS patients with many microbial ligands that were structurally unrelated to the MBP epitope recognized by those T cell clones (5, 6, 34, 35). Thus, it has become evident that simple sequence alignment will not suffice to identify microbial ligands for autoreactive T cells. In the EAE model, an improved method to identify microbial ligands for murine MBP-specific T cells was used: after careful definition of MHC and TCR contact residues within the immunodominant MBP epitope, database searches were performed that were based on these structural characteristics, allowing nonhomologous amino acids at the “non-contact-residues.” A number of microbial and viral peptides fulfilling the search criteria were identified, and some of these peptides induced EAE in mice (7, 24). However, this “knowledge-based” approach requires laborious analysis of the contact residues of an individual epitope with MHC and TCR. Therefore, we wished to examine an alternative approach to identifying microbial ligands for autoreactive TCRs. In earlier work, we had used the spot-synthesis technique for peptides (36, 37) to identify multiple ligands for mAbs (38). Here, we have used peptide spot synthesis for global amino acid replacements of the MBPAc1–11 epitope, which is immunodominant in mice of the H-2u haplotype. We identified 61 microbial mimic peptides that activated MBPAc1–11-specific T cells. Several of these peptides induced EAE in mice that are transgenic for a MBPAc1–11-specific TCR (11).

Mice transgenic for a TCR that recognizes MBPAc1–11 bound to I-Au (11) were crossed onto TCR α-chain knockout mice (39), resulting in mice carrying only αβ T cells specific for MBPAc1–11 (T+α) (40, 41), and were obtained from Dr. Juan Lafaille (Skirball Institute, New York, NY). Mice were bred at our animal facility in specific pathogen-free conditions and checked for TCR expression by flow cytometry with anti-Vβ8-PE (MR5–2, PharMingen, San Diego, CA) and anti-CD4-FITC Abs (GK1.5). All animal experiments were performed according to institutional and state guidelines.

Cellulose-bound peptides were prepared by automated spot synthesis (Abimed, Langenfeld, Germany; Software DIGEN, Jerini Biotools, Berlin, Germany) with the use of Whatman No. 50 cellulose membranes (Whatman, Maidstone, U.K.) as described before (36, 37). Peptides were N-terminally acetylated using acetanhydride and diisopropylethylamine. For synthetic reasons, the peptides contained an additional C-terminal glycine residue. Peptides were cleaved from the solid support by treating the cellulose with ammonia vapor for 5 h. Each spot was eluted in 200 μl double-distilled H2O resulting in an approximately 150–200 μM peptide solution. For titration experiments and in vivo analysis, peptides were conventionally synthesized according to standard Fmoc machine protocols with a multiple peptide synthesizer (Abimed). The following peptides were synthesized (given in single letter code, with Ac denoting N-terminal acetylation): MBPAc1–11 (AcASQKRPSQRSK); pep200 (AcANMQRQAVPTL; Escherichia coli, Salmonella typhimurium, Haemophilus influenzae, Buchnera aphidicola); pep378 (AcASMNRPNLVAL; Mycobacterium tuberculosis); pep383 (AcASMSRPVKQLK; E. coli, S. typhimurium); and pep387 (AcASQARQLADSY, E. coli). Purity of the peptides was determined by HPLC and composition monitored by MALDI-TOF mass spectroscopy.

Single-cell suspensions were prepared from spleens in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 50 μM 2-ME (complete RPMI, Sigma, St. Louis, MO) as described (14). For proliferation, cells were cultured in 96-well plates at 1 × 106/ml with 5 μl peptide spots (∼0.5–1.5 μM), with conventionally synthesized peptides at concentrations indicated, or with complete RPMI alone, at 37°C in 5% CO2. Proliferation was measured by an 18-h incorporation of 1 μCi [3H]thymidine on day 3. Stimulation indices (SI) were determined as cpm of peptides divided by cpm of cells cultured with medium alone (range for background values 1400–2100 cpm for the various experiments). SIs ≥15 were considered positive. For cytokine determination, cells were cultured in complete medium at 5 × 106/ml with 5 μl peptide spots, or at 1 × 106/ml for dose-response analysis with conventionally synthesized peptides as indicated. Supernatants were collected at 48 h for analysis by sandwich ELISA. IFN-γ, TNF-α, and TGF-β were determined with commercially available kits according to manufacturer’s instructions (Genzyme Diagnostics, Cambridge, MA). IL-4, IL-5, IL-10, and IL-2 were determined as described (42). The lower detection limit for each ELISA was as follows: IFN-γ, TNF-α, and IL-4, 50 pg/ml; IL-2 and TGF-β, 0.1 ng/ml; IL-5, 10 U/ml; and IL-10, 0.3 ng/ml.

Mice were injected s.c. at 2 sites at the base of the tail with 200 μg MBPAc1–11 or mimic peptides emulsified in CFA in a total volume of 0.2 ml. PT (200 ng; Life Technologies, Gaithersburg, MD) was injected i.v. 24 and 48 h after immunization. Age- and sex-matched control mice received PBS or CFA plus PT. Mice were examined every 1–2 days for clinical signs of EAE which was scored as follows: level 0, healthy; level 1, limp tail; level 2, partial hind leg paralysis; level 3, complete hind leg paralysis; level 4, front leg weakness; level 5, moribund. Data are represented as mean EAE of each group. Animals were sacrificed when their score reached 4–5, and their score was kept at 5 for the remainder of the experiment.

Peptides prepared by spot synthesis (37) were used for a substitution analysis of MBPAc1–11 in which each position of the peptide was substituted with all 20 naturally occurring amino acids. The resulting 220 peptides and synthesized spots of MBPAc1–11 were tested for induction of proliferation of T+α spleen cells in vitro (Fig. 1,A). SIs ≥15 were considered positive. The substitutional analysis identified the amino acid substitutions tolerated at each position of the peptide. This revealed the binding motif (supertope) and thus the structural requirements for T cell recognition for the transgenic TCR (Fig. 1,B). Arginine at position 5 (R5) could not be substituted with any other amino acid. At each of the other positions of the peptide, at least one substitution was tolerated (Fig. 1,A). Alanine at peptide position 1 (A1) could be substituted only by serine (A1S) and P6 could be replaced only by glutamine. S2, Q3, and S7 could each be replaced by several other amino acids, and positions 8–11 could be taken by any of the naturally occurring amino acids (Fig. 1 B). The supertope was used to screen the SwissProt and TREMBL databases (software ExPasy) (43), and 832 peptides of microbial origin were identified that contained the supertope.

FIGURE 1.

A, Substitutional analysis of MBPAc1–11 (AcASQKRPSQRSK). Each position of the epitope was substituted by all 20 naturally occurring amino acids. N-terminally acetylated peptides were prepared by spot synthesis and T+α T cells tested for proliferation at a peptide concentration of ∼1 μM. SI are shown in the figure. Dark boxes indicate SI ≥ 15. Values in the top line represent the wild-type (wt) peptide; all other values correspond to single substitution analogues. B, The “supertope” resulting from the substitution analysis, i.e., the amino acids allowed at each individual position of the 11-mer epitope. Bracketed residues indicate the allowed substitutions; X = all amino acids; braced residues represent all amino acids except those in the brackets.

FIGURE 1.

A, Substitutional analysis of MBPAc1–11 (AcASQKRPSQRSK). Each position of the epitope was substituted by all 20 naturally occurring amino acids. N-terminally acetylated peptides were prepared by spot synthesis and T+α T cells tested for proliferation at a peptide concentration of ∼1 μM. SI are shown in the figure. Dark boxes indicate SI ≥ 15. Values in the top line represent the wild-type (wt) peptide; all other values correspond to single substitution analogues. B, The “supertope” resulting from the substitution analysis, i.e., the amino acids allowed at each individual position of the 11-mer epitope. Bracketed residues indicate the allowed substitutions; X = all amino acids; braced residues represent all amino acids except those in the brackets.

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The 832 peptides containing the supertope were prepared by spot synthesis and assayed for the induction of proliferation in T+α spleen cells in vitro. Of the 832 microbial peptides, 61 induced proliferation of the T+α cells (SI ≥ 50; SI for MBPAc1–11 = 80). The microbial mimic sequences, listed in decreasing order of SI, and the organism(s) containing the corresponding protein are shown in Table I. The mimic peptides had anywhere from 5 to 9 amino acid substitutions compared with MBPAc1–11. There was no statistically significant correlation between the number of conserved amino acids and the SI (correlation coefficient, 0.08; p > 0.05). Therefore, other factors such as the topology of the peptide-MHC complex must determine the antigenic strength of the individual mimic peptides. Six peptides sharing only 2 amino acids with the original MBPAc1–11 sequence activated the T+α T cells (SI ≥ 50). Of the 61 mimics 52 had both R5 and P6 conserved and 45 of the mimics had A1 conserved. Altogether, A1, S2, Q3, and P5 were more frequently conserved than expected (p < 0.01) from the numbers of possible amino acids in the supertope. In contrast, K4, and SQRSK7–11, were conserved at the expected random frequencies. R5 could not be substituted at all (Fig. 1 A).

Table I.

Microbial peptides that induce proliferation (SI ≥ 50) in T+α cellsa

PeptideSequenceOrganismPeptideSequenceOrganism
042 AAMARPVKRQA Pneumocystis carinii 745 SSQLRPATNGS Candida albicans 
051 AAMLRPIIEAN Escherichia coli 100 AAQRRPSRPFR Herpesvirus saimiri 
146 AHHVRPPALVV Propionibacterium freudenreichii 087 AAQNRPSGPRK Agrobacterium tumefaciens 
064 AAQARPVVDER Aspergillus niger; Aspergillus ficuum 403 ASQNRPRDDVQ Aspergillus parasiticus 
038 AAHSRPVRLRY Bacillus subtilis 375 ASMKRPLFEFS Treponema pallidum 
019 AAFYRPNEVNL Chlamydia trachomatis 443 ATHYRPRSAYR Alcaligenes faecalis 
061 AAQARPRPVAV Herpes simplex virus (type 1/strain 17) 381 ASMRRPARTFC Bovine herpesvirus type 1 
111 AAQYRPDELAR Mycobacterium tuberculosis 726 SSHYRPTNEAE Trypanosoma cruzi 
108 AAQTRPNGALG Newcastle disease virus 366 ASHWRPTSANY Sphingomonas aromaticivorans 
156 AHQLRPGWSPP Leishmania major 736 SSQFRPIHRKL Reclinomonas americana 
063 AAQARPVKTVI Mycobacterium tuberculosis 411 ASQVRPQGRPA Streptomyces coelicolor 
076 AAQHRPAAQHR Schizophyllum commune 471 ATQYRPDQLAK Mycobacterium tuberculosis 
030 AAHLRQRPSLD Pseudomonas aeruginosa 684 SSFFRPILLQD Borrelia burgdorferi 
097 AAQQRQPAHLL Streptomyces kasugaensis 409 ASQSRPAPFLI Haemophilus influenzae 
705 SSFYRPTQPGS Mycobacterium tuberculosis 185 ANHLRPVRSGK Haemophilus influenzae 
129 ACQCRPTSDAV Acinetobacter calcoaceticus 376 ASMLRQHGLPA Bacillus stearothermophilus 
006 AAFHRPKRFFG Bacillus subtilis 364 ASHQRQRAFAQ Mycobacterium tuberculosis 
041 AAHWRPALAGM Acetobacter xylinum 120 ACFTRPARWTL Mycobacterium tuberculosis 
085 AAQMRPDIEIV Leishmania mexicana 032 AAHNRQHFVAH Alcaligenes eutrophus 
109 AAQVRPLLPGT Streptomyces coelicolor 347 ASFLRPGTEQI Rhodobacter sphaeroides 
378 ASMNRPNLVAL Mycobacterium tuberculosis 574 SHQIRPVCGQR Mycobacterium paratuberculosis 
706 SSHARPAFKGL Helicobacter pylori 077 AAQHRQIVADF Mycobacterium tuberculosis 
741 SSQIRPLLQTA Entamoeba histolytica 540 SAQSRPSSNVG Simian 11 rotavirus 
106 AAQTRPMIHGG Newcastle disease virus 505 SAHYRPPPNLN Saccharomyces cerevisiae  
500 SAHLRPLTDMM Leishmania major 226 ANQTRPADIAA Yersinia enterocolitica 
107 AAQTRPNGAHG Newcastle disease virus 002 AAFDRQPIAVG Western equine encephalomyelitis virus 
383 ASMSRPVKQLK Escherichia coli; Salmonella typhimurium 722 SSHNRQREQPT Human papillomavirus type 7 
112 AAQYRQLGYWQ Vibrio cholerae 746 SSQLRPDTASHaemophilus influenzae 
543 SAQVRPGNRSReovirus 007 AAFIRPVPSSEscherichia coli 
183 ANFYRPITMQR Escherichia coli 521 SAQARPTPKSRhodococcus fascians 
   740 SSQIRPKKALK Cryptococcus neoformans 
PeptideSequenceOrganismPeptideSequenceOrganism
042 AAMARPVKRQA Pneumocystis carinii 745 SSQLRPATNGS Candida albicans 
051 AAMLRPIIEAN Escherichia coli 100 AAQRRPSRPFR Herpesvirus saimiri 
146 AHHVRPPALVV Propionibacterium freudenreichii 087 AAQNRPSGPRK Agrobacterium tumefaciens 
064 AAQARPVVDER Aspergillus niger; Aspergillus ficuum 403 ASQNRPRDDVQ Aspergillus parasiticus 
038 AAHSRPVRLRY Bacillus subtilis 375 ASMKRPLFEFS Treponema pallidum 
019 AAFYRPNEVNL Chlamydia trachomatis 443 ATHYRPRSAYR Alcaligenes faecalis 
061 AAQARPRPVAV Herpes simplex virus (type 1/strain 17) 381 ASMRRPARTFC Bovine herpesvirus type 1 
111 AAQYRPDELAR Mycobacterium tuberculosis 726 SSHYRPTNEAE Trypanosoma cruzi 
108 AAQTRPNGALG Newcastle disease virus 366 ASHWRPTSANY Sphingomonas aromaticivorans 
156 AHQLRPGWSPP Leishmania major 736 SSQFRPIHRKL Reclinomonas americana 
063 AAQARPVKTVI Mycobacterium tuberculosis 411 ASQVRPQGRPA Streptomyces coelicolor 
076 AAQHRPAAQHR Schizophyllum commune 471 ATQYRPDQLAK Mycobacterium tuberculosis 
030 AAHLRQRPSLD Pseudomonas aeruginosa 684 SSFFRPILLQD Borrelia burgdorferi 
097 AAQQRQPAHLL Streptomyces kasugaensis 409 ASQSRPAPFLI Haemophilus influenzae 
705 SSFYRPTQPGS Mycobacterium tuberculosis 185 ANHLRPVRSGK Haemophilus influenzae 
129 ACQCRPTSDAV Acinetobacter calcoaceticus 376 ASMLRQHGLPA Bacillus stearothermophilus 
006 AAFHRPKRFFG Bacillus subtilis 364 ASHQRQRAFAQ Mycobacterium tuberculosis 
041 AAHWRPALAGM Acetobacter xylinum 120 ACFTRPARWTL Mycobacterium tuberculosis 
085 AAQMRPDIEIV Leishmania mexicana 032 AAHNRQHFVAH Alcaligenes eutrophus 
109 AAQVRPLLPGT Streptomyces coelicolor 347 ASFLRPGTEQI Rhodobacter sphaeroides 
378 ASMNRPNLVAL Mycobacterium tuberculosis 574 SHQIRPVCGQR Mycobacterium paratuberculosis 
706 SSHARPAFKGL Helicobacter pylori 077 AAQHRQIVADF Mycobacterium tuberculosis 
741 SSQIRPLLQTA Entamoeba histolytica 540 SAQSRPSSNVG Simian 11 rotavirus 
106 AAQTRPMIHGG Newcastle disease virus 505 SAHYRPPPNLN Saccharomyces cerevisiae  
500 SAHLRPLTDMM Leishmania major 226 ANQTRPADIAA Yersinia enterocolitica 
107 AAQTRPNGAHG Newcastle disease virus 002 AAFDRQPIAVG Western equine encephalomyelitis virus 
383 ASMSRPVKQLK Escherichia coli; Salmonella typhimurium 722 SSHNRQREQPT Human papillomavirus type 7 
112 AAQYRQLGYWQ Vibrio cholerae 746 SSQLRPDTASHaemophilus influenzae 
543 SAQVRPGNRSReovirus 007 AAFIRPVPSSEscherichia coli 
183 ANFYRPITMQR Escherichia coli 521 SAQARPTPKSRhodococcus fascians 
   740 SSQIRPKKALK Cryptococcus neoformans 
a

The Swissprot and TREMBL databases were searched for microbial peptides containing the supertope depicted in Fig. 1. Sixty-one of the peptides identified induced proliferation in T+α cells with SIs ≥50.

Next, we compared the dose requirements for the activation of MBPAc1–11-specific T cells by microbial mimic peptides. Four peptides were selected for further analysis. On the basis of the results obtained with the peptides prepared by spot synthesis, we chose two highly stimulatory peptides (pep378 and pep383, SI ≥ 50; see Table I), and two peptides with low stimulatory capacity (pep200 and pep387, SI < 10; not included in Table I). These peptides and MBPAc1–11 were synthesized conventionally and analyzed for the induction of proliferation and cytokine production in T+α cells in a dose-response analysis. pep378 and pep383 induced proliferation comparable to that of MBPAc1–11 (Fig. 2,A). At concentrations ≥100 μg/ml, pep200 induced low proliferation of the T+α cells, whereas pep387 did not induce proliferation of the T+α cells. Thus, the results obtained with conventionally synthesized peptides confirmed the results obtained with these peptides prepared by spot synthesis. pep378 and pep383 induced stronger IL-2 production than MBPAc1–11 (Fig. 2,B), and neither pep200 nor pep387 induced IL-2 production in T+α cells (Fig. 2,B). IFN-γ was induced by MBPAc1–11 and pep383 in similar amounts (Fig. 2,C), the dose-response curve for pep378 was slightly shifted to higher concentrations, whereas neither pep200 nor pep387 induced IFN-γ production. Small amounts of TNF-α were induced by MBPAc1–11, pep378, and pep383 (Fig. 2 D), whereas none of the peptides induced IL-4, IL-10, or TGF-β (data not shown).

FIGURE 2.

Comparison of MBPAc1–11 and microbial peptides in T+α spleen cell cultures. A, Proliferation of the T+α cells in response to the different N-terminally acetylated peptides ([3H]thymidine incorporation). IL-2 (B), IFN-γ (C) and TNF-α (D) concentrations were determined by ELISA at 48 h. No IL-4, IL-5, or IL-10 was detected in these cultures. Results shown are the mean of triplicate wells and are representative of three independent experiments.

FIGURE 2.

Comparison of MBPAc1–11 and microbial peptides in T+α spleen cell cultures. A, Proliferation of the T+α cells in response to the different N-terminally acetylated peptides ([3H]thymidine incorporation). IL-2 (B), IFN-γ (C) and TNF-α (D) concentrations were determined by ELISA at 48 h. No IL-4, IL-5, or IL-10 was detected in these cultures. Results shown are the mean of triplicate wells and are representative of three independent experiments.

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To test whether the microbial mimic peptides that activated T cells in vitro could also induce EAE, we immunized T+α mice with these peptides. Mice were immunized with 200 μg of MBPAc1–11, pep383, pep378, pep200, or pep387. All mice received PT i.v. at 24 and 48 h after immunization and were observed for at least 35 days postimmunization for the development of EAE (Fig. 3). Mice immunized with MBPAc1–11 showed clinical onset of EAE at day 8 (mean value; range, 7–9 days) and rapidly progressed to final stages by day 12. Immunization with the mimic peptides pep383 and pep378 induced EAE in 8 of 8 and 6 of 8 mice, respectively (data pooled from two independent experiments). Both onset and progression of disease with pep383 were delayed as compared with MBPAc1–11 or pep378. Control mice received PBS in CFA and PT and remained healthy for the duration of the experiments (50 days). Of the two peptides identified that induced low proliferation of transgenic T cells in vitro, pep200 and pep387, neither induced EAE (even when the observation period was extended up to 60 days; data not shown). Pertussis toxin was necessary to facilitate EAE and mice that were immunized with MBPAc1–11 or mimic peptides without PT did not develop EAE (data not shown). In agreement with other studies (11, 41, 44), we observed that all T+α mice progressed to full EAE (score 5) rapidly after displaying clear signs of onset of EAE (score 2).

FIGURE 3.

EAE induction with MBPAc1–11 or microbial peptides. T+α mice were immunized with MBPAc1–11, or one of the following N-terminally acetylated peptides: pep383 (S. typhimurium; E. coli), pep378 (M. tuberculosis), pep200 (S. typhimurium; E. coli; H. influenzae; B. aphidicola), pep387 (E. coli) (200 μg), or PBS in CFA s.c. (day 0). All mice received 200 ng PT i.v. at 24 and 48 h postimmunization. Mice were examined every 1–2 days for clinical signs of EAE. Data are represented as mean EAE of each group (±SEM). Data shown are from one representative experiment (from two independent experiments) containing four to five mice per group. Arrows indicate PT administered i.v. on days 1 and 2. Animals were sacrificed when their score reached 4–5, and their score was kept at 5 for the remainder of the experiment.

FIGURE 3.

EAE induction with MBPAc1–11 or microbial peptides. T+α mice were immunized with MBPAc1–11, or one of the following N-terminally acetylated peptides: pep383 (S. typhimurium; E. coli), pep378 (M. tuberculosis), pep200 (S. typhimurium; E. coli; H. influenzae; B. aphidicola), pep387 (E. coli) (200 μg), or PBS in CFA s.c. (day 0). All mice received 200 ng PT i.v. at 24 and 48 h postimmunization. Mice were examined every 1–2 days for clinical signs of EAE. Data are represented as mean EAE of each group (±SEM). Data shown are from one representative experiment (from two independent experiments) containing four to five mice per group. Arrows indicate PT administered i.v. on days 1 and 2. Animals were sacrificed when their score reached 4–5, and their score was kept at 5 for the remainder of the experiment.

Close modal

Global amino acid substitution of the immunodominant encephalitogenic epitope MBPAc1–11 allowed us to define the structural motif (supertope) recognized by the MBPAc1–11/I-Au-specific TCR transgenic, Cα−/− T lymphocytes used in this study (11, 41, 45). The supertope recognized by the T+α T cells confirms and extends previous findings on the recognition of variants of the MBPAc1–11 epitope by I-Au-restricted T cells. Using different T cell hybridomas, T cell clones, or intact mice, others have identified lysine at position 4 of the original peptide (K4) and R5 as the MHC contact sites of MBPAc1–11 and Q3 and P6 as the putative TCR contact sites of the MBPAc1–11/I-Au complex (46, 47, 48, 49, 50, 51, 52, 53, 54, 55). Most of this work was performed with alanine substitutions in the MBPAc1–11 epitope. Our systematic analysis in which every residue of MBPAc1–11 was replaced by every naturally occurring amino acid revealed that the MHC contact site R5 could not be replaced by any other amino acid without destroying recognition by the T+α T cells, whereas L4 could be replaced by any of the amino acids. This is in agreement with earlier studies, which had demonstrated that substitution of L4 with several different amino acids can dramatically increase MHC binding (46, 49, 50, 53, 54). However, increased MHC binding was not always associated with improved T cell activation in vitro, and several of the peptides with substitutions at position 4 abolished T cell activation of individual T cell hybridomas or clones (27, 46, 48, 50, 51, 53, 55). Thus, the T+α T cells differ from some of the other T cell clones and hybridomas studied to date in that all the substitutions for K4 induced strong T cell proliferation (Fig. 1). As expected, only few substitutions were possible at the TCR contact sites. At position 5 P5Q was the only possible substitution. Moreover, only 9 of the 61 mimic peptides had the P5Q substitution, significantly less than expected for a chance distribution (p < 0.01; Table I). Similarly, whereas the supertope analysis had shown that phenylalanine, histidine, and methionine could each substitute for Q3, 31 of the 61 mimic peptides maintained glutamine at position 3, significantly more than expected for a chance distribution (p < 0.01). In addition to the known MHC and TCR contact residues, we also found A1 and S2 significantly (p < 0.01) more frequently conserved than expected by chance (Table I). In contrast, we found 16 different amino acids to be tolerated at position 7 and all naturally occurring amino acids at positions 8–11. Furthermore, no amino acid was overrepresented at any of these positions in the 61 mimic peptides. This is in agreement with earlier studies that had shown that alanine substitutions at positions 7–11 did not influence MHC binding or T cell recognition (27, 49, 54, 56).

A search of the SwissProt and TREMBL databases for peptides containing the supertope shown in Fig. 1 B yielded 832 potentially cross-reactive peptides of microbial origin. However, only 61 of the 832 peptides induced proliferation of the T+α T cells. Wucherpfennig et al. (5) used structural criteria to search a protein database for microbial mimics of MBP89–94, the immunodominant epitope in HLA-DR2+ MS patients. Of 129 peptides fulfilling the set criteria that were synthesized, only 7 activated at least 1 of the 5 DR2-restricted human T cell clones tested in that study. Why do so many peptides that fulfill carefully designed structural criteria fail to induce T cell activation? One explanation is that some combinations of amino acid substitutions that are allowed individually will be “forbidden” when combined in one peptide sequence. This has been observed in a recent study in which MBP-specific human T cell clones were tested for reactivity with random peptide libraries (34). Furthermore, Reay et al. (28) have shown that changing residues apparently not involved in MHC or TCR contact can nevertheless have dramatic consequences on T cell activation. Thus, neither a detailed knowledge about the MHC and TCR contact sites of an epitope nor a global substitution analysis as performed in the work described here can exactly predict those peptides that will activate a cross-reactive TCR. Importantly, either of these approaches will not only predict T cell reactivity with peptides that are nonstimulatory but also miss several peptides that are stimulatory for the TCR in question. Substitutions that are “forbidden” if considered individually can be compensated for by additional substitutions at other positions of an antigenic peptide that enhance T cell activation (28, 34). Thus, it is very likely that our supertope analysis has missed some microbial peptides capable of stimulating the T+α T cells.

25 of the 61 microbial mimic peptides that activated the T+α T cells had four native MBP residues. Gautam et al. (49) have shown earlier that a peptide with only 4 native MBP-residues could activate T cell hybridomas specific for MBPAc1–11/I-Au. Extending these data, we found 18 peptides (see Table I) that had 3 native MBP residues and 6 peptides that had only 2 native MBP residues among those mimics that induced SIs ≥50 in the T+α T cells. Thus, in addition to viral peptides that have been shown earlier to activate MBPAc1–11/I-Au-specific or MBP87–99/I-As-specific T cells (7, 24), we demonstrate here that bacterial peptides with as little as 2 or 3 conserved MBP residues can activate MBPAc1–11/I-Au-specific T cells. This is similar to findings obtained with human MBP87–99-specific T cell clones; microbial mimics with as little as 3 native MBP-residues were shown to activate such clones (5). In one case, a peptide not sharing a single residue with the original MBP87–99 sequence was found to stimulate human T cell clones raised against MBP (34).

The animal model EAE permits testing of microbial peptides for encephalitogenicity. Previous work had shown that some altered peptides could still induce T cell activation but not EAE when injected into susceptible mice (46, 47, 49). Previous findings had also indicated that at least 5 native MBP residues need to be present in a mimic peptide for the peptide to induce EAE after immunization of susceptible mice (24, 49). Extending these earlier reports, we found that a mimic peptide containing only 4 native MBP residues (pep 378) could induce EAE when injected into susceptible mice (Fig. 3). Both of the encephalitogenic peptides examined in our study had A1, S2, R5, and P6 conserved. Our findings that at least 61 microbial mimic peptides can activate the in T+α T cells in vitro and that a fraction of these mimic peptides can also induce EAE in the T+α mice support recent evidence coming from extensive analyses of Ag recognition by individual T cells (5, 6, 27, 28, 29, 30, 31, 32), or the structural analyses of TCR-peptide-MHC complexes (33), demonstrating that TCR recognition of Ag is degenerate. In fact, it has recently been suggested that a single TCR might productively interact with as many as 106 different ligands (57). How do these findings relate to the “molecular mimicry” hypothesis (8)? Our data presented here (and those of B. Maier and T. Kamradt, unpublished observations) and those of others (5, 6) indicate that peptide molecular mimicry at the level of T cell activation is a frequent event. We consider it very likely that T cell cross-reactivity between a microbial peptide and a self peptide alone is not sufficient to induce autoimmune disease (9). In fact, in preliminary experiments we could not induce EAE in T+α mice via infection with S. typhimurium, the bacterium from which the encephalitogenic mimic pep383 is derived (J. L. Grogan, U. E. Schaible, and T. Kamradt, unpublished observations). Such preliminary observations, however, must be interpreted with great caution because all the peptides used in our studies were N-terminally acetylated. Earlier work had shown that N-terminal acetylation of MBP1–11 is essential for T cell recognition. It was proposed that the positively charged amino terminus revealed by removal of the N-terminal acetyl group was responsible for the observed elimination of the proliferative activity (58). Wraith et al. (46) found that unacetylated MBP1–11 with a K4A substitution (MBP1–11[4A]) effectively activated T cell hybridoma 1934.4 despite its decreased binding to I-Au. Therefore, the N-terminal acetyl group is an important determinant in interactions with I-Au but not absolutely necessary for interaction with the TCR. This notion was further supported recently. Lee et al. reported on an unacetylated but NH2-terminally extended MBP1–11 peptide (OVA-MBP). This peptide induced IL-3 production in an MBPAc1–11-specific T cell clone yet failed to trigger full T cell proliferation (54). Finally, acetylated MBP1–11 variants have been reported that induce T cell proliferation in vitro but not EAE in vivo (46, 47, 49). Therefore, it is impossible to predict from our in vitro and in vivo data that were obtained using N-terminally acetylated MBP1–11 whether the nonacetylated or N-terminally extended natural peptides would have similar effects. Current work in our laboratory addresses the questions whether the mimic peptide sequences are processed naturally and whether the naturally processed peptides are encephalitogenic. In addition to this aspect which is specific for the MBPAc1–11 system, a multitude of mechanisms usually prevents the induction of autoimmunity. For cross-reactive T cells to induce autoimmunity, neither the microbial peptide nor the self peptide should be a cryptic epitope (59); the self Ag must be present at high enough concentrations and the T cells at high enough numbers (60); the T cells must receive the “right” costimulatory signals (61), to produce the “right” set of cytokines (1, 40, 62), to migrate to the site where the self Ag is expressed (45, 63, 64), and must escape immunoregulation (41, 44). Nevertheless, molecular mimicry remains an attractive hypothesis for the pathogenesis of autoimmunity. It is, for example, conceivable that microbial Ags, even if they do not trigger disease directly, help maintain the memory T cell pool specific for a particular autoantigen. Furthermore, recurrent infections possibly even with different microbes could bring the number of autoreactive T cells over a critical threshold such that autoimmune disease will finally become manifest.

We have shown that the definition of a supertope by global amino acid substitution can identify multiple microbial mimic peptides that activate an encephalitogenic TCR. Peptides with only 2 native MBP-residues are sufficient to activate MBPAc1–11-specific T cells in vitro and EAE can be induced by immunizing mice with a mimic peptide with only 4 native MBP residues. The data show that molecular mimicry at the level of TCR cross-reactivity is a frequent event.

We thank Dr. J. Lafaille for the MBPAc1–11-TCR-transgenic and TCRα−/− breeders; Dr. A. O’Garra for generous gifts of mAbs; Dr. U. E. Schaible for providing the helpful discussions and advice on the infection experiments; Dr. O. Liesenfeld for helpful discussion; and Maja Affeldt, Grit Czerwony, Kristine Hagens, Berit Hoffmann, and Christiane Landgraf for excellent technical assistance.

1

This work was supported by the Alexander v. Humboldt-Stiftung, the Berliner Senatsverwaltung für Wissenschaft und Kultur, the Bundesministerium für Bildung, Wissenschaft, Forschung and Technologie, the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 421), and the Fritz-Thyssen-Stiftung.

3

Abbreviations used in this paper: EAE, experimental autoimmune encephalitis; MBP, myelin basic protein; MS, multiple sclerosis; pep, peptide; PT, pertussis toxin; SI, stimulation index.

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