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Experimental Allergic Neuritis in Lewis RatsInduces T Cell Anergy and Prevents A Complementary Peptide Vaccine That

NakashimaShigeru Araga, Masahiro Kishimoto, Satoko Doi and Kenji

http://www.jimmunol.org/content/163/1/4761999; 163:476-482; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/163/1/476.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1999 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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A Complementary Peptide Vaccine That Induces T CellAnergy and Prevents Experimental Allergic Neuritis inLewis Rats1

Shigeru Araga,2 Masahiro Kishimoto, Satoko Doi, and Kenji Nakashima

We have developed and described a new method of altering T cell-mediated autoimmune diseases by immunization with thecomplementary peptide against T cell epitopes. The complementary peptide (denoted NAE 07-06) to the bovine P2 protein,residues 60–70 (denoted EAN 60–70), was tested in the Lewis rat model of experimental allergic neuritis (EAN). Immunizationwith NAE 07-06 induced polyclonal and monoclonal Abs that inhibited the proliferation of the P2-specific T cell line, stimulatedwith EAN 60–70, and recognized Vb, but not Va, of TCRs. Proliferation of T cells treated with anti-NAE 07-06 Abs could bepartially restored by treatment with rIL-2, in accordance with an anergy model. A homologous sequence was found between NAE07-06 and the VDJ junction of the TCRb-chain from an EAN 60–70-specific T cell line. Rats preimmunized with NAE 07-06 invivo before EAN induction showed less disease severity clinically and histologically. These data suggest a new therapeutic ap-proach for T cell-mediated autoimmune disorders through the induction of anti-TCR Abs with complementary peptide Ags. TheJournal of Immunology,1999, 163: 476–482.

N eurological diseases include many disorders caused byautoimmune T and B cells. Guillain-Barre syndrome(GBS)3 is the popular disorder of the peripheral nervous

system the central nervous system counterpart of which is multiplesclerosis (MS). Some cases of GBS show a long term bed-riddenstate requiring respiratory assistance and long lasting severe para-paresis. Immunomodulatory therapies such as corticosteroid orplasmapheresis are effective for GBS. However, such immunosup-pressive treatments are nonspecific and have severe side effectssuch as allowing opportunistic infection. In theory, anti-Id Absreactive with disease-causing Id Ab or clonotypic T cells representideal therapeutic agents. However, it is difficult to know the ap-propriate Id Ab or clonotypic T cell to use for the induction ofanti-Id Ab. We have previously reported a technique that over-comes this problem by actively inducing anti-Id Ab with a peptideimmunogen rather Id Ab (1, 2). Our approach is based on theobservation that peptides specified by complementary nucleotidesequences can specifically bind to each other, apparently as a resultof their having complementary shapes (3–5). These interactingpeptides, with presumed complementary shapes, can in turn inducethe formation of interacting pairs of polyclonal or monoclonal Idand anti-Id Abs the combining sites of which are complemen-tary (6, 7).

Experimental allergic neuritis (EAN) provides an opportunity totest this new approach for the treatment of T cell-mediated auto-immune disorders. EAN is an animal model of GBS and is causedby immunization with bovine P2 protein (8). Histological exami-nation shows infiltration of lymphocytes and demyelination in theperipheral nerve, similar to that seen in GBS. Passive transfer withP2 protein-sensitized T cells, but not with Abs against the myelincomponents including P2 protein, demonstrate the cell-mediatedorigin of EAN (9–11). As demonstrated in experimental autoim-mune encephalomyelitis (EAE), an animal model of MS, Th1 lym-phocytes against the P2 protein play a major role in developmentof EAN (12). Bovine P2 protein, residues 53–78, can induce theEAN clinically and histologically in Lewis rats (13, 14). Further-more, a recent study showed that the minimal epitope was locatedin the bovine P2 protein, residues 60–70 (15, 16). In this article,we demonstrate that a complementary peptide for this T cellepitope induced polyclonal and mAbs against TCR on P2-reactiveT cells. The mAbs specifically recognized TCR Vb and induced Tcell anergy in the EAN model. Furthermore, we have demonstratedin vivo protection against EAN using the complementary peptideas a vaccine.

Materials and MethodsBovine peripheral myelin purification

Bovine peripheral myelin was prepared from bovine dorsal roots accordingto the method of Cammer (17) and stored at280°C until used.

Complementary peptide design for bovine P2 protein, residues60–70

Bovine P2 protein was reported to induce EAN in Lewis rats (8). However,the nucleotide sequence for bovine P2 protein has not yet been reported. Incontrast, nucleotide sequences for the P2 protein in rabbits and mice areavailable (8, 18). Furthermore, the amino acid sequence of P2 protein,residues 60–70 (denoted EAN 60–70) are conserved in bovine, rabbit, andmouse. Using the rabbit nucleotide sequence, we designed a complemen-tary peptide (denoted NAE 07-06) for EAN 60–70 peptide. Namely, theamino acid sequence of NAE 07-06 peptide, was derived by 59 to 39assignment of amino acids to the nucleotide sequence complementary tothe mRNA of rabbit P2 protein encoding EAN 60–70 peptide (Fig. 1).

Division of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tot-tori University, Yonago, Japan

Received for publication January 28, 1999. Accepted for publication April 8, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by Grants 07807065 and 09670657 from the JapaneseMinistry of Education, Science, and Culture to S.A.2 Address correspondence and reprint requests to Dr. S. Araga, Division of Neurol-ogy, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, 36-1Nishimachi, Yonago, 683-8504, Japan.3 Abbreviations used in this paper: GBS, Guillain-Barre syndrome; MS, multiplesclerosis; EAN, experimental allergic neuritis; EAE, experimental autoimmuneencephalomyelitis; KLH, keyhole limpet hemocyanin; EAMG, experimental auto-immune myasthenia gravis.

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00

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As expected, the complementary peptide NAE 07-06 has an inverted hy-dropathic profile compared with the target peptide, EAN 60–70 (Fig. 2).The amidated form of the peptide, EAN 60–70, and NAE 07-06 werecommercially synthesized with F-moc chemistry and were purified by re-verse phase HPLC (Kurabo Biomedical, Tokyo, Japan). An amidated con-trol peptide denoted PBM 9-1 was similarly synthesized and purified. PBM9-1 is a complementary peptide for the first 9 residues of human myelinbasic protein and has the sequence NH2-Arg-Ser-Leu-Leu-Ser-Gly-Gly-Leu-Pro-NH2 (7).

Coupling of a peptide to keyhole limpet hemocyanin (KLH)

A peptide used for immunization in an experiment was coupled to KLHusing 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (19).Peptides coupled to KLH were purified on a gel filtration column, dialyzedagainst PBS, and stored at220°C until used.

Immunization

Female Lewis rats age 6 weeks was obtained from Charles River JapanBreeding Laboratories (Seiwa Animals, Fukuoka, Japan). The rats weredivided into two groups (A and B) with six animals per group. Beforechallenging with EAN 60–70 peptide, group A was preimmunized twicewith 200 mg of NAE 07-06 peptide coupled with KLH in AjuPrime Im-mune modulator (Pierce, Rockford, IL). To elicit EAN, all rats were thenimmunized with 200mg of EAN 60–70 peptide, emulsified in CFA. Pre-immunization with 200mg PBM 9-1 peptide coupled with KLH in Aju-Prime Immune modulator served as the control group (group B).

Clinical evaluation of EAN

Rats were weighed and assessed for clinical signs. The severity of EANwas graded as follows: 0, no abnormality; 1, limp tail; 2, monoparesis orparaparesis; 3, monoplegia or paraplegia; 4, tetraparesis or tetraplegia; 5,moribund or death (20).

Histological examination

For histological examination and to assess the filtration of mononuclearcells, the sciatic nerves were removed from different groups of rat 3 weeksafter inoculation and were stained with hematoxylin and eosin. The sever-ity of cell infiltration was graded as follows: 0, no abnormality; 1, cellularinfiltration adjacent to a vessel; 2, cellular infiltration in immediate prox-imity to a vessel; 3, cellular infiltration around a vessel and in more distantsites (20). Slides were evaluated in a blinded manner.

Preparation of rat Abs to NAE 07-06 peptide

For preparation of anti-complementary peptide Ab, three female Lewis rats(8 wk old) were immunized three times at 3-wk intervals with KLH-cou-pled NAE 07-06 complementary peptide in an AjuPrime Immune modu-lator at a concentration of 1 mg/ml. For controls, the rats were immunizedwith PBM 9-1 control peptide as mentioned above. After checking the titerof Abs against both peptides, sera were collected by cardiac puncture fromrats under ether anesthesia. Samples were pooled and stored at220°Cuntil used.

Monoclonal Abs

A mAb (denoted NAE3, IgG2a,k) was obtained from a fusion from ratsimmunized with NAE 07-06 peptide, coupled to KLH using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride methods as mentionedabove. Spleen cells were fused with the mouse myeloma cell line X63Ag8.653 by using a 50% (v/v) polyethylene glycol solution (Sigma, St.Louis, MO). Hybridomas were selected for monoclonality against NAE07-06 peptide by five serial limiting dilutions and were grown in protein-free medium (PFHM-II; Life Technologies, Gaithersburg, MD). mAb

CTCR8 (IgG2b,k), against the complementary peptide forTorpedoace-tylcholine receptor, residues, 100–116, was used for control mAb (21).mAbs were purified by ammonium sulfate precipitation, followed by pass-ing over a protein G column (HiTrap affinity column, Pharmacia Biotech,Uppsala, Sweden). The purity and reactivity were checked by ELISA andelectrophoresis. mAb CTCR8 was used as a control mAb and has beendescribed (21).

Biotin-labeled mAb NAE3

MAb NAE3 was first oxidized with sodiumm-periodate (Sigma) at a con-centration of 10 mM in labeling solution (0.1 M sodium acetate, pH 5.5) for20 min at 4°C in the dark. The oxidation reaction was stopped by addingglycerol, followed by dialysis against labeling solution at 4°C overnight.Finally, Biotin-LC-Hydrazide (Pierce) was added at a final concentration of5 mM for 2 h at room temperature. Biotin-labeled sample was dialyzedagainst PBS, containing 0.1% sodium azide, and stored at 4°C until used.

Bovine peripheral myelin-reactive T cells

Female Lewis rats (8 wk old) were immunized into the rear footpads with2 mg of purified bovine peripheral myelin solution emulsified with an equalvolume of CFA. Eight days after immunization, rats were sacrificed underether anesthesia. The inguinal and popliteal lymph nodes were removedaseptically. The lymphocytes were collected by passing through a stainlessmesh. The tissue debris and dead cells were removed by Ficoll-Hypaquecentrifugation and resuspended in complete medium (RPMI 1640, 10%FCS, 53 1025 M 2-ME, 100 U/ml penicillin, 100 mg/ml streptomycin,0.25 mg/ml Fungizone, 10 mM HEPES (pH 7.0), 10 mM nonessentialamino acids, and 20 mML-glutamine, and 10 mM sodium pyruvate). Mac-rophages were depleted by a plastic plate adherence method.

Irradiated spleen cell preparation

Normal Lewis rats (8 wk old) were killed, and spleens were removedaseptically. The splenocytes were collected by Ficoll-Hypaque centrifuga-tion and suspended in the above medium. The splenocytes were irradiatedwith 3000 rad. Then, irradiated splenocytes were incubated in completemedium with 100mg/ml EAN 60–70 peptide for 1 h in ahumidified 5%CO2 incubator. These irradiated cells were used for educated APC.

EAN 60–70-specific T cell line

Lewis rats were immunized s.c. with 2 mg purified bovine peripheral my-elin, emulsified with an equal volume of CFA. Eight days later, their drain-ing popliteal and inguinal lymph nodes were removed, and a single-cellsuspension was made. The primed lymph node cells were stimulated in thepresence of EAN 60–70 peptide at a concentration of 100mg/ml and ir-radiated APC for 3 days, followed by a 7-day coincubation with irradiatedAPC alone. After three passages through the above procedure, T cell lineswere expanded by EAN 60–70 peptide and irradiated APC in the presenceof 10% IL-2 without Con A (Genome Therapeutics, Waltham, MA).

For control cell lines, Lewis rats were immunized s.c. with 50mg OVA,emulsified with an equal volume of CFA. This was followed by an ordinarypreparation procedure for T cell line as mentioned above.

Inhibition of bovine peripheral myelin-reactive lymphocyteproliferation by anti-NAE 07-06 Abs

The sensitized T cells (23 105/well) and educated APC (53 105/well)were coincubated with the indicated concentrations of either anti-NAE

FIGURE 2. Hydropathic profile of bovine P2 (EAN 60 –70) andits complementary peptide (NAE 07-06). Hydropathic score is basedon Kyte and Doolittle values. Scale: most hydrophobic,14.5, to leasthydrophobic,24.5.

FIGURE 1. Bovine and rabbit peripheral nerve myelin P2 protein, res-idues 60–70 (denoted EAN 60–70), and their complementary peptide(NAE 07-06).

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07-06 serum or anti-PBM 9-1 serum for 4 days at 37°C in a 5% CO2

incubator. Cultures for proliferation assay were harvested after 4 days withthe final 16 h, composed of pulse labeling with 0.5mCi/culture [3H]TdR(Amersham, Arlington Heights, IL).

Inhibition of EAN 60–70-reactive T cell line proliferation bymAb NAE3

To test the inhibition of proliferation by mAb NAE3, before coincubationof educated APC (53 105/well) and sensitized lymphocytes (23 105/well), educated APC or sensitized lymphocytes were preincubated withmAb NAE3 or control mAb (CTCR8) at a concentration of 1mg/well for1 h at room temperature and then were washed with complete mediumtwice, followed by the proliferation assay. OVA-specific T cell lines wereused as control cell lines.

Measurement of IL-2 production

P2 residues, 60–70-specific T cells (23 104), and APC (23 105) werecocultured with 10mg/ml EAN 60–70 peptide in the absence or presenceof mAb NAE3 (1 mg/ml) for 3 days of culture. The supernatants werestored at220°C until used. The amount of IL-2 in the supernatant wasdetermined by bioassay using CTLL-2 cells (22). The activity of the IL-2was determined by the proliferation of the CTLL-2 line as described above.Human rIL-2 was kindly provided by Takeda Chemical Industry (Osaka,Japan) and was used as standard.

Restoration of mAb NAE3-induced T cell anergy by coculturewith exogenous rIL-2

EAN 60–70 specific T cells (23 105) and irradiated APC (23 106) werecocultured with indicated amount of EAN 60–70 peptide in the presence ofmAb NAE3 for 3 days of culture. Then, NAE3-treated EAN60–70-specificT cells were restimulated with fresh irradiated APC and EAN 60–70 pep-tide in the absence or presence of rIL-2 (50 U/ml) for 5 days. To demon-strate the Ag specificity of the restoration, PBM 9-1 peptide was used forrestimulation. Finally, ordinary proliferation assays were done.

Flow cytometric analyses of T cell line

T cell line reactive to EAN 60–70 peptide was analyzed with the use ofFACScan (Becton Dickinson, Mountain View, CA) with propidium iodideand FITC-labeled anti-CD3 (W3/13), anti-CD4 (W3/25), or anti-CD8(OX-8) (Serotec, Oxford, U.K.). EAN 60–70-specific T cell clones werealso analyzed by biotin-labeled NAE3, followed by FITC-labeled avidin(Becton Dickinson) or Abs to NAE 07-06, followed by biotin-anti-rat IgG(Jackson ImmunoResearch Laboratories, West Grove, PA) and FITC-la-beled avidin (Becton Dickinson). All T cell lines were phenotyped to beCD31, CD41, and CD82 (data not shown).

Immunoblotting with mAb NAE3

EAN 60–70-specific T cells or OVA-specific T cells were lysed in 10 mMTris-HCl buffer, pH 7.4, containing 1% (w/v) Nonidet P-40 (Sigma), 150mM NaCl, 1 mM EDTA, supplemented with the protease inhibitors 2

mg/ml leupeptin, 2mg/ml aprotinin, 4 mM 4-(2-aminoethyl)benzenesulfo-nyl fluoride, hydrochloride (Boehringer Mannheim, Indianapolis, IN), and20 mM iodoacetamide. The lysate was then treated with streptavidin aga-rose (Life Technologies) to preclear lysate. The precleared lysate was re-duced with 100 mM DTT. We used this preparation as total soluble mem-brane proteins. Furthermore, part of the lysate was dialyzed against 10 mMTris-HCl buffer, pH 7.4, containing 1% (w/v) Nonidet P-40, 150 mM NaCl,1 mM EDTA to remove excess DTT. The lysate was immunoprecipitatedwith mAb R73 (IgG1, Serotec) using agarose anti-mouse Ig (Sigma) toremove the TCR-Vb molecule. We used this preparation as R73-preclearedlysate. These total soluble membrane samples and R73-precleared lysatewere analyzed by 10% acrylamide gel and then electrotransferred to poly-vinylidene difluoride membrane (Bio-Rad, Richmond, CA). After blockingwith 10% skin milk in PBS, membrane were incubated with either biotin-labeled mAb R73 or biotin-labeled mAb NAE3 at the concentration of 10mg/ml in TPBS (PBS, containing 0.1% Tween) using a 10-well-slot appa-ratus (CosmoBio, Tokyo, Japan). After being washed in TPBS, membraneswere incubated with alkaline phosphatase-labeled streptavidin. Finally,bands were detected with CDP-Star Western blot chemiluminescence re-agent (DuPont NEN) on exposure to x-ray films.

The blot of soluble membrane proteins of OVA-specific T cells waschecked by reactivity against either mAb R73 or mAb NAE3. This wasthen followed by an ordinary Western blotting technique as mentionedabove.

TCR Vb chain analysis

TCR Vb chain usage was analyzed by the RT-PCR method. RNA wasprepared from cloned T cell lines with TRIZOL LS reagent (Life Tech-nologies). cDNA was synthesized from total RNA using SuperScript II

FIGURE 3. A, Immunoblots of membrane protein from an NAE 60–70T cell line. Membrane were blotted as described inMaterials and Methodsand stained with mAbs R73 (lane a) or NAE3 (lane b).Lane cis an R73precleared lysate developed with NAE3.B, Immunoblots of membraneprotein from an OVA-specific T cell line. Membranes were blotted asdescribed inMaterials and Methodsand stained with mAbs R73 (lane a)or NAE3 (lane b).

FIGURE 4. Inhibition of bovine peripheral myelin-sensitized lympho-cyte proliferation by Ab to NAE 07-06. Purified bovine peripheral myelin-sensitized lymphocytes were incubated with irradiated macrophages,pulsed with EAN 60–70 peptide and the indicated dilutions of rat antiserato NAE 07-06 or PBM 9-1. Proliferation assays were done as described inMaterials and Methods. Each point represents the mean6 SEM; 100% of[3H]thymidine uptake represents 85,740 cpm.

FIGURE 5. Inhibition of EAN 60–70-specific lymphocyte proliferationby pretreatment of APC or EAN 60–70-sensitized lymphocytes withNAE3. EAN 60–70-sensitized lymphocytes, depleted of macrophages(sen-T), were coincubated with EAN 60–70 or EAN 60–70 educated (ed)irradiated APC. In each assay, sensitized T cells or educated APC werepreincubated or coincubated with 1 mg/ml of either mAb NAE3 or CTCR8,followed by the proliferation assay described inMaterials and Methods.Sensitized T cells and APC (nonloaded with EAN 60–70) gave a back-ground incorporation of 1015 cpm. No inhibition was induced on prolif-eration of OVA-sensitized lymphocytes (sen-OVA), pretreated with mAbNAE3. Each bar represents mean6 SEM; 100% of [3H]thymidine uptakerepresents 67,960 cpm.

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RNase H2 reverse transcriptase (Life Technologies) and a random hex-amer. The cDNA was then amplified using an antisense Cb and specificprimers for Vbs (Vb 1, Vb 2, Vb 3.3, Vb 4, Vb 5.1, Vb 6, Vb 7, Vb 8.1,Vb 8.2, Vb 8.3, Vb 8.5, Vb 9, Vb 10, Vb 11, Vb 12, Vb 13, Vb 14, Vb15, Vb 16, Vb 17, Vb 18, Vb 19, Vb 20) (23) in a typical PCR reactionfor a total of 40 cycles consisting of 94°C/1 min, 54°C/1 min. ending witha 10-min extension at 72°C. PCR products were size-selected using a 2%agarose gel. PCR products were directly ligated into pGEM-T vector (Pro-mega, Madison, WI). V-D-J genes were identified by comparisons to pre-viously published sequences in the EMBL-GDB (European BioInformaticsInstitute) and LASL-GDB (GenBank, National for Biotechnology Infor-

mation) with the use of genetic MaC/CD software (Software Development,Tokyo, Japan). Rearranged V-D-J sequences of each T cell clone weredetermined by sequencing at least 10 isolates.

Statistical analysis

Statistical analysis was conducted with the two-tailed Studentt test and thetwo-tailed Mann-Whitney test. A significant difference was considered tobe p 5 0.01.

ResultsAbs and mAb to NAE 07-06 peptide recognize a Vb of EAN 60–70-specific T cell line

In EAN, Th1 cells are reported to be effector T cells. This T cellepitope is located in the bovine P2 protein, residues 53–78 (14).Recently, the minimal epitope was reported to be located in thebovine P2 protein, residues 60–70 (15). Therefore, rats were im-munized with purified bovine peripheral myelin, and the sensitizedT cells were pulsed with APC, educated with an appropriate con-centration of EAN 60–70 peptide. Flow cytometric analysisshowed that these cells were CD41CD82 lymphocytes (data notshown).

To test whether or not mAb NAE3 recognize TCR, EAN 60–70-sensitized T cells were lysed, followed by electrophoresis andWestern blotting to polyvinylidene difluoride membrane. Fig. 3Ashows that NAE3 immunostained cell surface proteins from this Tcell line which reacted with one recognized as well by mAbagainst rat TCRab (mAb R73). Precleared lysate with mAb R73shows no visible bands around 38 kDa by mAb NAE3. The spec-ificity of TCR molecules was demonstrated by the ability of mAb

FIGURE 6. A, Inhibition of proliferation of A6 clone by pretreatmentwith NAE3. The A6 clone was coincubated with EAN 60–70-educatedirradiated APC in the absence or presence of NAE3. Each bar representsmean6 SEM; 100% of [3H]thymidine uptake represents 44,700 cpm.B,Flow cytometric analysis of A6 clone using NAE3. A6 clone was stainedwith biotin-labeled NAE3 followed by FITC-labeled avidin. Open histo-gram, control for isotype Ab staining. The same results were obtained fromH10 clone.

FIGURE 7. Inhibition of IL-2 secretion of EAN 60–70 specific T cellline by NAE3. EAN60–70 specific T cell line was cocultured with irradi-ated APC educated with EAN 60–70 and NAE3. IL-2 secretion was mea-sured as described inMaterials and Methods. Each bar represents mean6SEM; 100% of proliferation of CTLL-2 cell line was 126,445 cpm.

Table I. Sequence homology between NAE 07-06 and the VDJ junctional regions from EAN 60–70-specific clonesa

Peptide orClones

Abs to NAE 07-06Reactivity (a)

NAE3Reactivity (b) Amino Acid Sequence Homology (%)

NAE 07-06 1 1 S L D G E L K — —F F F F E ‚ ‚

A6 clone 1 1 — — S L D G G A R — — 64‚ F E ‚ F ‚

H10 clone 1 1 — — P L N T E V — — — 54F F ‚ E

B5 clone 1 2 — — S — D W T G N — — 36F ‚ ‚ ‚

1H7 clone 1 2 — — S P G T H T E — — 36F ‚ F

2A6 clone 1 2 — — S G R G A Q F — — 27

a T cell clones were stained by Abs against NAE 07-06, followed by biotin-anti-rat IgG and FITC-avidin (a), or were stained by biotin-labeled NAE3, followed by FITC-avidin(b). T cell clones were analyzed using FACScan flow cytometric analysis (Becton Dickinson). VDJ regions of the indicated clone were aligned from N/Db into Jb. The sequencewas then compared with that of NAE 07-06. Significant homology was found with the VDJ junction from A6 and H10 clones.

F, identical amino acid;E, conservative substitution;‚, amino acid with a similar hydropathy.

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R73 but not mAb NAE3 to immunostain a TCR from a rat OVA-specific T cell lines (Fig. 3B). Recently, mAb R73 has been re-ported to recognize a constant region of rat TCRb-chain (24).Thus, these results suggest that NAE3 recognized a TCR-b mol-ecule from EAN 60–70-specific T cells.

We next examined whether or not Abs against the NAE 07-06inhibited the proliferation of EAN 60–70-specific T cells. Fig. 4shows that rat antiserum to NAE 07-06 caused a dose-dependentinhibition of the proliferation of bovine peripheral myelin-reactivelymphocytes. The specificity of the inhibition was demonstrated bythe inability of antiserum to control peptide PBM 9-1 with aninverted hydropathic profile relative to that of myelin basic proteinresidues 1–9, to block the bovine peripheral myelin-reactive lym-phocytes. Fig. 5 similarly shows that mAb NAE3 block the pro-liferation of EAN 60–70-sensitized lymphocytes by pretreatmentof EAN 60–70-sensitized T cells with mAb NAE3, not by pre-treatment of EAN 60–70-educated APC with mAb NAE3. Thecontrol mAb CTCR8 showed no inhibition. No inhibitory effectwas demonstrated on the proliferation of OVA-specific T cell linesusing mAb NAE3. Not unexpectedly, the inhibition of prolifera-tion of T cell lines by mAb NAE3 was weaker than that by poly-clonal Abs against NAE 07-06. This is probably due to the rec-ognition by mAb NAE3 of only a subpopulation of the total T celllines reactive to EAN 60–70 peptide. In fact, the ability of inhi-bition by mAb NAE3 was demonstrated in only two of five cloneswe established from the aforementioned T cell lines, whereas Absto NAE 07-06 induced an inhibitory effect on all clones (Fig. 6A).These two clones (denoted A6 and H10) were also stained by mAbNAE3 using FACScan analysis (Fig. 6B, Table I). All clones werefurther studied for analysis of Vb usage in the TCR. Taken to-gether with Western blot analysis, mAb NAE3 specifically recog-nized a TCRb molecule of EAN 60–70-sensitized T cells andblocked the proliferation of these cells. IL-2 production was alsoinhibited by treatment with mAb NAE3 (Fig. 7). The proliferationof these T cells was partially restored by following coculture withEAN 60–70 peptide and rIL-2, not with PBM 9-1 and rIL-2 in theculture (Table II). These results suggest that Abs to NAE 07-06 ormAb NAE3 induced T cell anergy.

To ascertain whether there was restricted Vb region usage in theTCR that was recognized by mAb NAE3, two clones (H10 and A6clones) were analyzed. These clones were found to utilize Vb 8family, which have a unique VDJ region (PLNTEV and SLDG-GAR). A homology check showed that these VDJ region se-

quences are similar to that of NAE 07-06 (Table II). Taken to-gether with the results of Western blotting, these results suggestthat immunization with NAE 07-06 can induce polyclonal andmonoclonal Abs that block the proliferation of EAN 60–70-spe-cific T cells, in accordance with T cell anergy through recognitionof their TCR-b.

Prevention of EAN

Because we found that Ab to NAE 07-06 recognized Vb of EAN60–70-sensitized T cells and blocked their proliferation in vitro.We tested whether preimmunization with NAE 07-06 could inducepolyclonal Abs to this peptide and block the development of EAN.Preimmunization with NAE 07-06 lessens the severity of diseasewhen compared with animals preimmunized with control peptide,PBM 9-1 (Table III). This effect was demonstrated in animals inwhich disease was induced by EAN 60–70 peptide (Fig. 8). Thesame effect was also demonstrated in animals in which disease wasinduced by purified bovine peripheral myelin (data not shown).These results support the notion that the T cell epitope in EAN islocated in bovine P2 protein, residues 60–70. Histological exam-inations showed that no visible cell infiltration was detected insciatic nerves of rats (EAN induced by EAN 60–70) preimmu-nized with NAE 07-06. The control peptide, PBM 9-1, did nothave this effect (Fig. 9). These results suggest that clonal expan-sion of EAN-specific T cells are completely blocked by polyclonalAbs to NAE 07-06. Furthermore, these polyclonal Abs did notinfluence the total subset of T cells in the peripheral lymphocyteschecked by flow cytometry using W3/13, W3/25, and OX8 mAbs

FIGURE 8. Protection from EAN by preimmunization with NAE06–07 peptide. Rats were preimmunized with complementary peptide(NAE 07-06) or control peptide (PBM 9-1) and then challenged with bo-vine P2 protein, residues 60–70 peptide (EAN 60–70) (A andB). The ratsin group A are preimmunized with complementary peptide (NAE 07-06);the rats in group B are preimmunized with control peptide (PBM 9-1). Therats were observed for 12 wk after challenge of EAN induction. Clinicalscores are explained inMaterials and Methods. Each dot represents theaverage score of six rats. P, preimmunization; C, EAN challenge.

Table II. Restoration of NAE3-induced T cell anergy by stimulation with Ag and IL-2a

ExperimentPretreatmentwith NAE3 Stimuli IL-2 (U/ml)

Proliferation (cpm)(mean6 SEM)

a 1 EAN 60-70 0 13282.36 1062.5b 1 EAN 60-70 50 42946.26 8766.3c 1 PBM 9-1 50 17709.86 2833.5d 2 EAN 60-70 50 88549.06 11954.1

a After anergy induction with EAN3, EAN 60-70-specific lymphocytes were cocultured with EAN 60-70-educated irradiated APC in the absence (a) or presence (b) of IL-2or with PBM 9-1-educated irradiated APC in the presence of IL-2 (c). EAN 60-70-specific lymphocytes without pretreatment with NAE3 were used as control (d).

Table III. Histological scoresa

Group Mean6 SD (n 5 6)

A 0.26 0.45B 2.26 0.45

a Rats were sacrificed 3 wk after inoculation with bovine P2 protein, residues60-70. Group A was preimmunized with NAE 07-06 peptide. Group B was sham-treated with PBM 9-1 peptide (control group). Each group consists of six rats.

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(Serotec) (data not shown). Namely, selective T cells (EAN 60–70specific T cells) were blocked.

DiscussionThe two-signal model for complete T cell activation has been pre-viously described and reviewed (25–27). In short, T cell anergywas described as the results of a lack of costimulation during initialTCR engagement with Ag (28). Namely, T cells do not proliferatewell in the absence of a costimulatory signal during an Ag pre-sentation (29). Recent investigations demonstrated that anergy ofregulatory T cells may play an important role in susceptibility toautoimmune disease (30). In T cell-mediated disorders such asMS, Th2 cells have regulatory function against the effector T cells(Th1). Thus, anergy of Th2 cells consequently activates and elicitsthe disease (30). Similarly, induction of T cell anergy of effectorcells could also be one of the ultimate therapeutic methods for Tcell-mediated disease. The procedures presented here demonstrateT cell anergy induction using a complementary peptide as animmunogen.

The immune network theory has classically been characterizedas physiological autoimmunity. The formation of anti-Id Abs couldregulate the formation of Id Abs (disease-causing Abs) in exper-

imental autoimmune myasthenia gravis (EAMG) (1). The essenceof the network theory is also applied to the T-B cell interaction.Namely, the formation of anticlonotypic Abs could recognize theparatope of TCR and could regulate the Th2 function to protectagainst the development of EAMG (21). Thus, anticlonotypic Absinduced by a complementary peptide could also protect against thedevelopment of EAN, which is caused by a Th1 response. Theanti-Id Abs that are complementary to the Id of the binding site forAg could mimic the structure of the Ag (mirror image). Thus, theseanti-Id Abs could enhance or suppress the idiotypic response tothat Ag. Zhou and Whitaker (31–33) have also had success withcertain aspects of this procedure in an EAE model. In our exper-iment, monoclonal and polyclonal Abs against the complementarypeptide were anti-Id Abs and blocked the proliferation of Ag-spe-cific T cell lines. Although Abs to NAE 07-06 were not detectedin the natural course of EAN using a ELISA (data not shown), itis possible that Abs to NAE 07-06 may be induced during thecourse of EAN.

As mentioned above, vaccination with certain CDRs of TCReffector cells is also reported to alter the regulatory T cells and toprotect against the EAE (34, 35). These regulatory T cells recog-nize the CDR of effector T cells and suppress the effector cell

FIGURE 9. Histopathology of EAN 07-06-treated orsham-treated EAN. Longitudinal section of sciaticnerves 21 days after inoculation bovine P2 protein,60–70 (A and B). EAN 07-06-pretreated rats showedless cell infiltration around a vessel (A). Sham-treatedrats showed a heavy mononuclear cell infiltration (B).Hematoxylin and eosin stain (3200).

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function (36). In our experiments, the complementary peptideshared homology with the VDJ junction of TCR-b. Thus, it ispossible that immunization with a complementary peptide couldalso induce the regulatory T cells and play a role in regulation ofthe disease. As demonstrated in a previous report, Th2 responseswere not completely ablated in EAMG with this technique becauseof the existence of other T cell epitopes on the acetylcholine re-ceptor. However, in the present experiments, Th1 responses werecompletely depleted and were nonexistent in the peripheral nervein EAN. Because immunization with a complementary peptide forEAN 60–70 peptide can block the EAN induced by whole bovinemyelin or bovine P2 protein 60–70, our experimental results sup-port the idea that bovine P2 protein 60–70 is the major epitope ofthe Lewis rat model of EAN. Furthermore, no occurrences of dis-ease were observed for 3 mo in our experiment.

Our goal is to achieve an ideal therapeutic procedure to correctaberrant immune responses in autoimmune diseases. The proce-dures presented here and elsewhere (1, 2, 21) may have wide util-ity of selective immunological suppression for T and/or B cells. Asmentioned in a previous publication (1), the procedure requires noknowledge of the paratope sequences of B or T cells. The role ofthe precise pattern of amino acid hydropathy in protein and peptideshape or structure allows one to construct peptides, which presum-ably assume shapes or structures complementary to disease-asso-ciated epitopes. Thus, vaccination with a complementary peptideinduces the production of antiidiotypic and anticlonotypic Abs thecombining sites of which are complementary to and therefore re-active with Ag receptors on disease epitope-specific T and B cells.Also, Ab-induced T cell anergy may provide more precise infor-mation for understanding the mechanism of T cell regulation andT-B cell interaction.

AcknowledgmentsWe thank Dr. J. E. Blalock for much helpful discussion.

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