Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology...

6
Downloaded by guest on August 27, 2021 Downloaded by guest on August 27, 2021 Downloaded by guest on August 27, 2021 Downloaded by guest on August 27, 2021 Downloaded by guest on August 27, 2021 Downloaded by guest on August 27, 2021 Downloaded by guest on August 27, 2021

Transcript of Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology...

Page 1: Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology Preventionofexperimentalautoimmunemyastheniagravisby manipulationofthe ...

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

Dow

nloa

ded

by g

uest

on

Aug

ust 2

7, 2

021

Page 2: Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology Preventionofexperimentalautoimmunemyastheniagravisby manipulationofthe ...

Proc. Natl. Acad. Sci. USAVol. 90, pp. 8747-8751, September 1993Immunology

Prevention of experimental autoimmune myasthenia gravis bymanipulation of the immune network with a complementarypeptide for the acetylcholine receptor

(anti-Idotpic anibodIes/complementary peptde)

SHIGERU ARAGA*t, ROBERT D. LEBOEUF*, AND J. EDWIN BLALOCK*$*Department of Physiology and Biophysics and Center for Neuroimmunology, University of Alabama at Birmingham, Birmingham, AL 35294-0005; andtDivision of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, Yonago City 683, Japan

Communicated by George K. Davis, March 1, 1993

ABSTRACT Myasthenia gravis (MG) and experimentalautoimmune myasthenia gravis (EAMG) are caused, in part,by the production of autoantibodles against the main immu-nogenic region, amino acids 61-76, of the a chain of theacetycholine receptor (AChR). Theoretically, induction ofanti-idltypic (Id) antibodies (Abs) should be a higly specifictreatment for the disease by virtue of their potential ability toneubrize Abs to the AChR. We have tested this idea byattempting to evoke such anti-Id Abs by Immunization with apeptide (termed RhCA 67-16) encoded byRNA complementaryto the Torpedo AChR main immunogenic regon and deter-mining whether such treatment will prevent the development ofEAMG. Immunization with RhCA 67-16, but not a controlpeptide termed PBM 9-1, was found to elicit the production ofanti-Id Abs that blocked recntion of native Torpedo AChRby its Ab. This anti-Id Ab actity was ablated by incubation ofthe anti-RhCA 67-16 serum with RhCA 67-16, but not PBM9-1, prior to the asay for Ab binding to AChR. The anti-IdAb-induclng activy of RhCA 67-16 was confimed by theability to produce a rat monocional Ab to RhCA 67-16 thatshowed anti-Id activity for polydonal rat Ab reactive withAChR residues 67-76. Most importantiy, RhCA 67-16 immu-nization also prevented the development of EAMG In Lewisrats challenged with Torpedo AChIR (25% incidence versus90% in the controls) and diinished the AChR Ab levels inanimals injected with low doses of AChR. Our results suggesta therapy for MG and perhaps other autoimune diseasesthrough the induction of anti-Id Abs by peptide immunogens.

Myasthenia gravis (MG) is an autoimmune disease that ischaracterized by fatigability and muscle weakness. Thisdisease and its animal model, experimental autoimmunemyasthenia gravis (EAMG), are the result of interferencewith neuromuscular transmission by autoantibodies againstthe nicotinic acetylcholine receptor (AChR) on muscle (1-3).In MG, global immunosuppressive techniques, such as ste-roid treatment and/or thymectomy, are widely used thera-pies and are quite effective (4). Unfortunately, the nonspe-cific nature of the immunosuppressive therapy is accompa-nied by many side effects. One way of getting around theseside effects would be to influence the immune system in sucha way as to cause a decrease in only disease-associatedanti-AChR antibodies (Abs). According to Jerne's (5) net-work theory, the production of such disease-causing anti-AChR Abs could possibly be regulated by altering the bal-ance between the idiotype (Id)-bearing anti-AChR Ab and theappropriate anti-Id Ab. In fact, a reciprocal fluctuation ofthelevels of anti-AChR Ab and anti-Id Ab was observed in thesera of MG patients (6), and passive immunization with an

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

anti-Id Ab suppressed EAMG (7). Thus, due to its highdegree of specificity, anti-Id Ab treatment would appear to bean ideal therapy for MG.Although such anti-Id Abs are usually administered pas-

sively after having first been induced by immunization withthe Id-containing Ab to AChR, we have developed a methodfor actively inducing anti-Id Abs by using a peptide ratherthan Id Ab as the immunogen (8, 9). This approach is basedon the observation that peptides specified by complementarynucleotide sequences can specifically bind to each other,apparently as a result of their having complementary shapes(10-13). These interacting peptides, with presumed comple-mentary shapes, can in turn induce the formation of inter-acting pairs ofpolyclonal (8, 14) or monoclonal (15-17) Id andanti-Id Abs whose combining sites are complementary. Fur-thermore, anti-Id monoclonal Abs (mAbs) generated by thisprocedure have been shown to suppress hybridoma produc-tion of Id Ab in vitro (17, 18). A prediction from these resultswould be that immunization with a peptide (termed comple-mentary peptide) encoded by a nucleotide sequence comple-mentary to that of a proteinaceous antigenic site mightrepresent a means to lower disease-causing Id-bearing Ablevels such as those in MG or EAMG. Most importantly, thiscould be done without any prior knowledge of or immuniza-tion with the Id Ab as is required to conventionally evoke ananti-Id Ab response. The elucidation of the amino acidresidues ofthe main immunogenic region (MIR) ofthe AChRa subunit against which EAMG-inducing Abs are directedhas afforded an opportunity to test this idea (19, 20). In thisreport, we demonstrate that immunization with a peptide(denoted RhCA 67-16) encoded by a nucleotide sequencecomplementary to that of the MIR (residues 61-76 of theTorpedo AChR a subunit) prevents the development ofEAMG in Lewis rats challenged with Torpedo AChR. Thisprotection is associated with the production of an anti-Id Abas well as a diminution in the levels ofAb to the AChR if theEAMG-inducing dose of antigen is low.

MATERIALS AND METHODSAChR Purification. AChR was purified from Torpedo cal-

ifornica electroplax organs (Pacific Bio-Marine, Venice, CA)by using detergent solubilization and affinity chromatogra-phy on cobra toxin-Sepharose 4B as described by Froehnerand Rafto (21). The affinity column was prepared by coupling50 mg of Naja naja siemensis toxin (Sigma) to CNBr-activated Sepharose 4B beads (Pharmacia) (22). Purified

Abbreviations: MG, myasthenia gravis; EAMG, experimental au-toimmune MG; AChR, acetylcholine receptor; Ab, antibody; mAb,monoclonal Ab; Id, idiotype (idiotypic); MIR, main immunogenicregion; KLH, keyhole limpet hemocyanin; CFA, complete Freund'sadjuvant; TCR, T-cell antigen receptor; MBP, myelin basic protein.MTo whom reprint requests should be addressed.

8747

Page 3: Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology Preventionofexperimentalautoimmunemyastheniagravisby manipulationofthe ...

Proc. Natl. Acad. Sci. USA 90 (1993)

AChR was analyzed for the presence of four intact subunitsby SDS/PAGE and was quantified by binding of 125I-labeleda-bungarotoxin (Amersham). The specific binding activity ofthis preparation was 5.8 pmol of 1251-labeled a-bungarotoxinbound per pg of protein. Purified Torpedo AChR was storedat -80°C until used. Prior to immunization, excessive TritonX-100 was removed from the purified AChR by passingthrough an Extracti-Gel D column (Pierce).

Peptide Synthesis. The amidated form of the complemen-tary peptide, RhCA 67-16, was synthesized on a Biosearchpeptide synthesizer, model 2500, using fluoren-9-ylmethox-ycarbonyl (Fmoc) chemistry and was purified by reverse-phase high-performance liquid chromatography. The aminoacid sequence of the complementary peptide, RhCA 67-16,corresponding to the Torpedo AChR equivalent of the MIR,was derived by 5' to 3' assignment of amino acids to thenucleotide sequence complementary to that of TorpedoAChR mRNA encoding amino acids 61-76 (23) (Fig. 1). Forimmunization purposes, the peptide was coupled to keyholelimpet hemocyanin (KLH; Calbiochem) with glutaraldehyde(24) and was stored at 4°C until used.An amidated control peptide denoted PBM 9-1 was simi-

larly synthesized, purified, and used to produce rat antisera.PBM 9-1 is a complementary peptide for the first nineresidues of human myelin basic protein (MBP) and has thesequence NH2-Arg-Ser-Leu-Leu-Ser-Gly-Gly-Leu-Pro-NH2(16).

Immunization. Female Lewis rats aged 6 weeks wereobtained from Charles River Breeding Laboratories. In thefirst two experiments (Exps. 1 and 2 in Table 1), rats weredivided into four groups with six animals per group. Beforeimmunization with purified native Torpedo AChR, eachgroup was immunized five times with either the indicatedamounts of Torpedo AChR 61-76 complementary peptide(RhCA 67-16)-KLH conjugate in complete Freund's adju-vant (CFA; Sigma), KLH plus CFA, CFA alone, or phos-phate-buffered saline (PBS) alone. To elicit EAMG, all ratswere then immunized with various doses of purified nativeTorpedo AChR emulsified in an equal volume of CFA.Immunization and dose schedules are indicated in the appro-priate figures. Since the first two experiments showed nosignificant differences either in clinical scores or AChR Abtiters between the three control groups (i.e., KLH plus CFA,CFA, or saline), we used the KLH plus CFA-pretreatedgroups as the control for the third experiment.

Clinical Scoring. Rats were observed daily and weighedtwice a week. Clinical signs were scored on a scale rangingfrom 0 for normal, + for weak grip and cry, to + + + forsevere generalized weakness (25). Serum was collected fromthe tail vein under ether anesthesia, and samples were storedat -20°C until used.Ab Assays. Ab against the Torpedo AChR was determined

by an indirect ELISA. Briefly, 96-well flat-bottomed plates(Costar) were coated with the AChR at 1 ug/ml in PBS at 4°Covernight. Plates were then washed with PBS and incubatedwith 4-fold serial dilutions ofserum samples overnight at 4°C.The plates were then incubated with alkaline phosphatase-labeled goat Ab against rat immunoglobulins (Fisher Biotech,Atlanta, GA) at room temperature for 2 hr. After washingwith PBS, p-nitrophenyl phosphate (Sigma) was added, andthe color development was measured at 405 nm on a Bio-Rad

EIA reader model 2550. Ab titers were calculated by linearregression estimation and are presented as a logarithmicvalue above that prior to immunization. Ab titers againstRhCA 67-16 were determined as mentioned above exceptcoating was with peptide at 10 pg/ml.

Preparation of Rat Abs to Torpedo AChR 61-76 Comple-mentary Peptide (RhCA 67-16) and Native Torpedo AChR. Forpreparation of anti-complementary peptide Ab, three femaleLewis rats aged 8 weeks were immunized five times withKLH-coupled complementary peptide emulsified with anequal volume of CFA. Anti-native Torpedo AChR Ab wasprepared by immunization ofrats with 10 pg ofnative purifiedTorpedo AChR at 0 and 35 days. Ab titers were checked byan indirect ELISA. Blood was collected by cardiac punctureunder ether anesthesia. Sera were pooled and stored at-20°C until used.mAbs. A mAb (denoted TCM 240, IgG2b/K) was obtained

from a fusion from rats immunized with RhCA 67-16 coupledto KLH according to published procedures (26). Rat spleencells were fused with mouse myeloma cell line X63 Ag8.653(kindly provided by J. F. Kearney, University ofAlabama atBirmingham) by using a 50%6 (vol/vol) polyethylene glycolsolution (Sigma). Hybridomas were selected for monoclon-ality by three serial limiting dilutions and were grown inprotein-free medium (PFHM-II; GIBCO/BRL). TCM 240was selected for reactivity to RhCA 67-16 and was subse-quently found to bind to the complementary peptide forAChR residues 67-76. A control mAb, F28C4 (IgG-2a/A),against human MBP 1-9 was kindly provided by John N.Whitaker and Shan-Ren Zhou and has been described (17).mAbs were purified by ammonium sulfate precipitation fromprotein-free medium followed by dialysis against PBS.

Inhibition of Torpedo AChR Ab Binding to Native TorpedoAChR by Ab to Complementary Peptides. The Torpedo AChRantiserum was diluted in PBS and was coincubated with serialdilutions ofantisera to RhCA 67-16, pooled normal rat serum,rat antisera to PBM 9-1, TCM 240, or F28C4 on an AChR-coated plate at 4°C overnight. This was then followed by anordinary ELISA.

Statistical Analysis. All statistical analyses were done withthe two-tailed Student t test, two-tailed Mann-Whitney two-sample test, and Fisher's exact test.

RESULTSIn Vitro. Fig. 2 shows that antiserum to RhCA 67-16, but

not normal rat serum or rat antiserum to control peptide(PBM 9-1), caused a dose-dependent inhibition ofthe bindingof Torpedo AChR Ab to the native Torpedo AChR. Thefinding that about 30% of the Ab reactivity toward the AChRcould be inhibited by Ab to RhCA 67-16 is in agreement withprevious data showing that the Ab response to the nativeTorpedo AChR is of limited heterogeneity and particularlydirected to the amino acid residues to which RhCA 67-16 iscomplementary (19, 20). Specificity of the inhibition wasfurther demonstrated by the ability ofRhCA 67-16, but not acontrol peptide (PBM 9-1), to completely abrogate the inhib-itory effect of Ab to RhCA 67-16 on the binding ofAChR Ab(Fig. 3). These results strongly suggest that immunizationwith RhCA 67-16 induced an anti-Id Ab response againstId-bearing Abs specific for the Torpedo AChR, possibly

AChR 61-76 NH2-I D V R L R W N P A D Y G G I K

5'-ATT-GAT-GTG-AGG-CTT-CGC-TGG-AAT-CCA-GCC-GAT-TAT-GGT-GGA-ATT-AAA-3'3'-TAA-CTA-CAC-TCC-QGA-GCG-ACC-TTA-GGT-CGG-CTA-ATA-CCA-CCT-TAA-TTT-5'

RhCA 67-16 N I H P K A P I W G I I T S N F-NH2

FIG. 1. Torpedo AChR 61-76 and its complementary peptide, RhCA 67-16.

+ RNA- RNA

8748 Immunology: Araga et aL

Page 4: Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology Preventionofexperimentalautoimmunemyastheniagravisby manipulationofthe ...

Proc. Natl. Acad. Sci. USA 90 (1993) 8749

*- NRS-0<)- Ab to RhCA 67-16-0-D- Ab to PBM 9-1 E

c 12LO0t 1.0*0

@ 0.8C

.0O 0.6-.04

- 3 - 2 - 1Dilution (I1o0g)

0

-0--- TCM240* F28C4

n.nI.l * .* . .* * . . .* . * I

0 1 1 0 100 1000Concentration

FIG. 2. Inhibition of anti-AChR Ab binding to the native AChRby Ab to RhCA 67-16. Fifty microliters of a 1:500 dilution of serumcontaining anti-AChR Ab was incubated with 50 id of the indicateddilution of either normal rat serum (NRS), rat serum containinganti-PBM 9-1 Ab, or rat serum containing anti-RhCA 67-16 Ab. AnELISA for native Torpedo AChR was then done as described inMaterials and Methods. Each point represents the mean ± SD.

residues 61-76. Furthermore, the high percentage ofblockingof recognition of the AChR suggests that much of the anti-IdAb was apparently directed against the paratope, or combin-ing site, of the Id-bearing anti-AChR Ab, presumably againstresidues 61-76.To conclusively demonstrate that RhCA 67-16 was able to

induce anti-Id Ab against Ab to the MIR of the AChR,hybridomas were made from rats immunized with this com-plementary peptide. One such hybridoma, TCM 240, se-creted Ab reactive with the amino-terminal residues ofRhCA67-16 that are complementary to AChR residues 67-76 (S.A.and J.E.B., unpublished data). TCM 240, but not a mAb toMBP 1-9 (F28C4), was able to block the recognition ofAChRa-chain residues 67-76 by rat antisera to native TorpedoAChR (Fig. 4). Thus, RhCA 67-16 not only induced anti-IdAb, it caused the production of an anti-Id mAb that wasdirected toward the paratopes of the subpopulation ofAbs inpolyclonal antisera to AChR that recognize residues 67-76 ofthe AChR a subunit.In Vivo. To test whether the induction of anti-Id Ab by the

complementary peptide to the MIR would prevent the de-

30 -

C

.2 20-

._

C

I

--- RhCA 67-16 peptide

* PBM 9-1 peptide

I . . . .B... . . . . .... . . .. - - ..-I

0 .1 1 10 100Concentration (gg/ml)

FIG. 3. Blocking ofthe anti-Id activity ofanti-RhCA 67-16 serumwith RhCA 67-16. Fifty microliters of a 1:500 dilution of serumcontaining anti-AChR Ab and 25 p1 of a 1:2 dilution of serumcontaining anti-RhCA 67-16 Ab were coincubated with 25 of theindicated concentration of either RhCA 67-16 peptide or PBM 9-1peptide. An ELISA for native Torpedo AChR was then done. Eachpoint represents the mean ± SD.

FIG. 4. Inhibition of anti-AChR Ab binding to AChR 67-76peptide by mAb TCM 240. Fifty microliters of a 1:100 dilution ofserum containing anti-AChR Ab was incubated overnight at 4°C with50 A4 of the indicated concentration of either mAb TCM 240 orF28C4. This was followed by an ordinary ELISA on a plate coatedwith AChR 67-76. Each point represents the mean ± SD.

velopment of EAMG, rats were immunized with a RhCA67-16-KLH conjugate and were then challenged with nativeTorpedo AChR. In three separate experiments, there was avery marked overall lowering of the incidence (4/16 or 25%immunized with RhCA 67-16 versus 35/39 or 901% in thecontrol) and severity of the disease (Table 1). The threeexperiments were set up such that each successive trialemployed an increasing initial dose ofAChR in order to speedthe development of the disease and intensify the severity(Table 1). In each instance, immunization with the comple-mentary peptide was quite effective in providing protectionfrom the disease. Not unexpectedly, the disease incidence inthe peptide-treated group appeared to increase with thehighest dose of AChR. Additionally, as expected, we ob-served a positive relationship between dose of AChR andseverity and onset of EAMG symptoms.The increased severity and rapidity of the EAMG due to

higher challenge doses of AChR was paralleled by a fasterand more robust Ab response to the native Torpedo AChR(Fig. 5). The increasing dose of receptor appeared to antag-onize the suppressive effect of RhCA 67-16 treatment on theAb response to the AChR. Since there is not a parallel degreeof increase in the incidence of disease, we suspect that thehigher Ab titers may reflect a response to epitopes on theAChR in addition to the MIR, and these antigenic sites would

Table 1. Clinical incidence and severity of EAMG in Lewis ratsNo. of animalswith EAMG/ Severitytotal animals

Exp. Immunization (%o) 0 + ++ +++

1 RhCA 67-16 1/5 (20)* 4 1 0 0Control 16/17 (94) 1 12 4 0

2 RhCA67-16 1/6 (16)** 5 1 0 0Control 14/16 (87) 2 9 4 1

3 RhCA 67-16 2/5 (40) 3 2 0 0Control 5/6 (83) 1 1 2 2

Cumulative RhCA 67-16 4/16 (25)*** 12 4 0 0Control 35/39 (90) 4 22 10 3

Rats were immunized with the RhCA 67-16-KLH conjugate orKLH plus CFA (control) and challenged with native Torpedo AChR.Initial AChR doses were 5 pg (experiment 1), 10 pg (experiment 2),or 20 pg (experiment 3). *, Significantly different from the controlgroup atP < 0.0033; **, significantly different from the control groupat P < 0.0043; ***, significantly different from the control group atP<0.00001.

Ec

to0

00C0.000.0.4

1.2 -

1.1 -

1.0

0.9

0.8 -

0.7 -

0.0 i- 4

(gg/m 1)

* | s w w w w

n I................................ul

Immunology: Araga et aL

v

Page 5: Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology Preventionofexperimentalautoimmunemyastheniagravisby manipulationofthe ...

Proc. Natl. Acad. Sci. USA 90 (1993)

4.5-

3.5 -

2.5 -

1.5 -

5.5

4.5

V

.0

t 3.5--.00

2.5

5.5 -

4.5 -

3.5 -

2.5 -

1.5-

5 5 10 10 10

v v+

-0-- - RhCA67-16+-@ Control

0

10

5 10 15

10 10

-0--- RhCA 67-16* -Control

0 5 10

20 20 20

-0-- - RhCA 67-160 Control

0 10 15Weeks

FIG. 5. Effect of increasing doses of Torpedo AChR on thedevelopment of anti-AChR Ab in Lewis rats preimmunized withRhCA 67-16. Animals were preimmunized with the complementarypeptide or controls as described in Materials and Methods. InitialAChR immunization doses were 5 (A), 10 (B), or 20 (C) Ug injectedat the times denoted with vertical arrows. Each point represents themean ± SEM. A, B, and C correspond to experiments 1, 2, and 3 ofTable 1, respectively. *, P < 0.01; **, P < 0.001; +, P < 0.05; ++,P < 0.02.

not be disease related or affected by Ab to RhCA 67-16.Alternatively, but less likely, the anti-AChR Ab in vivo couldbe coupled with the anti-Id Ab. The complex would theneffectively be dissociated by the AChR in the ELISA. Theformer possibility is much more likely and fits well with whatis known of the disease. That is, although the degree of

severity of EAMG can be increased with a larger dose ofAChR, severity does not correlate well with overall Ab titersto the intact AChR in either EAMG orMG. This suggests thatmany Abs are made to sites on the AChR that are not diseaserelated. Of course, the total complex Ab response to a verylarge antigen with multiple epitopes, like the AChR, wouldnot be expected to be markedly inhibited by anti-Id Abs forAbs recognizing a single epitope.

DISCUSSIONAn ideal treatment for an autoimmune disease such as MGwould be one that specifically affects the immunologic reac-tivity leading to the disorder without compromising theimmune system's ability to react to foreign antigens. Anti-IdAbs, of course, represent such specific agents, and harness-ing the immune system itselfto correct autoimmune disorderswould seem to be the method of choice. The problem,however, is that for this method to be effective the disease-causing Abs or T cells must have a common characteristicsuch as a shared or cross-reactive Id for Abs or restricted Vgene usage for the T-cell antigen receptor (TCR). Unfortu-nately, even in a situation where there is a restriction ofTCRVgene usage, as is seen in the rat model for multiple sclerosis,experimental allergic encephalomyelitis, administration ofmAb to the Vp chain (27) or immunization with peptidesequences that define this family ofTCRs apparently resultsin deletion of an entire family of T cells expressing thedisease-associated Vp gene (28). In contrast, in a B-cell-mediated disorder such as MG or its model EAMG, one isconfronted with multiple Ids on the various disease-causingMIR-directed Abs, and thus it is difficult to know theparticular Id Abs that should be used for the elicitation oftherapeutic anti-Id Ab (7). Thus for both T- and B-cell-mediated autoimmune diseases, there is a lack of specificityto the immunotherapy in spite of a great deal of detailedinformation on the etiology of the disorder.The present procedure would seem to represent a more

precise type of targeting of an Ab response that might beparticularly useful for the treatment of autoimmune diseasesin general. This specificity apparently results from the abilityofa complementary peptide immunization to evoke an anti-IdAb response specifically targeted to the most importantshared element of disease-causing Abs or T-cell receptors,the combining sites that define their antigen specificity. Forexample, even though multiple Ids may be expressed on Absto the MIR of the AChR, this population of Abs will sharespecificity in binding the MIR and will be inhibited by thepopulation of Abs against the complementary peptide to theMIR. In other words, the complexity of the response to theMIR will be matched by an equally complex response to itscomplementary peptide. Furthermore, this apparent abilityof anti-complementary peptide Abs to recognize differentAbs with shared antigen specificity has now been extended todifferent recognition molecules with the same specificity. Inthis case, mAbs to a complementary peptide for the first nineamino acids ofMBP [MBP-(1-9)] recognized not only mAbsto MBP-(1-9) but also the TCR from CD4-positive T cellsspecific for this same peptide (29). In addition, these anti-complementary peptide Abs blocked functions mediated bythis TCR and inhibited adoptive transfer of experimentalallergic encephalomyelitis by MBP-(1-9)-specific T cells (29).In general, the results from the previous (29) and presentstudies provide a specific approach to manipulating theimmune network that may be useful in the design of addi-tional therapies for the treatment of autoimmune diseases ofT- and B-cell origin, and more specifically the present resultssuggest an alternative or additional therapy for MG.

We thank Diane Weigent for expert editorial assistance. This work

I

1 .5 m I I I

i I I I

8750 Immunology: Araga et aL

5.51 +

* *

Page 6: Prevention - PNASProc. Natl. Acad. Sci. USA Vol. 90, pp. 8747-8751, September 1993 Immunology Preventionofexperimentalautoimmunemyastheniagravisby manipulationofthe ...

Proc. Natl. Acad. Sci. USA 90 (1993) 8751

was supported in part by National Institutes of Health GrantsDK38024 and NS29719 and a grant from the Muscular DystrophyAssociation to J.E.B. and National Institutes of Health GrantCA54290 to R.D.L. S.A. was kindly supported in part by NationalInstitutes of Health Grant NS29719 and as a Visiting AssistantProfessor by funds from the Faculty ofMedicine, Tottori University.

1. Fambrough, D. N., Drachman, D. B. & Satyamurti, S. (1973)Science 182, 293-295.

2. Patrick, J. & Lindstrom, J. (1973) Science 180, 871-872.3. Sugiyama, H., Benda, P., Meunier, J. C. & Changeux, J. P.

(1973) FEBS Lett. 35, 124-128.4. Genkins, G., Kornfeld, P., Paptest, A. G., Bender, A. N. &

Matta, R. J. (1987) Ann. N. Y. Acad. Sci. 505, 505-513.5. Jerne, N. K. (1974) Ann. Immunol. C 125, 373-389.6. Dwyer, D. S., Bradley, R. J., Urquhart, C. K. & Kearney,

J. F. (1983) Nature (London) 301, 611-614.7. Souroujon, M. G. & Fuchs, S. (1987)Ann. N. Y. Acad. Sci. 505,

676-682.8. Smith, L. R., Bost, K. L. & Blalock, J. E. (1987) J. Immunol.

138, 7-9.9. Blalock, J. E., Whitaker, J. N., Benveniste, E. N. & Bost,

K. L. (1989) Methods Enzymol. 178, 63-74.10. Bost, K. L., Smith, E. M. & Blalock, J. E. (1985) Proc. Natl.

Acad. Sci. USA 82, 1372-1375.11. Blalock, J. E. (1990) Trends Biotechnol. 8, 140-144.12. Clarke, B. L. & Blalock, J. E. (1991) in Antisense Nucleic

Acids and Proteins: Fundamentals and Applications, eds.vander Krol, A. R. & Mol, J. N. M. (Dekker, New York), pp.169-186.

13. Jarpe, M. A. & Blalock, J. E. (1993) in Peptides: Design,Synthesis, and Biological Activity, eds. Basava, C. & Anan-thaamaiah, G. M. (Springer, New York), in press.

14. Pascual, D. W. & Bost, K. L. (1989) Peptide Res. 2, 207-212.

15. Whitaker, J. N., Sparks, B. E., Walker, D. P., Goodin, R. &Benveniste, E. N. (1989) J. Neuroimmunol. 22, 157-166.

16. Zhou, S.-R. & Whitaker, J. N. (1990) J. Immunol. 145, 2554-2560.

17. Zhou, S.-R. & Whitaker, J. N. (1992) Clin. Immunol. Immu-nopathol. 63, 74-83.

18. Zhou, S.-R., Whitaker, J. N. & Dwyer, D. (1990) J. Neuroim-munol. 29, 113-124.

19. Barkas, T., Gabriel, J. M., Mauro, A., Hughes, G. J., Roth, B.,Alliod, C., Tzartos, S. & Ballivet, M. (1987)J. Biol. Chem. 263,5916-5920.

20. Tzartos, S., Kokla, A., Walgrave, S. L. & Conti-Tronconi,B. M. (1988) Proc. Natl. Acad. Sci. USA 85, 2899-2903.

21. Froehner, S. C. & Rafto, S. (1979) Biochemistry 18, 301-307.22. Wofsy, L. & Burr, B. (1969) J. Immunol. 103, 380-382.23. Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani,

Y., Hirose, T., Asai, M., Inayama, S., Miyata, T. & Numa, S.(1982) Nature (London) 29, 793-797.

24. Reichlin, M. (1980) Methods Enzymol. 70, 159-165.25. Lennon, V. A., Lindstrom, J. M. & Seybold, M. E. (1975) J.

Exp. Med. 141, 1365-1375.26. Linddell, J. E. & Cryer, A. (1991) in Cell Fusion, Hybrid

Selection, and Cloning: A Practical Guide to MonoclonalAntibodies, eds. Linddell, J. E. & Cryer, A. (Wiley, Chich-ester, U.K.), pp. 85-104.

27. Acha-Orbea, H., Mitchell, D. J., Timmermann, L., Wraith,D. C., Tausch, G. S., Waldor, M. K., Zamvil, S. S., McDevitt,H. 0. & Steinman, L. (1988) Cell 54, 263-273.

28. Hashim, G. A., Vandenbark, A. A., Diamanduros, T., Car-valho, E., Srinirasan, J., Jones, R., Vainiene, M., Momrson,W. J. & Offner, H. (1990) J. Immunol. 144, 4621-4627.

29. Zhou, S.-R. & Whitaker, J. N. (1993) J. Immunol. 150, 1629-1642.

Immunology: Araga et aL