Experimental allergic encephalomyelitis in Cynomolgus Monkeys: quantitation of T-cell responses in peripheral blood.

Experimental Allergic Encephalomyelitis in

Cynomolgus

Monkeys

Quantitation

of T

Cell Responses in Peripheral Blood

L. Massacesi, N. Joshi, D. Lee-Parritz,*A. Rombos, N.L. Letvin,*and S.L. Hauser DepartmentofNeurology, Massachusetts General Hospital, Boston, Massachusetts 02114; and *NewEnglandRegionalPrimateResearch Center, Southborough, Massachusetts 01772

Abstract

Chronic

relapsing-remitting

experimental allergic encephalo-myelitis(EAE)wasinduced incynomolgusmonkeysby a

sin-gle immunization with a homogenate of human brain white

matter(BH) inadjuvant. Proliferative T lymphocyteresponses toBH,tomyelin basic protein (MBP), but not toproteolipid protein, weredetected in peripheral blood mononuclear cells

(PBMC)of allanimals and persisted until theirdeath or,in surviving animals,for>

10

mopostimmunization. Responses ofhigher

magnitude

tendedto beassociated with

fatal,

com-pared with

nonfatal, episodes

of clinical EAE.Thefrequency of MBP-reactiveTcellsinPBMC of

animals

withacute EAE was

quantitated withasoftagarcolonysystem;theratio ofTcells

thatproliferated

specifically

toMBP wasestimatedatbetween

5 and 20per

10'

PBMC.Asimilarfrequency of peptide-speci-ficTcells wasestimatedfrom PBMC ofmonkeys immunized withasynthetic

14-mer

peptide

corresponding

to aregionnear

the carboxy terminus of MBP.

Thus,

autoantigen-reactive

T

cellscanbedetected in thecirculation

throughout

thecourseof chronicEAE,are

predictive

of disease

severity,

andoccurat a

frequency similar to thatestimated to be present in humans with multiple sclerosis. (J. Clin. Invest. 1992.

90:399404.)

Key words:experimentalallergicencephalomyelitis * multiple

sclerosis*myelin basic protein

Introduction

Experimental

allergic

encephalomyelitis (EAE)1

isan

autoim-mune

disease of

thecentralnervoussystem

(CNS)

thatis

me-diated,

inmost

species studied, by

Tcells reactive with

myelin

basic

protein (MBP) ( 1, 2).

Becauseof

pathologic

similarities between EAE and the human

demyelinating disease, multiple

sclerosis

(MS),

EAE has

long

served as amodel system for

testing

of

potential

new

therapies

for MS

patients.

Trials in

EAE have for the mostpart relied upon

clinical

end

points

(e.g.,

severity

of

paralysis)

in theassessmentof disease

severity

Address reprint requests to Dr. Hauser, Neuroimmunology Unit, Warren324,MassachusettsGeneralHospital,Boston,MA02114.

Receivedfor publication20September1991and inrevisedform9 January1992.

1.Abbreviations usedin thispaper: BH, humanbrain white matter homogenate; CFA, complete Freund'sadjuvant; CSF, cerebrospinal fluid;EAE,experimental allergic encephalomyelitis;MBP,myelin ba-sicprotein;MS,multiplesclerosis;PLP,phospholipid protein.

(3). Other proposed markers of theEAE process,forexample,

thequantitation of CNS inflammation (4-6)orthe measure-mentof various nonspecific immuneparameters (7, 8), have notgained widespreaduse.Some markers correlate poorly with theclinical illnessscore

(4-6)

and may

require

sacrifice of the

subjects.

These

limitations

are

particularly evident

instudies of chronicrelapsing-remitting EAE,the most appropriate model systemfor humanMS (9).

The current experimentswere designedto assess the

hy-pothesis

thatantigen-specific immuneresponsesmeasuredin peripheral blood providean informative markerofthe EAE process. Antigen reactivity from lymph node and splenic T

cells has been studied extensively in EAE, whereas little is knownof the T cellresponse

by

peripheral

blood mononuclear

cells (PBMC). By contrast, in humans with MS, PBMC re-sponseshave beencharacterized indetail.Inparticular,recent

studies indicate thatsomePBMCresponses to MBP are char-acteristic of MS ( 10-14); for example, a high frequency of

reactive cells (12) or the preferential recognition ofcertain

MBP

epitopes

(14)maybepresent.

Chronic relapsing-remitting EAE has been described in nonhuman primates (15-19), and these speciesare particu-larlyattractiveasMS modelsbecauseoftheir closephylogeneic relationshiptohumans,theirlargesize, and theiroutbred

char-acteristics. We describe in thisreport animproved model of

EAEin thecynomolgus monkey. Relapsing-remitting disease

canbeinduced insomeanimals with onlyone

injection

and in the absence

of corticosteroid

treatment( 16, 18). We

demon-stratethat PBMCTcellresponsescorrelatewith disease

sever-ity.

Furthermore,wefind that thefrequency of PBMC reactive

to MBPinEAEis similartothatdescribed in some

patients

with MS ( 12). Thus, inachronicorgan-specific autoimmune

disease,

PBMCresponsesreflect disease

severity,

andtheir mea-surement

provides

a

quantitative

markerof the

underlying

au-toimmunestate.

Methods

Induction ofEAE in Macacafascicularis. Macacafasciculariswere maintained in the New EnglandRegionalPrimate Research Center colonies. The animals used in thisstudywerecaredfor in accordance with theguidelines ofthe CommitteeonAnimalsofthe Harvard Medi-calSchool and thoseoftheCommitteeonCare and UseofLaboratory

Animals of the Institute of Laboratory Animal Resources, National ResearchCouncil.Amaximumof10 ml ofblood every other weekor1

mlofcerebrospinalfluid(CSF)obtainedby lumbar punctureon two

occasionswastakenfrom each animal.

Atotalof 18 animalswereimmunized with 200 mgof fresh-frozen postmortem humanbrain whitematterhomogenate(BH)emulsified withcomplete Freund'sadjuvant (CFA) containingkilled

Mycobacte-riumtuberculosis (H37Rastrain, 3 mg/ml). Intradermalinjections

(totalvolumeof 0.5mlperanimal)weredivided in four sitesonthe dorsalaxillaryandinguinal region.Threeof these animalswere

preim-munized withasynthetic 14-merpeptidecorresponding to a region

J.Clin.Invest.

© TheAmericanSocietyfor Clinical Investigation, Inc. 0021-9738/92/09/0399/06 $2.00

nearthecarboxylterminus(e.g.,amino acids 156-169) of MBP, in eitherCFA/H37Raorincomplete Freund's adjuvantsupplemented

withmuramyl dipeptide(2

Ag);

EAE was induced in these animalsby immunizationwithBH/CFA-H37Ra.

Manyanimalsdevelopedinjectionsite ulcerations2-5wk postim-munization.Inthese cases,cephalexin(20 mg/kg twice daily for 10 d) wasadministeredintramuscularlytopreventsecondaryinfection.

ClinicalscoringofEAE. Animalswereobserved dailyforsigns of disease. Thefollowing scoringsystemwasemployedtocategorizethe diseasestate:

Score Clinicalstate

0 Normalneurologicalexam

I Lethargy,anorexia,weight loss

2 Ataxia,tremor

3 Blindness,paraplegia,hemiplegia

4 Quadriplegia,quadriparesis

5 Moribund

Proliferation studies. PBMC werepreparedfromheparinized ve-nousbloodwithin 3 hof venipuncturebydensity gradient centrifuga-tion using dextrandiatrizoate(d 1,080).PBMC(2X 01 perwell)were

culturedinatotal volumeof200 ul in round-bottom microtiter plates (FalconPlasticsOxnard, CA)in culture medium consisting of RPMI 1640supplementedwith 10% Controlled Processed Serum Replace-ment-2 (Sigma Pharmaceuticals, St. Louis, MO), 100 mM L-gluta-mine(GibcoLaboratories,GrandIsland, NY), 25 mM Hepesbuffer (Sigma),and0.1%penicillin-streptomycin(Gibco).Tosomewells the followingantigens(final concentration)wereadded:(a) human MBP (50 ug/ml)preparedby the methodofDeibleretal.(20);(b)human phospholipid protein(PLP)apoprotein (10

;gf/ml),

preparedbythe method of Lees and Sakur (21 ); (c) human BH,0.1%,sonicatedand irradiated;(d)asynthetic14-merpeptidecorrespondingtotheregion 156-169(FKLGGRDSRGGSPM)ofhumanMBP(100ug/ml); (e) concanavalinA(ConA)(4Mg/ml)(Sigma);(I) phytohemagglutinin

(PHA;4,ug/ml) (Sigma).After5 dofincubation,[3H]thymidine( 1 MCi/ml) was added for another 16h,cellswereharvested,and thymi-dine incorporation measuredinascintillation counter.Proliferative responseswereexpressedasstimulationindexes,definedasthe stimu-lated culturecountsperminute perunstimulatedculture counts per minute intriplicatewells.

Soft agarcolonyquantitation of antigen-reactive Tlymphocytes.

ThismethodwasmodifiedfromSrednietal.(22).PBMC(2X

106/

ml)werecultured for 3 d inmediumsupplementedwith 10% human ABserumin the presence or absenceofMBP(50 ug/ml). Cells were thenpelletedandresuspendedinmediumsupplementedwith20%

hu-man ABserum, agar was added (0.32% finalconcentration) in flat bottomwells, and thesuspensionwasoverlaidon asecond layerofagar (0.5%) containingmediumsupplementedwith 20% humanABserum and 50 ug/ml MBP. The number ofcolonieswere counted under blindedconditions between 6 and 11 d later usinganinverted micro-scope.The numberofMBP-specificcolonies was estimated by subtrac-tion of the number ofcolonies in the MBP from those in control wells.

Results

Clinical EAE in

cynomolgus monkeys. Signs

ofEAE

developed

inall 15animals immunizedwithBH.Theclinical coursetook

theform ofanacuteprogressive illness leadingtodeath within 6-12d(n=. 11)ortocomplete recovery followedby the devel-opmentofa

relapsing-remitting

chronic disease(n = 4). The

time ofonsetoftheinitialillnessrangedfrom day 17 to day 55

afterimmunization.AttacksofEAEaccompaniedby aclinical

score

of

4 or greaterwere

invariably associated

with afatal outcome. No

difference

in the

degree of

CSF

pleocytosis

differ-entiated fatal

from

nonfatal

attacks

(Table I).

Inthefour

animals

that

survived

theinitial attack

of

EAE,

laterelapses,

numbering 1-6, occurred

in allcases.These re-lapses

resulted

in

death

in two

animals;

two late

survivors

(>

10

mo

postimmunization)

remain.

One

long-term survivor

had a

single

relapse

of

14 d

duration followed

by

complete

clinical

recovery. One

animal

developed

five

relapses of

vari-able

duration followed by

complete

recovery. Oneanimal

be-came

permanently blind

272 d

postimmunization.

Most

ani-mals

achieved complete clinical

neurologic

recovery

after

re-lapses that

did

not result in

death.

Fig.

1

summarizes

the

clinical

characteristics of

EAEinsix

representative

animals.

Several

clinical

syndromes

seen in these

monkeys

resem-bled

those presentin human

demyelinating

disorders.

These included

bilateral

optic neuritis

with markedvisual

disability

but

preserved

strength

and

coordination;

pure

ataxia,

acute

vomiting, and

instability

of

gait;

and

progressive

myelopathy

evolving

overseveral

days.

Sensitization

to EAE

by

MBP

156-169.

Three

animals

were

preimmunized

witha

synthetic peptide

corresponding

to

the

156-169

carboxy-terminal

region of

MBP. No

signs

of EAE

werenoted

during

a 14-mo

period of observation.

These

ani-malswerethen

immunized with

BH/CFA.

Asshownin

Fig. 2,

clinical

signs developed

in this group between 9 and11d

after

immunization, compared

to

with

onsetno

earlier

than

day

17 in the

animals

not

preimmunized

in

this

manner.The

clinical

coursein

peptide

preimmunized

animalswas

otherwise

identi-caltothatseenin

nonpreimmunized animals: either

a progres-sive

syndrome leading

todeath(n=2)or a

relapsing-remitting

course(n=

1).

Proliferative responses

tobrain

antigens. PBMC

prolifera-tive responses to BH, MBP, PLP, andmitogen (Con A or

PHA)wereassessed

prospectively.

Asillustrated in Fig. 1 for six

representative

animals,

proliferative

responsestoBHandto MBPweretypically absent before immunization and in the

early

postimmunization period,

butbecamepositiveat the on-set

of clinical

signs

of EAE.Inanimals with

chronic,

relapsing-remitting

EAE(Fig. 1,

lowerpanels), positive PBMC responses

to BH and MBP, once present,

persisted

forthe duration of study,up to 10 mo

postimmunization.

Stimulation

indexes in chronic EAE werelow,generally< 10times background val-ues.InbothacuteandchronicEAE,proliferative responsesto PLP weregenerally low or undetectable, although an

occa-sional animal did mount a

detectible

PLP-specific response (Fig. 1,bottom right).

Table I.Stimulation Index Correlations

PBMCstimulation index BH MBP CSF

cells/mm'

Nonfatal attacks 6.3±2.1 5.7±1.5 307±122 (n= 15) (n= 16) (n=7) Fatalattacks 13.0±3.6 8.2±3.4 352±158

(n

=

8)*

(n=

8)

(n=

7)

400 Massacesietal.

+ a 9-0 V. W -J 0 0 a I* 0e 04 I-aMSa7 ZNLW

S:

'I-0 0 0 0000 0 000rw0 0 0 0 om -a le W -MAP94r_ S 'no q 0 0 0 :n% -o In : fI o to IC C2 M 'UpuI 'o 4- -a V 0 01 v

I

I

a- -* a-S -e 4)CO . 4)8 -d° V to. 0 I-L cu

-

E

oC 0 Cu 0 a .eo oo Cu 0 .¢' -CX o

o

Cu8!

0

'.E

A

8

D} cd *; 0 C_ o _* Cu 0 o __ 0 o o sD5 Cdu 044) ° 0 0U v

A-0 0 0 u~~ ao * 4)d)4-06 0 0 0 000 0 0 cmeV cm o o a om 0 I* e -IV 0aipul&AMSL 0 0 0000 0 00 0 - 0 - - -ws 'UPwSWOW5 0 uO OE* O -C-D305- U lepIO uoo *"la ZAPM/ UORCIM I I I I III A ail II 0 0 0 0 0 0 0 v fw4 &a :u : l n t : n it' 0 co0 0 tm-W C.

5 4 3 2 1 0 5 4 3 2 1 0 5 4 3 2 0' I

t

fI

Lf llsss||sss

I.'

....L...J 0 10 20 30 40 50 60 70 80 90 100 DaysPi

Figure2.The clinicalcourseofEAE inthreecynomolgus monkeys

immunized with the 14-merMBPpeptidebeforereceivingasecond immunization with BH. Preimmunizationwasaccomplished using

either CFA(middle panel)orFreund'sadjuvant supplementedwith

muramyl dipeptide (topand bottompanels)asadjuvant.Clinical

signsofEAEwerepresent inallanimals between 9 and11d

postim-munization(PI).For eachanimal,the clinicalcourse(y-axis)is

il-lustratedfor the timeperiodafter immunization with whitematter

homogenate (x-axis).

Table I summarizes PBMCresponsestoBH and MBPin relationtotheonsetof fatalandnonfatalattacksof EAE. The magnitude ofthe proliferativeresponsetoBH and MBP varied

widely between animals. Immunizationwasassociated witha

fourfoldincrease in mitogen responsivenessandwiththe

devel-opment of antigen-specific PBMC proliferative responses. A

general correlationwasobserved betweenthemagnitudeofthe

responseto BH and the severity of individualattacks: higher stimulationindexes tendedtobeassociated with fataloutcome (Table I). No differences in the proliferativeresponseto

mito-gensdifferentiated fatal from nonfatal attacks (not shown).

Quantitation

of

the

circulating

Tcellresponse. Insix

ani-mals immunized with BH/CFA, the number of T cells specific toMBPwereenumerated fromperipheral blood byuse ofa soft agarcolony system. The frequency of MBP-specific T cells could be estimated atbetween 5 and 20 per 106 PBMC. As

shown in

Fig. 3,

the number of MBP colonies and the kinetics of theirappearance

postimmunization

in three individual ani-malscorrelated with themagnitude and kinetics ofthe prolifer-ativeresponseof whole PBMCtoMBP.

In thethree animalspreimmunized with the MBP 156-169

peptide, peptide-specific

colonies, enumeratedonday 55

pos-timmunization,

wereestimatedat 1, 6, and 9per106PBMC,

respectively. PBMC proliferative responses to this peptidewere generally loworundetectible, both inpeptide-preimmunized animals(datanotshown)and alsoinanimals immunizedonly

with BH/CFA (for example, Fig. 1,topright).

Discussion

Thecurrentreport representsthefirst

study

of

circulating

anti-gen-specific

Tcellsin

relapsing-remitting

EAE.The

large

size

of

theanimals and the chroniccourse

provided

the

opportunity

for serial

phlebotomy

andmeasurement of

antigen reactivity

during

thecourse

of

the

disease.

Specific proliferative

responses toBHandtoMBP werepresentin allanimalsattheonsetof clinical

signs of

EAE, and theseresponses

persisted

in

surviving

animals

for

the

duration of study. Strong

proliferative

re-sponses were associated

with fatal

attacks in mostcases.

Re-sponses toPLP,a

myelin protein capable of

inducing

EAEin

some

species (23),

were

generally

undetectable or

of

lower magnitude thanwerethoseto BH orMBP.

In

earlier

studies ofacuteEAEin various

species,

PBMC

responses to MBP weredetectedin some(24-27) but notin other

(28)

models. Inthe

guinea pig,

thisresponsewas

tran-sient andpresent

only

during

theacute

phase

of disease

(26).

Current

primate data, by

contrast, indicate that

antigen-reac-tive PBMCwerepresentin all

survivors of

acuteEAE,even

for

periods

upto10mo

postimmunization

and

long after

the

dis-solution of

a

visible

antigen

depot. The

persistence of

these

circulating

cellsmayhavecontributedtothe

high

relapserate

thatoccurred inthis

species.

A

soft

agarmethodfor the enumeration

of

antigen-reactive

Tcells

permitted

anestimate of the frequency of circulating

MBP-reactive

cellstobe made.In acuteEAE,between5and 20

per

106

PBMC

proliferated specifically

toMBP.

Unpublished

datafromourlaboratory alsoindicate thatalowerfrequency of MBP-reactiveTcells,

estimated

at1 per

106

PBMC,are present

in

clinically

stableanimals with

chronic

EAEstudied 6-18mo

postimmunization. The true clonalfrequency may be higher thanthese

estimates, assuming

limited

sensitivity

of theassay

(particularly

with respect to the CD8+ repertoire). On the otherhand, onlyafraction ofTcells thatproliferateto MBPin vitroarelikelytobeencephalitogenic invivo (29). These

con-siderations

notwithstanding,

it appearsthat, evenduringthe courseofacutefulminantEAE,few antigen-reactive cells are presentin

proliferation

assaysthattypicallyemploy

105

to

106

PBMCperwell.

Inhumanswith MS, smallincreases inPBMC responses to MBParereportedto

distinguish

patients from controls

(10-14). Onerecentestimate indicatedthat between27 and 52 per

106 PBMCwereMBP-reactiveasmeasured byMBP-induced

402 Massacesietal.

I

2

I

O MBP Stimulation

Index

MBP

Colonies/

IcfPBMC

Dws P/

Figure 3. Correlation be-tweenthe PBMCstimulation index and the frequency of MBP-specificT-cellcolonies. Datafrom threeimmunized animalsareshown. The stimulationindex, or the numberof colonies detected per106PMBC,is displayed

onthe y-axis and the day postimmunization isshown onthe x-axis. The shaded area representsthe onsetof clinicalsigns ofEAE.See text for details.

interferon-y

secretion (12). A lower estimate (1-5 per 106 PBMC)wassuggested inasecond study of MBPprecursorsin PBMC of MS patients (H.Offner and A. Vandenbar, personal communication). Thus, similar frequencies of circulating MBP-reactivecellsmaybepresentinboth MS and EAE.

Inthe related speciesRhesus (Macaca mulatta), EAEwas

induced by immunization with peptides correspondingtothe carboxy-terminal region of MBP (30-33). In cynomolgus monkeys,wefoundthatsigns of EAE didnotdevelop whena similarimmunizationregimenwasemployed, but thata mark-edly precociousonsetof EAEfollowedsubsequent immuniza-tion withBH/CFA-H37Ra. Thissuggeststhatpeptide immuni-zationmayhavesensitized the animalstoEAE. It issurprising that thesameregion of MBPmayplayarole ininducing EAE in most ifnot all individuals belonging to two distinct (al-though closely related) primate species.Ininbredrodents, the encephalitogenicTcellresponsetoMBPhas been showntobe strain andspecies specific, andtorecognize restricted regions of MBP inthegeneration ofanencephalitogenic response (re-viewed in reference 34). In H-2U mice (B10.PL and PL/J), for example,theamino terminus ofMBPis immunodominant for EAE, whereas in H-2' mice(SJL) the 89-101 regionis

domi-nant. Innonhuman primates extensive polymorphismof ma-jor histocompatibility complex (MHC)geneproducts is

pres-ent(35, 36) that might contributetodiversityintheresponse

toMBP.Heterozygosity for MHC haplotypesinmany individ-uals, coupled with additional MHC diversity resulting from interisotypyortranscomplementation, might broaden the po-tential forgeneration ofTcellresponsesagainst multiple

epi-topesofagiven antigen. In marmosetprimates, for example, wehave found that T-cell lines respondingtodifferentregions ofMBPcan mediatepassivetransfer EAE(Massacesi etal., manuscript in preparation), and in MSpatients nosingle re-gion of MBP has been identifiedasimmunodominant( 14,37, 38).Inmacaques,sensitization to EAEbythecarboxyterminal region of MBPsuggeststhat effectivepresentationofepitopes within thispeptidetoTcells is mediatedbyavariety of

differ-entMHChaplotypes.

In conclusion, relapsing-remitting EAE in cynomolgus monkeys is characterized by circulating T cell responses to

whitematterantigens that persistoverlong periods of time and thatbroadly mirror the clinicalillness.Furthermore, ourdata indicate thatachronicinflammatorydisease inprimatesmay result fromrelatively small increasesinthecirculatingpoolof cellsrespondingtothetargetantigen.Inrodents,intravenous

passive transfer ofasfewas30,000 cloned MBP-reactive T cells can produce EAE (39), demonstrating the remarkable effi-ciency ofsomeencephalitogenic T cell populationstomediate disease.Serialassessmentofautoantigen-reactive T cells from peripheral blood is thusaquantifiable immunologic marker of the diseaseprocessthatcanbeeasily appliedtotrials oftherapy inEAE, and similar strategiesmay proveuseful formonitoring of humans with MSor other T cell-mediated inflammatory diseases.

Acknowledgments

The authorsthank Dr. Marjorie Lees,whokindly provided purified proteolipid apoprotein, and Drs. Richard Fisherand C. Ramachandra ofBiogen,Inc.(Cambridge, MA),whosynthesized the 14-mer peptide

of MBPusedinthisstudy. The authors also wishtothank Leslie

Hop-perfor technicalassistance.

LucaMassacesi was apostdoctoral fellow oftheItalian Multiple

SclerosisSociety. StephenL.Hauser isaHarry Weaver Neuroscience

Scholar of the NationalMultiple Sclerosis Society (NMSS). This work

wassupported bygrantsfrom the NMSS, theMathersFoundation,and the Rita AllenFoundation.

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