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Role of the thymus in induction and transfer of

vaccination against adjuvant arthritis with a T

lymphocyte line in rats.

J Holoshitz, … , A Matitiau, I R Cohen

J Clin Invest.

1985;

75(2)

:472-477.

https://doi.org/10.1172/JCI111722

.

Adjuvant arthritis is an experimental disease of rats induced by immunization to antigens of

Mycobacterium tuberculosis. Our observation that arthritis could be induced in irradiated

rats by the A2 line of T lymphocytes in the absence of mycobacterial antigens suggested

that adjuvant arthritis is an autoimmune disease. Moreover, the A2 line could be used to

vaccinate unirradiated rats against the subsequent induction of adjuvant arthritis by active

immunization to Mycobacteria. In the present study we found that thymus cells obtained

from A2 vaccinated rats could transfer resistance to adjuvant arthritis to naive rats. This

indicates that the mechanism of resistance induced by A2 vaccination is probably

immunological and involves thymus-derived lymphocytes.

Research Article

Find the latest version:

(2)

Role of the Thymus in Induction and Transfer of Vaccination

Against Adjuvant Arthritis with

a

T

Lymphocyte Line in Rats

Joseph Holoshltz, Avraham Matitiau, and Irun R.Cohen

Department of Cell Biology, The Weizmann Institute ofScience, P.O. 26Rehovot, 76100; DepartmentofInternalMedicineB,

MeirHospital, Kfar Saba; and DepartmentofPediatricsB, KaplanHospital, Rehovot, Israel

Abstract

Adjuvant arthritis is an experimental disease of rats induced

by immunization to antigens of Mycobacterium tuberculosis.

Our observation that arthritis could be induced in irradiated rats by the A2 line of T lymphocytes in the absence of

mycobacterial antigens suggested that adjuvant arthritis is an autoimmune disease. Moreover, the A2 line could be used to

vaccinate unirradiatedratsagainstthe subsequent induction of

adjuvant arthritis by active immunization toMycobacteia. In the present study we found that thymus cells obtained from

A2 vaccinatedratscould transferresistancetoadjuvantarthritis

tonaive rats.This indicates that the mechanism of resistance

induced by A2 vaccination is probably immunological and

involves thymus-derived lymphocytes.

Introduction

Adjuvant arthritis (AA)' can be induced in rats by an

intra-dermal injection of killed Mycobacterium tuberculosis (MT) in oil in the form of complete Freund's adjuvant (CFA) (1).

AA presents someof the clinical and pathological features of

rheumatoid arthritis and has been considered as a model of

the human disease (2). From rats immunized with CFA, we

have isolated the A2 line ofT lymphocytes by virtue of its proliferative response to MT (3). The A2 line cells could induce arthritis following intravenous injection into irradiated recipient rats, which suggests that AA may be caused by an

autoimmune attack against joint antigensthatarecross-reactive

with MT (4). Moreover, A2 line cells could be used to

vaccinate unirradiated recipientratsagainstattemptstoinduce

active AAby subsequent inoculation with CFA (3).

In the studies reported here, weundertook tocharacterize

the mechanismsinvolved in A2 line-induced vaccinationagainst

AA.The major questions examinedwere:whatarethetemporal

and quantitative parameters ofvaccination; isthe presenceof

Dr.Holoshitzwassupported bythe SchreiberFund,Tel-AvivUniversity,

andbythe Israel Ministry ofHealth. Dr. Cohen is the incumbentof

theMauerbergerProfessorial Chair inImmunology.

Received for publication 21 June 1984 and in revised form 18 September1984.

1.Abbreviations used in thispaper:AA,adjuvant arthritis; BP, basic protein; CFA, complete Freund's adjuvant; Con A, concanavalin A;

EAE, experimental autoimmune encephalomyelitis; EAT, experimental

autoimmune thyroiditis; MT, Mycobacterium tuberculosis; OA, oval-bumin.

thymus orspleen required for the vaccination effect; and can

line-induced vaccination be adoptively transferred to naive rats?

Methods

Rats. Inbred Lewis rats were obtained from the Animal Breeding Center of this Institute. Unless otherwise stated, 2-mo-old female rats were used throughout thestudy.

Antigens.Heat-killedM. tuberculosis H37 Ra was purchased from

Difco Laboratories (Detroit, MI). The bacteria were ground with a mortar and pestle under sterile conditions, and suspendedeither in incomplete Freund's adjuvant to prepare CFA, or in phosphate-bufferedsaline(PBS)toprepareMTantigenfor in vitro experiments.

Awatersolublepreparation ofMT wasmadebysuspending 100 mg of groundMTindouble-distilledwaterfor 48 hat4VC with constant

stirring. Suspended MTfragments were removed from thewaterby

centrifugation twice at 2,000 rpm for 25 min. The supernatant

containinga watersolublefraction of MTwaslyophilizedand stored

at-20'C. Before use, thewatersoluble MT fractionwasdissolved in PBS.This materialwasusedonlyin theexperimentshown in Table III.ConcanavalinA(Con A)waspurchased from Bio-Yeda (Rehovot, Israel) and ovalbumin(OA) fromSigmaChemical Co., St.Louis,MO. Guineapigbasicproteinofmyelin (BP)waspreparedasdescribed(5). Culturemedium. All cell cultures used Dulbecco's modification of Eagle's medium (Gibco Laboratories, Grand Island, NY). Medium usedfor restimulation(proliferation medium)wassupplementedwith

I mM glutamine (Bio-Lab, Jerusalem, Israel), 2-mercaptoethanol

(5 X l0-5 M), gentamycin (40

gg/ml),

and 1% fresh autologous rat serum. The medium usedto maintain thepropagated celllinesand

clones inlong-term culture(propagation medium)wastheproliferation

medium supplemented with 15% (vol/vol)of supernatant of Con

A-stimulatedmouse splenocytesas asourceof T cellgrowth factor(5),

10% horseserum (GibcoLaboratories), I mMsodium pyruvate, and

nonessentialamino acids(Bio-Lab).

Linecells. Line A2 reactivetoMTwasisolated and maintainedas

described (3). A single cell suspension was prepared from pooled

lymph nodes removed from Lewisratsinjectedin thehindfootpads9 dearlier with CFA. The cellswereresuspendedinproliferationmedium

(5 X 106/ml) and plated in 10-ml petri dishes (Nunc, Kamstrup,

Denmark)in the presence ofMT(10

,g/ml).

After72 hof incubation

at 370C ina humidified atmosphere plus 7.5%C02, the cellswere

collected, washed three times by mild centrifugation using a table

centrifuge, resuspended in propagation medium (2 X 105/ml), and incubated for 48 h in 10-mlpetridishes. The cellswerethencollected,

washed three times,andrestimulatedbybeing resuspendedin

prolif-eration medium (2 X 105/ml) together with irradiated (1,500 rad) syngeneic thymocytes (15X

106/ml)

andMT(10 yg/ml)in 10-mlpetri

dishes. After 72 h the cells were collected, washed three times, and transferred for continuousgrowthin propagation mediumasabove. Cultures in propagation medium were refreshed every 34 d. Once every 2-4 wk, the line cellswererestimulatedbyincubation withMT

and irradiatedthymocytesfor 3 dasabove and then transferred back intopropagationmedium. Line A2wasmaintained in thismannerfor 8wkbeforestudyof itsantigenreaction (3)oritsabilitytomediate arthritisorinduce resistance. All A2line cells which have been used

throughoutourexperiments(3,4),includingthe present one,originated J.Clin.Invest.

© The American Societyfor ClinicalInvestigation,Inc.

0021-9738/85/02/0472/06 $1.00

(3)

fromthesamecommonpool of lymph node cells. Cloned subline A2b

wasisolatedand maintained as we described (4). LinesZla,anti-BP, andClaanti OAwereisolated and maintained as described (5).

Surgicalremovalofspleenorthymus.Thymectomyorsplenectomy of 4-8-wk-oldrats wasperformedunder ether anesthesia. Thymectomy

wasaccomplished by suction ofthegland through a midline incision madeinto the upperthorax. Splenectomy wasdone byexposing the

spleen throughalateral incision. The bloodvessels wereclosedby heat

or silkligation. Sham operations involved similar procedures except that the organs were not removed. Incisions were closed by metal

clips. Completenessofthymectomywasconfirmedatautopsy.

Induction ofactiveAA.Toinduce activeAArats wereinoculated

intradermallyatthe baseof the tail with 0.1 ml of CFAcontaining10

mg/mlMT(H37 Ra,purchasedfromDifco Laboratories),inincomplete

Freund'sadjuvant(DifcoLaboratories). Severityof arthritiswasassessed

as previously described (3) and the arthritis score computed as the

sum of thedegreeof inflammation of each of the four limbsgraded

on ascale ofonetofour. The maximum arthritisscore wasthus 16. Vaccinationusing line cells. Beforebeing inoculated,line cellswere

restimulated with theirrespective antigensin the presence of irradiated

(1,500 rad)thymusaccessory cells for 3 d asdescribed (3-6). Atthis

time,theirradiatedthymusaccessory cells have died anddisintegrated,

sothat>90% of the culture iscomposedoflymphoblasts. Restimulation ofline A2 wasdone routinely before vaccination toobtain the large

numbersof line cells needed for theexperimentsand becauseactivation

wasessential for vaccinationbyline cellsagainst experimental

autoim-muneencephalomyelitis(EAE) (6)oragainst experimentalautoimmune

thyroiditis(EAT) (7).Thelymphoblastswerecollected, separatedfrom residual accessory cells and from cellular debrisby density gradients

in someexperiments (seeTableI),washedtwice inPBS, andinjected

in 1 mlof PBS into the tail vein of nonirradiated Lewisrats.Insome

experiments(seeTableI),controlrats wereinoculated withirradiated

thymus accessory cells that had been incubated for 3 d with MTin the absence ofline cells. At varioustimes afterinoculation the rats

weretestedfor resistancetoactive induction ofAAby challenge with CFA. Vaccination was evident by a decrease in incidence and/or

severity of AA compared with control groups. Rats that did not

developarthritiswere not included in computation of themean day

ofonsetandmeanarthritisscore.

Adoptive transferofvaccination. Ratswereirradiated with 150 rad of gamma irradiation (3) and then inoculated intravenously with various numbers ofthymus orspleen cells orwith 1.5 ml ofserum

obtained fromratsthat had beenvaccinated 1 moearlierbyinoculation with2 X 107A2orZia line cells. Therecipientrats werechallenged

on thesamedaywith CFAtoassay theirsusceptibilitytoAA.

Results

Doseresponse

of

line-inducedvaccination.Todefinethenumber of A2 line cells neededtovaccinate

recipient

rats

against

AA,

we inoculated rats with various numbers of line A2 cells at

day

0 and

challenged

them with CFA 35 d later. As can be

seen in Table

I,

inoculation of I X

106

cells had no

effect,

5X

106

cells

produced

amoderate decrease inclinical severity, while 10X

106

cellscaused a marked decreaseinbothincidence and

severity

of disease.

Complete protection against

AA was

achieved

by

vaccination with 20 X

106

lineA2 cells.

Time

of

onset and duration of vaccination. The time

required

for line-induced protection to be generated was assessed

by challenging recipients

of 2 X 107 A2 cells with CFA at various intervals after inoculating the cells. Table II shows that while some

degree

ofprotection was found 3 d

after

inoculating

2 X 107 A2 cells, full protection wasfound from the 16th d on, andpersistedfor at least 180 d.

MT carry-over is not

responsible for

vaccination. It has been shown thatadministrationof small doses ofMT

antigens

TableI. Dose-ResponseofLine A2-induced Protection Against AA

AA inducedwithCFA atday 35 A2line cells

injected intravenously Meanday Meanarthritis

at day 0 (X10') Incidence of onset score

±SD ±SD

0 5/5 14.2±0.4 13.6±0.9

1 5/5 17.0±0.7 12.0±0.7

5 5/5 17.4±1.5 7.4±1.1

10 1/5 19.0 2.0

20 0/5

-3-mo-oldmaleLewisrats wereinoculated intravenously with various

numbersofA2 line cells andtheir susceptibilityto AA wastestedby immunizingthemwith CFA 35 d later.

can induce resistanceto AA (8). Therefore, a series of experi-ments were designed to test whether the vaccination effect could be attributed to MT antigens carried-over with the A2

linecellsintotherecipientrats.The results of theseexperiments

are tabulated in Table III. Groups 2 and 3 illustrate that

density gradient separation of residual accessory cells that

might have carried processed MT did not interfere with the ability of line A2 to vaccinate rats against AA. This was

confirmed by experimental group 4, which was successfully vaccinated with A2 line cells that had been activated by the mitogen Con A and were therefore free of any possible contamination with MT. Group S shows that water soluble MT was aseffectiveas particulate MT. Hence, incubation of A2 withparticulate material was not a prerequisite for vacci-nation.Group 6 shows that incubating the arthritogenic clone of A2 (4) with MT did not lead to successful vaccination,

although this clone responded to MT (4) and could have carried over any MT antigens picked up in culture. Group 7

illustrates thatvaccination could not beeffected byirradiated accessory thymocytes incubated with MT in the absence of

line A2. Therefore, the vaccination effect was seen to be a functionofthe A2linecells and was independent ofpossible

MTcarry-over.

Adoptive transfer ofvaccination. To learn whether the state of vaccination could betransferred,weirradiatedrecipientrats

TableII. TimeCourseofLineA2-inducedProtection Against AA

AA

Challenge with CFA

afterinoculation Mean day Mean arthritis

withline A2 (d) Incidence of onset score

±SD ±SD

Notinoculated 10/10 13.9±1.0 8.9±0.9 0 9/10 14.1±1.2 8.7±1.8

3 3/5 14.7±0.6 4.7±0.6

16 0/5

30 0/5

52 0/5

180 0/12

(4)

TableIII. Carryover ofMT is NotResponsible forVaccination

Inoculum

CFAchallenge Irradiated Antigen/ Separation of percentage Group Tcells thymocytes mitogen thymocytes incidence of AA

1 None None None No 90

2 A2 Yes MT No 0

3 A2 Yes MT Yes 0 4 A2 Yes Con A No 0

5 A2 Yes MT-H20 No 0

6 A2b Yes MT No 83 7 None Yes MT No 100

Cells(2X 105/ml)of lineA2 orclonedsublineA2b wereincubated withMT (10pg/ml),watersoluble extractofMT(MT-H20; 50 tg/ml),orConA(2.5

pg/ml)in the presenceof accessory cells in the form ofirradiated(1,500 rad) thymocytes for 72 h.Control cultures includedirradiatedthymocytes, which wereincubatedwith MT in the absenceof linecells.After72h the cultures werecollected and washed toeliminatecelldebris and accessory cells. Cultures in group 3 weresubjectedtodensity separation (5). The blasts were counted, and 2X101blastsor anequivalentnumberofirradiatedthymocyteswere

in-jectedintravenously intogroupsof6-10nonirradiated Lewisrats.After 35d,

alltheratswerechallengedwithCFAand observedforthe appearanceofAA.

with 150 rad and inoculated them intravenously with 1.5 ml

ofserum or with 109 spleen or thymus cells obtained from

normal donor rats orfromdonors thathad beenvaccinated 1 mo earlier by intravenous inoculation with 2 X 107 A2 line

cells. The recipients were challenged with CFA to induce active AA. Fig. 1 showsthat therecipients ofserumfrom A2

vaccinated rats showed enhanced expression of activeAA. In contrast, resistance toAA wastransferred by thymus cells of donors that had been vaccinated with line A2. Spleen cells

obtainedfrom vaccinatedratsand

thymus

orspleen cells from control ratshad no effecton

development

ofAA. Resistance

to AA was notdetectable

following

transfer intononirradiated recipients (not shown).

Fig.2showsadose-responseexperiment in which

recipients

were inoculated with 1 X

108,

2.5X

108,

5X

108,

or 1 X

109

donor thymus cells ofratsthat had been vaccinated

against

AAusingA2line cells. Agroupofratswasinoculated with5

X 0I thymuscellsobtained fromratsthat had beenvaccinated

against

EAE by inoculation with Zla anti-BP T

lymphocyte

line cells (9). Resistance to AA was evident in rats that had

10I Figure

1.

Transfer of

resis-tance toadjuvant arthritis

8 by thymus cells of

vacci-W nated donors.Groupsof 10

0 rats eachwere irradiated

6 with 150rad and then in-oculated

intravenously

with

1 4 1.5mlofserum

(closed

4 ~ At / \ squares)orwith109 spleen

--... (closed circles)or

109

thy-2 i / _ mus cells (closedtriangles)

N.

/sobtained fromratsthathad

0 4 beenvaccinated I moearly

10 14 IS 22 26 30 34 38 42 with A2line cells. Control

DAYS

ratsreceived nocells(open squares)orwereinoculated with 109spleen cells(opencircles)or109 thymuscells (opentriangles)obtained from naivedonorrats.The arthritisscore wasrecordedovertime.

10 _ Figure2. Dose response of transfer of resistance to ad-juvantarthritis. Groups of 8 10 rats wereirradiated with

0 /C\ \ _ 150 rad and thennot

inoc-n6 ulated

(open circles)

or

in-0

1/ < \ - oculatedwith 5 X lO8

thy-Cr

11 fm

't \

muscells obtainedfrom

/

4_rar + \ _ donor rats that had been

s////s\O

vaccinated

I moearlier

2 - o* r ^ Nu with Zla anti-BP line cells K,0/a/ \ *- (open

squares).

Other

o0 14 18 > groups ofrats received

109

10 14 18 22 26 30 34 38 42 (closedtriangles), 5X 108

DA Y S (opentriangles), 2.5 X

10i

(closedcircles),or 108(closed squares) thymus cells obtained from

ratsthat had been vaccinated 1 moearlier with A2 line cells. The arthritisscorewasrecordedovertime.

received 5 X I0O or 1 X I09thymuscellsobtained from donors that had been vaccinated against AA. Thymus cells (5 X 108)

from rats that had been vaccinated against EAE rather than protect, actually seemed to enhance the development ofAA. Thus, the degree of protection appeared to be related to the

number ofthymus cells transferred from rats that had been

specifically vaccinatedwith A2 line cells.

Presence of thymusnotrequiredfor successful vaccination. The above results suggested that the thymus gland contained cells that could transfer resistance to AA. It was therefore important to learn whether the presence ofthe thymus was essential for successful vaccination against AA. Accordingly,

rats were either thymectomized, splenectomized, or sham

operatedat the age of4

wk,

and some rats were inoculated with 2 X I07 A2 cells 45 d later. To assay susceptibility to AA, all the rats were challenged with CFA 80 days after

surgery. Table IV tabulates the results which illustrate two

findings. First, regarding rats that had not been vaccinated

with A2 line cells, splenectomy had no influence on the

susceptibilityof the rats toAA,while adultthymectomyeither alone or in combination withsplenectomy hastened the onset

Table IV. Presenceof ThymusorSpleenIs Not

Required forInductionofVaccinationAgainstAA

AA inducedbyCFA atday80

Percentage

Treatment Line A2 incidence Meanday Meanarthritis atday0 atday45 (nrats) ofonset score

2X107 ±SD ±SD

Sham surgery Yes 0(15) -

-No 94(16) 13.5±1.4 9.0±1.6 Thymectomy Yes 15(13) 16.5±0.7 2.5±0.7

No 100(9) 10.9±1.2 13.7±2.4

Splenectomy Yes 20(5) 14.0 4.0

No 100(5) 14.2±0.4 9.4±1.8

Thymectomyplus

splenectomy Yes 20(10) 15.0±0 2.0±0

No 100(10) 12.0±0.7 11.6±1.7

Lewisrats weresurgicallytreatedat4wkof age(day 0),andsome were

(5)

ofdisease (day 10.9 comparedto day 14.4) and increased the severity ofAA (arthritis score 13.7compared to9.2). Second, ratsthat had undergone either thymectomy, splenectomy, or both procedures acquired resistance to AA as a result of vaccination with A2 line cells. Thus, although the induction ofAA seemed to be enhanced in adult thymectomized rats,

such rats could stillbe vaccinated successfully against AAby inoculation of A2 line cells. Hence, adult thymectomy per-formed before vaccination did not interfere with acquisition ofresistance. Therefore, the presence of the thymus and/or spleen was not essential for the induction of the protective mechanism.

Latethymectomy of vaccinated rats abolishes resistanceto

AA. Despite successful vaccination of thymectomized rats, the

finding that thymus cells of vaccinated rats could transfer

resistance to AA suggested that the thymus might house the cells responsible for resistance after they were induced. We therefore vaccinated 1-mo-old rats by inoculating them with A2 or control Cla (anti-ovalbumin) line cells, and then removed thethymus 3 or 35 d later. 45 dafterthe thymectomy, the rats were challenged with CFA to test their resistance to AA. Table V shows that recipients of Cla line cells were susceptibletoinduction ofAA whethertheywere or werenot

thymectomized. Incontrast, theratsthat hadbeen inoculated with A2line cells wereresistanttoAAprovidedthatthey had been sham thymectomized orthymectomized only 3 d after vaccination. However, rats that had been thymectomized 35 d after vaccination with A2 line cells manifested a high incidence of severe arthritis. Therefore, late thymectomy led tolossof acquired resistancetoAA.Hence, the cellsresponsible

for acquired resistance could be generated in the absence of the thymus, but, given a thymus, they seemed to be housed

there.

Discussion

Resistance to active induction of autoimmune disease is achievable by inoculating experimental subjects with cells of

long term lines ofTlymphocytes specifically reactive against the relevant antigens. We have called this phenomenon

vac-cination by its analogy to the use of attenuated microbial pathogens to induce specific resistance (10). Vaccination has been successful in three experimental diseases: EAE, EAT, andAA.

For example, inoculationof rats with anti-BP linesproduced EAE, and inoculation of mice with anti-thyroglobulin lines produced EAT. However, upon irradiation (1,500 rad), the line cells no longer mediated disease, but animalsreceivinga

single inoculation of cells acquired resistance toinduction of

EAE byimmunization with BP(9)or toinduction of EATby immunization with thyroglobulin (7). In the case of

line-mediated AA, arthritiswasproduced by A2 line cellsonlyin

recipients that had beenirradiated (750 rad), and therefore to

avoid disease itwas notnecessarytoirradiatethe A2line,but

onlyto desist from irradiatingthe recipientrats(3). Whyline

A2 mediatesarthritis in irradiated but not in intact rats is not

known. It has been shown that irradiation augments the

severity of actively induced AA (11) and that some rats

subjectedtototallymphoidirradiation spontaneously develop arthritis (12). Therefore, it is conceivable that natural

mecha-nisms of resistance to AA andtoline-induced arthritis existin

intact rats, and that these mechanisms can be neutralized by irradiation. Hence, vaccination might work by strengthening these mechanisms. The point of this report, however, is that

induced resistance is associated with the thymus and can be transferred.

Whether or not AAis anautoimmune disease has been a

controversial question. Severalhypotheseshave been proposed: that the arthritis results from animmune response to Myco-bacterialantigens settlinginthejoints after immunizationwith

CFA (13);that thephysical injection of CFA sensitizes therat to endogenous connective tissue antigensin the skin (14); or

that there existscross-reactivity between antigenicdeterminants

ofMycobacteriaandantigensinthejoints (15).The induction of arthritis using anti-MT line cells in the absence of CFA

injection supports the latter hypothesis (4). Weare presently

characterizingtheself-antigenthatis the target of the immune attackonthejoints,butasyetcanonlysay that it isprobably

notcollagentype11(4).

Inthe present study weinvestigated factors involved inthe

Table V. Thymectomy35 DAfterInoculation of Line A2 Eliminates Line-induced Protection

Treatment of recipient rats AAinduced by CFA 45 daftertreatment

Inoculation of T Daysafterline Percentage incidence

cellline inoculation Treatment (n rats) Mean dayof onset Meanarthritis score

±SD ±SD

A2 3 Thymectomy 13 (8) 17.0 2.0

Sham thymectomy 0(13)

35 Thymectomy 83(6) 13.4±1.1 9.4±1.7

Sham thymectomy 0 (5)

Cla 3 Thymectomy 100(5) 12.0±1.6 10.9±1.8

Sham thymectomy 100(6) 14.2±1.5 8.7±1.4

35 Thymectomy 100(6) 11.2±0.8 12.5±1.6

Sham thymectomy 100 (6) 14.0±2.3 9.0±1.9

(6)

acquisition ofresistance to AA induced by vaccination with A2 line cells. Vaccination against AA was found to be a function of thenumber of A2 line cells (Table I), and required time to become established (Table II). We have observed that resistance to AA becomes solid by- 10 dafter inoculation of A2 line cells. Once it sets in, resistance seemed to persist (Table II). Asillustrated in Table III,vaccinationcould not be explained by possible carryover of MT antigens. Line A2 did not need to beincubated with MT to induce resistance, and incubation of clone A2b or of thymus accessory cells alone with MT did not produce vaccination. Moreover, A2 line cells were as effective after incubation with water soluble MT as they were afterincubation with particulate MT.

Theimportanceof the thymus in vaccination was indicated by the transfer ofresistance by thymus cells and not by spleen

cells or serum obtained from vaccinated rats (Figs. 1 and 2). Inthis study,transfer of vaccination was successful when the recipient rats were irradiated with 150 rad. Transfer was unsuccessful when the recipients were not irradiated (not

shown).Whylymphocytetransfer is enhanced by mild

irradia-tion isunknown, but several mechanisms have been proposed

including the creation of "space" in lymphoid organs by depletion of recipient lymphocytes (16).

Vaccination has been shown to be antigen-specific in AA

(3), EAE(17), and EAT (7), andits transfer by thymus cells, probably T lymphocytes, suggests that resistance is mediated

by an immunological mechanism. We have found that the

specificityofvaccinationwasdirected bythe finespecificity of

the line cells used forvaccination, which suggests thepossibility that the antigen-specific line cells immunized the recipients

against

the receptors needed to

recognize

the

self-antigens

criticalfor the autoimmune disease(17). Although anti-receptor

or anti-idiotypic immunity might serve to regulate

autoim-munity (18, 19), other mechanisms are conceivable and the

question remains to be resolved. For

example,

clone A2b, although

arthritogenic,

could not vaccinate

against

AA

(4),

andseveral

encephalitogenic

clones have been isolated that do notvaccinateagainstEAE

(in preparation).

Bethatasit may, the present study shows that the thymus can be involved in

acquired resistanceto

autoimmunity. However,

the roleofthe

thymus inthis

resistance, although major,

was not exclusive.

Vaccination against AA could be obtained in rats that had been

thymectomized,

eventhoughadult

thymectomy

seemed toincreasethe

severity

ofAA inthis

study

(Table III)

and in others(11).

Nevertheless, by

35 d after

vaccination,

resistance

could beabolishedby removingthethymus.Thisimpliesthat

resistance can be induced and maintained outside of the

thymus, but in the presence of the

thymus

the resistance

mechanismmay settle there and hence becomes vulnerable to thymectomy.

The thymus has been looked upon as an organ devoted

primarily tothedifferentiation ofT

lymphocytes

and discon-nected from immune responses which take place in the

pe-riphery. However, the results of

investigations

carried out in

this laboratory indicate that the

thymus

receives and houses

antigen-activated

Tlymphocytes from the

periphery

and may

perform

important

functions in

regulating

autoimmune

re-sponses. The return to the thymus of potential effector T

lymphocyteswasdemonstratedbyinoculatingratswith radio-labeled lines of Tlymphocytes specifically reactive against BP

or against foreign antigens. About 1% of the inoculated line cells accumulated in the thymus and persisted there formonths

(6, 20). Anti-BP line cells recovered from the thymus were found to be capable of mediating EAE in normal recipient

rats. Thus, autoimmune, potential effector lymphocytes

per-sisted in clinically well rats.

Another study involved rats that had acquired resistance toinduction of a second attack of EAE subsequent to recovery from primary EAE induced by immunization to BP(21). It

was found that this form of acquired resistance too was associated with the appearance in the thymus, and less so in the spleen, of cells that specifically inhibited the proliferative responses in vitro of anti-BP effector T lymphocyte line cells. Afunction for the thymus in regulating EAE wasalsosuggested by the finding that adult thymectomy of rats increased their susceptibility to progressive or relapsing EAE (22). Whatever the cellular mechanisms of resistance turn out to be, the presentfindings indicatethat it is feasible using T lymphocyte line cells to manipulate the state of resistance to induction of what appears to be autoimmune arthritis. The clinical

impli-cation of these studies isclear(10, 19).

References

1. Pearson,C. M. 1956. Development of arthritis, periarthritis and

periostitis in rats given adjuvant. Proc. Soc. Exp. Biol. Med. 91:95-101.

2. Pearson, C. M. 1964. Experimental models in rheumatoid disease. Arthritis Rheum. 7:80-86.

3.Holoshitz,J., Y.Naparstek,A.Ben-Nun, and I. R. Cohen. 1983. Lines of T lymphocytes induce or vaccinate against autoimmune

arthritis. Science(Wash. DC). 219:56-58.

4. Holoshitz, J., A. Matitiau, and I. R. Cohen. 1984. Arthritis induced inratsby clonedTlymphocytes responsivetomycobacteria

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5.Ben-Nun, A., and I. R. Cohen. 1982.Experimentalautoimmune

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6.Naparstek,Y.,A.Ben-Nun,J.Holoshitz,T.Reshef,A.Frenkel,

M. Rosenberg, and I. R. Cohen. 1983. Tlymphocytelinesproducing

orvaccinating againstautoimmuneencephalomyelitis(EAE):functional activation inducesPNAreceptors and accumulation in the brain and thymus of line cells. Eur. J. Immunol. 13:418-423.

7.Maron, R., R.Zerubavel,A.Friedman,and I.R.Cohen. 1983. T lymphocyte line specific for thyroglobulin produces orvaccinates

againstautoimmunethyroiditisinmice. J. Immunol. 131:2316-2322. 8. Perper, R.J., andA.L.Oransky. 1972. Serum factorprotecting

against experimental arthritis. Nature NewBiology (Lond.). 238:23-25.

9. Ben-Nun, A.,H.Wekerle, andI. R.Cohen. 1981. Vaccination

against autoimmune encephalomyelitis withT lymphocyte line cells reactiveagainstmyelinbasicprotein.Nature(Lond.). 292:60-61.

10. Cohen, I. R., A. Ben-Nun, J. Holoshitz, R. Maron, andR.

Zerubavel. 1983. Vaccinationagainstautoimmune diseaseusinglines

of autoimmuneTlymphocyte.Immunol. Today.4:227-230.

11. Kayashima, K., T. Koga, and K. Onoue. 1976. Role ofT

lymphocytes in adjuvant arthritis. I. Evidence for the regulatory

functions ofthymus-derived cells in theinduction ofthe disease. J.

Immunol. 117:1878-1882.

12. Trentham, D., E. Brahn, W. Williams, W. J. McCune, and J.E.Belli. 1984. Connective tissue diseasecan developinratseither

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13. Ginsburg, I. 1977. Can chronic and self-perpetuating arthritis in the human be caused by arthrotropic undegraded microbial cell wallconstituents? Aworkinghypothesis.Rheumatol.Rehabil. 16:141-149.

14. Steffen, C., and G. Wick. 1971. Delayed hypersensitivity reactions to collagen in rats with adjuvant-induced arthritis. Z. Immunol. Forsch. BioL. 141:169-180.

15. Mori, T., and S. Innami. 1971. Immune cross reaction among mycobacterium leprae, BCG and human bone and cartilage. Int. J. Lepr. 39:89-90.

16. Bernard, C. C. A., J. Leydon, and I. R. Mackay. 1976. T cell necessity in the pathogenesis of experimental autoimmune encephalo-myelitis in mice. Eur. J. Immunol. 6:655-660.

17.Holoshitz, J.,A.Frenkel, A.Ben-Nun, and I. R.Cohen. 1983. Autoimmune encephalomyelitis (EAE) mediated orprevented by T

lymphocyte lines directed against diverse antigenic determinants of

myelinbasic protein. Vaccination is determinant specific. J. Immunol. 131:2810-2813.

18. Jerne, N. K. 1974. Towardsanetwork theory of the immune system.Ann.Immunol. (Paris.). 124C:289-373.

19. Cohen, I. R. 1984. Autoimmunity: physiologic and pernicious. Adv. Intern. Med. 29:147-165.

20. Naparstek, Y., H. Holoshitz, S. Eisenstein, T. Reshef, S. Rappaport, S.Chemke, A. Ben-Nun, and I. R.Cohen. 1982. Effector

Tlymphocyteline cells migrate to the thymus and persist there. Nature (Lond.). 300:262-264.

21. Ben-Nun, A., and I. R. Cohen. 1982. Spontaneous remission andacquired resistancetoautoimmune encephalomyelitis (EAE)are

associated withsuppression of T cell reactivity: suppressedEAEeffector

References

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