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:
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 celllinesandclones 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 incubationat 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-mlpetridishes. 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
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 neededtovaccinaterecipient
ratsagainst
AA,we inoculated rats with various numbers of line A2 cells at
day
0 andchallenged
them with CFA 35 d later. As can beseen in Table
I,
inoculation of I X106
cells had noeffect,
5X
106
cellsproduced
amoderate decrease inclinical severity, while 10X106
cellscaused a marked decreaseinbothincidence andseverity
of disease.Complete protection against
AA wasachieved
by
vaccination with 20 X106
lineA2 cells.Time
of
onset and duration of vaccination. The timerequired
for line-induced protection to be generated was assessedby challenging recipients
of 2 X 107 A2 cells with CFA at various intervals after inoculating the cells. Table II shows that while somedegree
ofprotection was found 3 dafter
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 ofMTantigens
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
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 effectondevelopment
ofAA. Resistanceto AA was notdetectable
following
transfer intononirradiated recipients (not shown).Fig.2showsadose-responseexperiment in which
recipients
were inoculated with 1 X
108,
2.5X108,
5X108,
or 1 X109
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 Tlymphocyte
line cells (9). Resistance to AA was evident in rats that had
10I Figure
1.
Transfer ofresis-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
with1 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)
orin-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 moearlier2 - o* r ^ Nu with Zla anti-BP line cells K,0/a/ \ *- (open
squares).
Othero0 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 fromratsthat 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 aftersurgery. 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
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
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 torecognize
theself-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, althougharthritogenic,
could not vaccinateagainst
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 inacquired resistanceto
autoimmunity. However,
the roleofthethymus inthis
resistance, although major,
was not exclusive.Vaccination against AA could be obtained in rats that had been
thymectomized,
eventhoughadultthymectomy
seemed toincreasetheseverity
ofAA inthisstudy
(Table III)
and in others(11).Nevertheless, by
35 d aftervaccination,
resistancecould beabolishedby removingthethymus.Thisimpliesthat
resistance can be induced and maintained outside of the
thymus, but in the presence of the
thymus
the resistancemechanismmay 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 thepe-riphery. However, the results of
investigations
carried out inthis laboratory indicate that the
thymus
receives and housesantigen-activated
Tlymphocytes from theperiphery
and mayperform
important
functions inregulating
autoimmunere-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).
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