Eur. J. Immunol. 1988.18: 1993-1999 Induction of resistance in transferred EAE 1993

Deming Sun, Avraham Ben-Nunn and Hartmut Wekerle

Max-Planck-Society, Clinical Research Unit for Multiple Sclerosis', Wurzburg and Department of Cell Biology, The Weizmann Institute of Science', Rehovot

Regulatory circuits in autoimmunity: recruitment of counter-regulatory CD8' T cells by encephalitogenic CD4' T line cells

In this study, pretreatment of Lewis rats with a syngeneic encephalitogenic T cell line (S1) was found to be able to constantly induce resistance to the subsequent induction of transferred experimental autoimmune encephalomyelitis (tEAE). This treatment was capable of protecting recipient animals for at least 2-4 months. Here we show an enhanced suppressor T(anti-S1) cell activity, which can be readily detected in the lymphoid organs of animals which recovered from S1-induced tEAE, or from rats pretreated with attenuated (irradiated, fixative treated or water-lysed) S1 cells. Anti- S1 cells, which uniformly express the CD8 phenotype, were selectively stimulated to grow and expand into lines by confronting primed lymphoid cells with irradiated S1 cells in culture. The proliferative response of anti-S1 cells was independent of myelin basic protein and antigen-presenting cells, and the responses against unrelated encephalitogenic T cell lines were minimal. It was also found that none of the mono- clonal antibodies tested (including CD8 and MHC class I antigen-specific antibodies) was able to block Sl/anti-S1 interactions. These cells are functionally suppressive to the proliferation of S1 cells in vitro, are specifically cytolytic directed against the EAE-inducing S1 cells and are able to antagonize encephalitogenic capacity of S1 cells in vivo. In vivo elimination of the CD8+ T subset from Lewis rats, using a combined treatment of thymectomy and OX-8 antibody injection before the initial cell transfer, totally blocked the induction of resistance. Our experiments document that induction of functionally active suppressor T cells is responsible for the induced resistance observed in tEAE.

1 Introduction

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease which can be induced actively in Lewis rats by a single injection of myelin basic protein (MBP) emulsified in complete Freund's adjuvant (CFA), or produced passively by adoptively transferring per- manent encephalitogenic T cells specific for MBP [l, 21.

Lewis rats which have recovered from actively induced EAE usually develop resistance against subsequent reinduction of the disease [3-6]. Induction of resistance has also been achieved by injecting animals with attenuated autoaggressive T cells as a vaccine [7, 81.

However, even repeated vaccination did not protect Lewis rats from transferred EAE (tEAE) [8, 91. In this study, we describe a rat encephalitogenic T cell line (Sl), which con- stantly induces tEAE. Quite remarkably, the animals which recovered from S1-mediated tEAE were consistently and specifically protected from reinduction of disease for at least 2-4 months. In an attempt to isolate and characterize the

[I 71041

This unit is supported by funds from the Hermann and Lilly Schil- ling-Stiftung.

Correspondence: Deming Sun, Max-Planck-Society, Clinical Research Unit for Multiple Sclerosis, P.O. Box 6120, D-8700 Wurzburg, FRG

Abbreviations: APC: Antigen-presenting cells IL 2: Interleukin 2 mAb: Monoclonal antibody(ies) Con A. Concanavalin A MBP Myelin basic protein MHC: Major histocompatibility complex EAE: Experimental autoimmune encephalomyelitis tEAE: Trans- ferred EAE

effector cells responsible for the protection of the animals, a functionally active, CD8+ T lymphocyte population (anti-S1 cells) was found in the lymphoid organs of the animals which had recovered from S1-induced tEAE. These cells can be readily detected by an in vitro proliferation assay. They selec- tively respond to the stimulation by S1 cells, and were func- tionally active in antagonizing S1-mediated tEAE in vivo. The possibility that these suppressor cells, functionally specific for the challenging encephalitogenic T cells, may be responsible for the induced resistance in tEAE is discussed.

2 Materials and methods

2.1 Animals and reagents

Inbred Lewis rats were obtained from the animal breeding facilities of the Max-Planck Institute of Immunobiology (Freiburg, FRG). Cells were cultured in RPMI 1640 medium (Gibco, Eggenstein, FRG) supplemented with 10% selected fetal calf serum (FCS; Gibco), 5 X M 2-mercaptoethanol (2-ME) and penicillin/streptomycin (100 pg/ml) in a humidified atmosphere with 5% COz at 37 "C. FCS was inactivated before use at 56 "C for 30 min. Supernatant containing interleukin 2 (IL 2) was prepared from cultures of concanavalin A (Con A)- stimulated spleen cells of C3WHeN mice. The supernatants were collected and tested for growth-promoting activities on freshly activated rat T cell lines, or IL 2-dependent mouse T cell lines.

The monoclonal antibodies (mAb) used in this study were from the the OXFORD series. OX-19 (CD5), OX-6 (mouse I-A equivalent), OX-18 [major histocompatibility complex (MHC) class I antigen specific] and OX-17 (mouse I-E equiva- lent) were purchased from Serotec/Camon (Wiesbaden,

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1988 0014-2980/88/12 12- 1993$02,50/0

1994 D. Sun, A. Ben-Nun and H. Wekerle Eur. J. Immunol. 1988.18: 1993-1999

FRG), OX-35 (CD4) and OX-8 (CD8) were derived from our own hybridoma cell cultures. Hybridoma culture supernatants and ascites were stored at -20 "C until use. Each lot of culture supernatant or ascites was tested by flow cytometry with nor- mal rat spleen T cells to ensure the presence of comparable concentrations of specific antibodies. Ovalbumin was pur- chased from Sigma (Deisenhofen, FRG). Purified protein derivative of tuberculin was obtained from the Statens Serum Institute (Copenhagen, Denmark).

MBP was prepared from guinea pig (GP) brain and spinal cord, as described [lo]. The purity of the preparations was examined by polyacrylamide gel electrophoresis. Synthetic MBP peptide 68-84 was obtained from Peninsula Lab. (St. Helens, GB) and peptide 45-67 from Nova Biochem (Laeufel- fingen , Switzerland).

2.2 Establishment of rat encephalitogenic T cell lines from spleen of MBP-immunized animal

Essentially the method of Ben-Nun et al. [l, 21 was used with some modifications. Briefly, a Lewis rat, 8 weeks old, was immunized in both hind footpads with 50 pg MBP emulsified in CFA containing 4 mg/ml Mycobacterium tuberculosis (H-37RA, from Difco, Detroit, MI). After 12 days the spleen was removed and a single-cell suspension prepared. Splenic T cells were separated from macrophages and B cells by absorp- tion with nylon wool. The cells (5 x 10') were first stimulated with 20 pg/ml MBP in a 100-mm petri dish in the presence of 2 X lo8 irradiated syngeneic thymocytes as a source of exogenous antigen-presenting cells (APC). After 3 days the activated lymphoblasts were isolated by Lymphoprep (Nycomed AS. Oslo, Norway) gradient centrifugation, and cultured in RPMI 1640 medium supplemented with 15% IL 2- containing medium (supernatant from Con A-stimulated mouse spleen cells). The T cell lines were maintained by periodical (about every 10 days) restimulation with MBP in the presence of irradiated syngeneic thymocytes used as APC.

2.3 Evaluation of EAE

Animals were scored daily for weight loss and clinical disease as follows: 0: no EAE; +: partial loss of tail tonicity; +: loss of tail tonicity; ++: unsteady gait and mild paraparesis; +++: hind limb paralysis and incontinence; + + + + : death.

2.4 T cell preparations

T cells were enriched by passage of unfractionated spleen or lymph node cells through a nylon wool column and, subse- quently, a Sephadex G-10 (Pharmacia, Freiburg, FRG) col- umn. The enrichment of the preparations was confirmed by staining the cells with a standard panel of mAb (OX-19, pan- T; OX-35, CD4; and OX-8, CD8) followed by a fluorescein isothiocyanate (F1TC)-conjugated goat anti-mouse Ig. Stained cells were analyzed by flow cytometry(0rtho 50 H, Ortho Diagnostics, Westwood, MA).

2.5 Isolation of anti431 T cells from S1-primed spleens

Responder splenic T cells were prepared from Lewis rats which had survived S1-induced tEAE for 3-8 weeks. A prolif-

eration assay was performed in 96-well flat-bottom microtiter plates, each well containing 2 X lo5 nylon wool-enriched splenic T cells with 4 X lo4 irradiated (1000 rad) S1 cells, for 3 days in the absence of exogenous antigens and APC. The cul- tures were then pulsed with 0.5 pCi = 18.5 kBq tritiated thy- midine [3H]dThd for 18 h , harvested and their incorporated [3H]dThd was determined. For recovery of proliferating cells, 4 x lo7 splenic responder T cells were co-cultured with 8 x lo6 irradiated S1 cells in a 100-mm petri dish. After 3 days the proliferating cells were isolated by Lymphoprep gradient centrifugation and cultured in IL 2 medium.

2.6 Thymectomy

Thymectomy of 4-wk-old female rats was carried out under ether anesthesia. Postmortem examination at the end of the experiments revealed no thymic remnants. tEAE was induced when all rats were 8 weeks old.

2.7 Flow cytometry analysis

Indirect immunofluorescence was performed by incubating 1 x lo6 cells with appropriate concentrations of antibodies for 30 min. After washing twice in phosphate-buffered saline con- taining 1% bovine serum albumin and 0.1% sodium azide (PBA), FITC-conjugated goat anti-mouse Ig was added for 30 min, the cells were then washed twice and the stained cells were analyzed by flow cytometry. Each sample was counted for 40000 cells, and cell number vs. fluorescence intensity (linear) was illustrated.

3 Results

3.1 Characteristics of encephalitogenic S1 cells

Line S1 recognizes the encephalitogenic epitope on MBP (MBP sequence 68-88) in the context of syngeneic MHC class I1 determinants, ignores a preceding nonencephalitogenic epitope (MBP sequence 45-67) and, like all our antigen- specific T cell lines, S1 cells express the CD4 phenotype. Intravenous transfusion of a sublethal dose of 2 x lo6 freshly activated S1 cells to syngeneic Lewis rats regularly induces both pathological and clinical EAE (Fig. lA) , which begins abruptly on day 4-5 after cell transfusion. The surviving ani- mals recover rapidly thereafter. A complete recovery from clinical symptoms, together with the beginning of weight gain, is usually observed on day 7-10 (Fig. 1B).

3.2 Development of resistance to S1-mediated tEAE

Lewis rats which had recovered from a first S1-mediated attack of tEAE were observed to be absolutely resistant to later reinduction of disease in a total of 100 rats studied. Resistance was also obtained when animals were pretreated with nonactivated S1 cells (S1 cells which were grown in IL 2- containing medium for 5-7 days after restimulation), irradi- ated S1 cells, S1 cells which were fixed by 0.5% paraformal- dehyde or water-lysed S1 cells (Table 1). It should, however, be noted that resistance to tEAE was not induced by all T cell lines. First of all, only MBP-specific encephalitogenic T line

Eur. J. Immunol. 1988.18: 1993-1999 Induction of resistance in transferred EAE 1995

3 I In*

2 I 1 ) .

1 I l l '

0

* + * + t i +

C L I N I C A L E C O R E E l CELL- INJECTED

0

/I71041( DAYS AFTER CELL INJECTION

Figure 1. Encephalitogenic activity of S1 cells. (A) Lewis rats were injected i.v. with graded doses of freshly activated S1 cells. The reci- pient rats were recorded daily for their clinical scores, as described in Sect. 3.4. (B) Kinetic observation on the weight loss vs. clinical scores in the recipient rats which were injected with 3 x lo6 (0-0) or 2 x1O6 (-0) s1 cells.

cells were observed to induce resistance. Pretreatment of the recipient rats with large doses (4 x lo6 - 10 X lo6) of freshly activated syngeneic T cells specific for ovalbumin neither induced EAE, nor protected recipient rats from subsequent induction of EAE (Table 1). Second, even among the encephalitogenic T line cells only three from a total of ten encephalitogenic T line cells tested were able to induce resist- ance. Kinetic studies revealed that induced resistance could protect treated animals from subsequent EAE for at least 2-4 months (data not shown).

3.3 S1 cells selectively stimulate CDS', anti41 cell proliferation in vitro

In an attempt to study whether induced resistance to tEAE was related to an enhanced suppressor cell activity in vivo, splenic T cells from animals which had recovered from tEAE were enriched by passage through nylon wool and Sephadex G-10 columns. The T cells were then coincubated in v i m with irradiated, resting S1 cells. A vigorous cell proliferation was observed in such cultures which started within 24 h, and reached its peak at day 3. The phenotypic characterization of the proliferative lymphoblasts as analyzed by flow cytometry revealed that > 95% of the proliferative splenic T cells (anti- S1) had the phenotypic profile of CD8 cells (OX-35-, OX@, OX-19+,OX-6+) [ll].

The proliferative response against unrelated encephalitogenic T cell lines (e.g. LPlClO and Zla) was minimal, and only a very low response was regularly observed in cultures with splenic T cells from naive rats as responders (Table 2.) Thus, suppressor T cell activity can be demonstrated in vitro in rats which had recovered from tEAE. Kinetic studies have shown that augmented suppressor cell activity appeared about 1 week after adoptive transfer of S1 cells, and was maintained at a high level for at least 8-12 weeks (Table 3). This course exactly parallels the resistance observed in the clinical course.

Table 1. Protective effect of S1 cells in tEAE Table 2. Organ distribution of anti41 activitya)

Treatment Induction of EAE" T cell Pretreatment Clinical Maximal EAEI lines of cells score weight loss total

(g)

- - +++ 40-50 20/20 s1 Activated blasts

(2 x 106) - - 0134 s1 Resting cells

s1 S1 0.5% Paraformaldehyde

(4 x 106) - - 0112

(4 x I d )

(4 x 106) - - 014

(1 x 10')

Irradiated - - 0112

fixed

Water-lysed - 014 - s1

LOA Activated blasts (4 x 106) +++ 30-40 414

a) Lewis rats were pretreated i.v. with 2 x lo6 - 10 X lo6 S1 cells, pretreated as indicated. Three to twelve weeks later the animals were challenged with an EAE-inducing dose (2 X lo6) of freshly activated S1.

Responder Stimulating T line cells T cells Zla LPlClO S1 ConA NoAg

+ APC

From EAE-recovered animal Spleen 2 X 16 859 1453 15046 168480 591 mLNCb) 1 X 106 911 2041 11793 140800 786

2 x 1 6 174 227 2094 173550 97 Thymus 1 X 106 208 178 1367 102814 57

2 x 16 139 165 656 106207 62

From normal animal Spleen 2 X 1 6 265 508 2410 165340 221 mLNC 1 X 106 667 534 2566 133867 196

2 x 16 346 275 573 156680 63 Thymus 1 X 106 130 123 787 113231 47

1 x 1 6 72 98 226 70911 73

a) Responses of lymphoid cells obtained from both EAE-recovered and normal Lewis rats were valued for their activities against 4 X lo4 irradiated encephalitogenic T cells as indicated. The incu- bation time was 3 days, and the cultures were labeled with tritiated thymidine for the final 18 h before harvest.

b) Mesenteric lymph nodes.

1996 D. Sun, A. Ben-Nun and H. Wekerle Eur. J. Immunol. 1988.18: 1993-1999

Table 3. Appearance and persistence of anti61 activity in postencephalitogenic rat spleen

Stimulator Delay after S1 injection") cells

1 wk 3 wk 8wk -

- 240 f. 33 345 * 75 577 f. 52 261 f. 32 a) Lewis rats had survived a tEAE episode 1-8 s 1 3362 f 315 18972 k 242 21264 f. 153 16018 k 227 wks earlier as indicated. Splenic cells de- Zla 307 k 30 844 k 95 552 f. 5 380 k 48 rived from immunized and nonimmunized Con A + APC 152381 f 1516 137036 f. 508 149534 f. 3848 158003 f. 183 animals were assayed for their responses LPlClO 696 f. 32 1971 2 178 3439 k 279 2263 k 199 against three different encephalitogenic T

cell lines (Sl, Zla and LPlC10).

Table 4. Proliferative response of anti41 cells is independent of MBP and APCa)

Sib) APC" Dose pf MBP (pgiml) in culture 10 1 .0 0.1

+ - 46330 rt 2010 47355 k 2094 43365 k 5484 + + 46898 If. 4757 NDd' ND

ND ND ND - -

A similar cell activity was also found, though with lower fre- quency, in mesenteric lymph nodes, as well as in the thymus (Table 2). The proliferative responses were independent of MBP and APC (Table 4). The T cell nature of anti41 cells was unequivocally established both by their membrane phenotype, and by the demonstration of full-length mRNA for the T cell receptor a- and 0 chains as reported before [l l] .

3.4 In vitro studies on the anti-S1 cells

Anti-S1 cells were first studied for their in vitro suppressive activities. They have strongly suppressive activities on the anti- gen-stimulated, as well as Con A-mediated S1 cell prolifera- tion in vitro. As few as 2 X lo3 anti-S1 cells totally abolish the proliferating capacity of 2 x lo4 responder T cells (data not shown). Anti-S1 cells were also found to be specifically cytoly- tic against S1 cells [11]. In order to identify the target epitopes on the labeled cells, antibody blocking tests were carried out, using mAb directed to different epitopes on the T line cells. Antibodies used in this study were 1: 100 diluted ascites, which were pretested either for their maximal blocking effect in an MBP-specific T cell proliferation assay (OX-6, W3/25), or for efficient binding to relevant T cells analyzed by flow cytometry, and they were confirmed to be free of toxic activities. The result was that none of the antibodies tested, including CD8 and MHC class I antigen-specific antibodies, was able to block the cytolytic activity (Table 5A), and the same was true in the proliferation essay (Table 5B). An antagonistic effect against the encephalitogenic S1 cells in vivo has been reported in a previous study [ 111.

3.5 In vivo elimination of CDS' cells prevents development of resistance

Two sets of experiments were done to learn whether in vivo elimination of CD8' cells is able to abrogate resistance or to prevent its development. In these experiments, all the Lewis

0

48299 f. 4570 33308 f. 2809

a) See legend to Table 3. b) 1000 rad irradiated.

d) Not done. 486 _t 31 c) lo6 Irradiated syngeneic tbymus cells.

rats were thymectomized at the age of 4 weeks. The animals were randomly divided into groups. In the first set of experi- ments, the animals were first injected with irradiated S1 cells. Two weeks later, OX-8 mAb (0.3 ml of ascitic antibody) was injected i.v. twice at an interval of 5 days, and this was fol- lowed by injection of an EAE-inducing dose of S1 cells. The

Table 5. mAb do not block the cytotoxic activity of anti-S1 cells (A) and the proliferation of anti41 cells (B)")

(A) mAb Target molecules Exp. 1 Exp. 2 Exp. 3

- - 59.5 51.5 54.2 ox -18 MHC class I antigen 49.5 49.6 52.8 0x4 CD 8 56.8 50.6 50.1 OX-6 MHC class I1 (LA)

OX-17 MHC class I1 (I-E) 58.3 50.3 51.1

W312.5 CD 4 58.0 50.2 47.9 Spontane- ous release 20.6 24.3 23.9

antigen 58.9 47.9 49.9

antigen

a) lob freshly activated anti-S1 cells were co-incubated with 3 X lo4 "Cr-labeled S1 cells for 20 h in the presence of indicated antibod- ies (throughout the assav).

NON OX-6 X-8 OX-17 OX-18 W3l25

See legend to Table 2.

proliferation of anti-S1 cells

46435 k 3042 48497 f 2406 53212 k 7467 54432 f. 3379 53826 f 2395 52097 rt 1684

Eur. J. Immunol. 1988.18: 1993-1999

Table 6. In vivo elimination of CD8+ T subset prevents the development of resistance

Induction of resistance in transferred EAE 1997

Pretreatment EAE in- EAEitotal Clinical duction

Thymectomy (wk 4) (wk5-6) (wk6) (wk7) (wk8)

OX-8 injab Vaccineb’ 0x4 inj.”

- - - + 818 - + - + 018 - - - + 6/6 - + - + 016 + + + + 515 - + + + 016

Table 7. Induction of resistance by early and late passages of the S1 T line

Passage numbers” Ability of inducing resistence 3 +++ 4 +++

13 +++ 14 +++

a,) Restimulation rounds of T line S1 before use in the experiment.

results shown in Table 6 indicate that thymectomy by itself neither changes the disease course, nor influences inducibility of resistance. In animals which had been vaccinated before OX-8 antibody treatment, elimination of CD8’ cells was not able to negatively affect the established resistance, although fluoresence-activated cell sorter analysis clearly demonstrated that CD8’ cells were thoroughly eliminated (Fig. 2 ) . The sec- ond trial was to eliminate CD8’ cells before vaccination. Thus, the thymectomized animals were first injected with two doses of OX-8 mAb, and were then vaccinated. Ten days later, they were challenged with an EAE-inducing dose of S1

piiq Figure2. In vivo elimination of CD8’ cells by injection of OX-8 mAb. Splenic T cell from normal (A) and OX-8-treated (B) Lewis rat were prepared as described in Sect. 2.5., and flow cytometry analysis was as described in Sect. 2.7.

score (Maximal)

++I+++

++/+++

+++

-

-

- a) Pre-vaccination treatment. b) 2 x lo6 2000 rad-irradiated S1 cells. c) Post-vaccination treatment.

cells. The animals in this group were not protected by the vaccination suggesting that elimination of CD8’ cells before vaccination totally prevents the induction of resistance.

4 Discussion

Cells possessing down-regulatory activities have been repeatedly found in the lymphoid organs of animals recovered from EAE [6, 12-17]. However, since there is more than one cell type or immune regulatory mechanism involved in immune suppression, caution is necessary when drawing con- clusions from studies on the suppressive effects of unseparated lymphoid cells. In this investigation, we succeeded in isolating T (anti-S1) cell lines from animals recovered from tEAE, which are able to specifically neutralize EAE-inducing encephalitogenic T cells in vivo and in vitro. These cells pos- sess suppressive properties in vitro, and specifically inhibit encephalitogenic action of the autoaggressive anti-MBP T cells in vivo. Recent studies from several groups have shown that pretreatment of animals with attenuated autoaggressive T line cells protected them against subsequent induction of autoim- mune diseases [7, 181, and T cells possessing suppressive activities have been repeatedly found [1&20].

We have found in the present study that pretreatment of Lewis rats with a permanent encephalitogenic T cell line (Sl) reliably induces resistance against subsequent induction of tEAE. Furthermore, induction of resistance is not limited to pretreat- ment of animals with activated S1 cells and thus induction of clinical EAE. “Vaccination” of animals with resting cells, paraformaldehyde-fixed or even water-lysed S1 cells was equally effective. From this observation we concluded that the induction of resistance in our system must be mediated by evoking a specific immune response in the peripheral immune system against the S1 cells. The induced resistance to tEAE is indeed associated with an induction of a suppressor T cell population in vivo, which can be readily detected in the lym- phoid organs of recovered or vaccinated animals, by using an in vitro proliferation assay (Table 2 ) . We isolated and cultured these suppressor T cells as long-term T cell lines and character- ized them functionally and phenotypically. The T cell identity of anti-S1 cells was established, first of all, by excluding the possibility that they might be cells possessing natural killer (NK)-like activity. A lack of spontaneous cytolytic activity against NK-sensitive targets (YAC-I) was demonstrated, which contrasted with the high NK activity of unfractionated rat spleen cells (data not shown). Phenotypically, concomitant expression of OX-8 and OX-19 membrane markers argue

1998 D. Sun, A. Ben-Nun and H. Wekerle Eur. J. Immunol. 1988.18: 1993-1999

against the NK nature of our cells [21, 221. Finally, full length mRNA of both the a- and f3 chain of the T cell receptor genes has been described previously [ll].

Our in vitro studies demonstrated that anti41 cells not only suppress proliferation of S1 cells, but also that they are specifi- cally cytolytic. The cytotoxic activity of anti41 cells was strictly directed against the EAE-inducing S1 cells, but not unrelated syngeneic T cell lines, including those with encephalitogenic properties [ 111. Adoptive transfer of anti-S1 cells to naive rats abolishes the encephalitogenic activity of the autoaggressive S1 cells [ l l ] . Our experimental data of both in vivo and in vitro studies indicate that the induction of resis- tance to S1 cells may be mediated through specific stimulation of functionally active suppressor T cells.

In order to identify the potential target structure on the encephalitogenic S1 cells, which might be recognized by the anti41 cells, we screened a panel of syngeneic T cell lines. None of the unrelated syngeneic T cell tested was able to stimulate anti41 cells to proliferate in vitro, and none of these line cells were lysed by anti41 cells in a cytotoxicity assay. Unexpectedly, none of a panel of mAb, including those specific for MHC class I, MHC class I1 and the CD8 molecule, was able to block the cytolytic activity, or interfered with induction of proliferation. The molecular basis of the Sl/anti- S1 interaction is still obscure. One possible target structure of Sllanti-S1 interaction could be neoantigen(s) appearing on T line cells during in vitro culture. These molecule(s) could induce a graft rejection response. However, we found that S1 cells of early line stage (S1 cells which have been frozen after third restimulation round in vitro) and those of later passages ( 14'h restimulation in vitro) induced resistance equally well (Table 7). Although these findings do not formally exclude the possibility that neoantigen(s) might arise in the early passages of the cells, these results, combined with the finding that anti- body specific for MHC class I antigens lack the blocking activ- ity on the proliferation and cytotoxic activity, may argue against a possible graft rejection mechanism underlying the S1-induced resistance. It would indeed be interesting to learn whether our anti-S1 suppressor cells have anti-idiotypic specificity for the T cell receptor of the encephalitogenic S1 cells, as suggested in other systems [23, 241. Our current experiments are aimed at identifying possible clonotypic molecule(s) of S1 cells using appropriate mAb.

Screening a panel of encephalitogenic T cell lines and clones, we found that the vaccinating potential is by no means a prop- erty peculiar to the S1 cell line which, incidentally, differs from the other encephalitogenic T cell lines by its origin from an immune spleen. We have observed that only three of a total of ten established encephalitogenic T cell lines tested are able to induce resistance in tEAE (unpublished data). However, the ability of encephalitogenic T cell lines to induce "resistance" varies among individual T lines, and the same is true for the duration of the resistance induced. S1-vaccinated animals were protected from tEAE induced by the same T cell line for at least four months; in the case of other lines, shorter durations were recorded.

In order to confirm the identity of CD8' cells as the cell ele- ment responsible for induced resistance in tEAE, we tried to eliminate CD8' cells in vivo, and thus abolish the established resistance or to prevent the development of resistance. Our data revealed that elimination of CD8' cells in vivo by thy-

mectomy and OX-8 mAb injection could totally block the initiation of resistance to tEAE. However, to abolish an already established resistance, elimination of CD8' cells has so far been unsuccessful. Two explanations are suggested: (a) OX-8 mAb treatment in the vaccinated animals, although able to eliminate CD8' cells, is not able to prevent the differentia- tion of precursors of functional active CD8' cells. In fact, in an additional study, we found that stimulation of CD8-depleted splenic T cells, derived from pretreated animals (but not naive animals), with S1 cells in vitro, was able to give rise to a predominantly CD8' cell proliferation (unpublished data) (b). Once a functionally specific suppressor T cell has been acti- vated in vivo, the cascade of intercellular reactions started might enable non-T suppressor cell elements to become func- tionally active. In fact, immune suppression circuits are known to be complex and involve various distinct cellular subsets [25-291. Taken together, our results suggest that CDS' T lym- phocytes are the major cell responsible for induced resistance in tEAE. Induction of resistance using well-defined encephalitogenic T cell lines or clones may offer a great oppor- tunity for studying the immunoregulatory mechanisms in EAE .

Received September 1, 1988; in final revised form October 20, 1988.

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