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Studies on the mechanism of suppression of experimental allergic encephalomyelitis induced by myelin basic protein-cell conjugates
CELLULAR
IMMUNOLOGY
88, 25 l-259 (1984)
Studies on the Mechanism of Suppression of Experimental Encephalomyelitis Induced by Myelin Basic Protein-Cell Conjugates RACHEL M. MCKENNA,’
BRIAN G. CARTER,
Allergic
AND ALEC H. SEHON
MRC Group for Allergy Research, Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada Received February 7, 1984; accepted May IO, 1984 The mechanism of suppression of experimental allergic encephalomyelitis (EAE) induced in Lewis rats by pretreatment with myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) was examined. Studies on the kinetics of the tolerance induction showed that pretreatment with MBP-SL suppressed EAE if given 7 but not 3 days before the diseaseinducing injection of MBP in Freund’s complete adjuvant. Treatment with cyclophosphamide 48 hr before administration of MBP-SL completely abolished the suppression of EAE. Transfer of lymph node and spleen cells from MBP-syngeneic erythrocyte conjugate (MBP-RBC) but not MBP-SL-pretreated rats resulted in suppression of disease in recipients subsequently given a disease-inducing injection of MBP. Administration of MBP coupled to SL from the histocompatible rat strain F344 resulted in suppression of the MBP-induced proliferative response of spleen cells from Lewis rats which had been given a disease-inducing injection of MBP. Taken together these results are consistent with the suppression of EAE induced by MBP-SL being mediated by suppressor T cells. o 1984 Academic RW, I”~.
INTRODUCTION It has been widely demonstrated that suppression of the humoral and cellular immune responses can be achieved by pretreatment with antigen coupled to lymphoid cells ( l-3). This suppression has been attributed to two distinct mechanisms, which may coexist (4-6), one involves T-cell tolerance (clone inhibition), the other active suppression by T suppressor (T,) cells. Antigen-lymphocyte conjugates have been used successfully to suppress the symptoms of autoimmune experimental thyroiditis (7) and collagen-induced arthritis (6). We (8) and others (9) have reported that experimental allergic encephalomyelitis (EAE) also can be suppressed using this approach, i.e., pretreatment of rats with conjugates of myelin basic protein (MBP) with lymhocytes or erythrocytes suppressed the disease. Furthermore, the suppression of both disease and perivascular cuffing in the spinal cord was found to correlate positively with the amount of MBP bound to the lymphocytes (8). In this report we provide evidence consistent with the suppression being mediated by T, cells. ’ Holder of fellowship from the Medical Research Council of Canada. 2.51 0008-8749/84 $3.00 Copyright Q 1984 by Academic Ptw, Inc. All rights of npmduction in any form reserved.
252
MC KENNA,
MATERIALS
CARTER, AND SEHON
AND
METHODS
Ruts. Female Lewis and F344 rats weighing 150-200 g were obtained from The Charles River Breeding Laboratories (Wilmington, Mass.) and were used for all experiments. Antigens. The method used to isolate MBP from whole central nervous system tissue was a modification of that described by Pitts et al. ( 10). Whole guinea pig (gp) spinal cords (Pel Freeze, Rogers, Ark.) were subjected to lipid extraction, followed by aqueous extraction, and then final acid extraction. The MBP was isolated from the acid extract by gel filtration through an ACA 54 (Ultrogel, Fisher Scientific, Canada) column (2.6 X 90 cm) equilibrated with 1 M glycine-HCl buffer (pH 2.8) containing 1 M urea. The individual fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the electrophoretie patterns were compared with those of rabbit MBP and gpMBP, appropriate fractions were pooled and tested for their encephalitogenic properties. Purified MBP gave one major band and one minor band and appeared to have a similar purity to samples of rabbit MBP and gpMBP which were kindly provided, respectively, by Dr. M. Kies, Myelin Chemistry Laboratory, NIMH (Bethesda, Md.) and Dr. C. Whitacre and Dr. P. Y. Paterson (Department of Microbiology and Immunology, Northwestern University, Evanston Ill.). The ovalbumin used was a five-times recrystallized preparation (ICN, Cleveland, Ohio). Coupling of MBP to spleen leukocytes (MBP-SL). Whole spleens were removed from healthy rats and dispersed into single-cell suspensions by gentle homogenization in a loose-fitting Teflon-glass homogenizer. Erythrocytes were removed by centrifugation through Ficoll-metrizoate (11). Leukocytes obtained from the interface were washed three times with RPM1 1640 and resuspended at a concentration of 1 X 108/ml. To this I-ethyl-(3dimethylaminopropyl)carbodiimide (EDCI; Stony Chemical Corp., Muskegon, Mich.), at a concentration of 5 mg/ml, and different amounts (0.4 to 50 mg/ml) of guinea pig MBP were added. The mixture was incubated for 30 min at room temperature on a rotator and then washed three times with RPM1 1640 to remove any unbound MBP. Cells were resuspended and the viability was established by dye exclusion. To determine the amount of MBP that was actually bound to SL in each individual experiment, a 100 ~1 sample of the cell reaction mixture was removed at the start of the incubation period and immediately a trace amount ( 10 ~1, approximately 0.5 pg) of ‘2’I-labeled MBP, which had been prepared by the chloramine-T method, was added to it (12). (Free iodine had been separated from bound iodine by passage through a Sephadex G-25 column.) This radioactive mixture was then treated in the same way as the original cell suspension and the number of counts that remained associated with the cells after extensive washing was compared to the number of counts originally added, and the percentage of cell-bound MBP was thus calculated. From the results of four different experiments in which gpMBP was coupled to SL it was evident that from 4.4 to 96.8 rg were coupled to 1 X lo7 cells. Coupling of MBP to rat erythrocytes(MBP-RBC). This reaction was carried out in essentially the same way as for coupling MBP to SL. Lewis rats were bled from the heart and the blood was collected into heparinized containers. The erythrocytes were washed three times with saline to remove the bufIy coat, counted, and adjusted to a concentration of 1 X lo9 cells/ml in RPM1 1640. To the cells EDCI, at a
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253
concentration of 50 mg/ml, and different amounts (0.03 to 3 mg) of gpMBP were added. The percentage of MBP bound to RBC was determined as previously described for SL. Up to 10.6 pg of protein was coupled to 1 X lo8 erythrocytes. Induction of EAE. Lewis rats were routinely immunized with a total volume of 0.1 ml of an emulsion, containing 50 pg gpMBP emulsified in a 1: 1 ratio in Freund’s complete adjuvent (FCA) supplemented with 4 mg/ml of Mycobacterium butyricum (Difco, Detroit, Mich.) and 0.05 ml of this emulsion was injected into each hind footpad. Clinical evaluation of EAE. Rats were observed daily for EAE and assigned a daily grade as follows: 1, tail tip limp plus weight loss; 2, whole tail limp; 3, unsteady gait, dragging one hind leg; 4, dragging both hind legs; and 5, paralysis with urinary and fecal incontinence. A rat that died during an experiment was assigned a grade of 5 until the end of the period of observation for the group. Each animal received a clinical grade, which was the total of all daily grades for the duration of the experiment. Clinical signs usually appeared lo-12 days after challenge with MBP in FCA, and the rats were close to recovery by Day 18. Animals were considered to have been severely ill if they had hind limb paralysis and urinary incontinence for 1 or more days. Lymphocyte proliferation assay. Popliteal lymph nodes were collected on Day 28 after injection of rats with MBP in FCA. Single-cell suspensions were made by passing the cells through coarse and fine wire-mesh screens into RPM1 1640 (Gibco Laboratories, Burlington, Ontario). The cells were washed three times and were resuspended, to a concentration of 0.5 X lo6 viable cells/ml in medium composed of RPM1 1640 supplemented with 5% fetal calf serum (Gibco Laboratories) and containing 100 kg/ml of streptomycin, 100 units/ml of penicillin (Difco), 1% glutamine, and 2 X lop5 M 2-mercaptoethanol; their viability was established using the trypan blue dye exclusion method. Cell cultures (100 ~1 each) were set up in quadruplicate in Falcon 96-well plates (Becton-Dickenson, Oxnard, Calif.) and maintained for 96 hr with an additional 100 ~1 of medium without antigen. Antigens used in proliferation assays were gpMBP, lysozyme (Miles Laboratories, South Africa), purified protein derivative of tuberculin (PPD) (Connaught, Willowdale, Ontario), and concanavalin A (Con A) (Pharmacia, Dorval, Quebec). Six hours before harvesting the cells, 1 &i of [3H]thymidine was added to each culture and the cells were then harvested onto glass-fiber filter papers. Incorporation of 3H was determined with a Beckman LS-300 liquid scintillation counter (Beckman Instruments, Irvine, Calif.) Statistics. Differences between clinical grades and A cpm were analyzed using Student’s t test. When an experiment was repeated more than twice, means for each group were compared using a paired t test. Differences between numbers of severely ill rats in control and experimental groups were analyzed using Fishers Exact Test. RESULTS Kinetic Studies on the Suppression of EAE with MBP-SL Rats (four per group) were pretreated iv with 1 X lo7 (OA)-SL either 3 or 7 days before induction of EAE, i.e., MBP/FCA. The results of three separate experiments are pretreated 3 days before induction of EAE with MBP-SL
MBP-SL or ovalbumin before administration of shown in Table 1. Rats showed no reduction in
MC KENNA,
254
CARTER,
AND SEHON
TABLE 1 Kinetic Studies on the Suppression of EAE with MBP-SL” Day MBP-SL administered
Cell type! (1 x 10’)
Clinical grade score
Number of rats with severe EAEC
A cpm (X104)d MBP
PPD
-3 -3
OA-SL MBP-SL
12.3 k 2.1 12.6 + 2.4
7112 6112
4.3 f 1.7 4.2 + 1.4
8.8 * 3.5 9.5 k 3.5
-7 -7
OA-SL MBP-SL
14.5 rt 2.7 9.5 + 3.1
12112 3112’
6.2 + 1.7 3.6 + 0.9’
10.2 + 4.2 9.5 + 6.1
’ Results expressed are the mean results of three different experiments. b Rats (4/group/experiment) were pretreated with OA-SL or MBP-SL iv either 3 or 7 days Before induction of EAE with 50 pg MBP/PCA. The mean amount of MBP Bound to 1 X 10’ SL was 136 pg. c Severe EAE is, as described under Materials and Methods. d A cpm refers to the difference in cpm Between LNC (from pretreated rats) cultured with antigen plus medium and those LNC cultured with medium alone. e P < 0.025.
severity of disease compared with animals pretreated with OA-SL while rats pretreated with MBP-SL 7 days before induction of disease showed statistically significant suppression of their clinical disease compared to controls. In order to avoid the possible complication that coupling reactions performed at different times may result in varying amounts of MBP being bound to the SL, all MBP-cell conjugates were prepared and administered to the rats on the same day. In addition to the studies on disease induction the effect of administration of MBP-SL at different times before immunization with MBP/FCA on the in vitro proliferative response of lymph node cells (LNC) from pretreated rats was examined. Animals pretreated with MBP-SL 3 days before immunization with MBP showed no reduction in their proliferative response to MBP, while animals pretreated with MBP-SL 7 days before induction of disease showed a statistically significant reduction in their proliferative response to MBP. The in vitro proliferative response to PPD was not affected by pretreatment of rats with MBP-SL either 3 or 7 days before immunization with MBP/FCA. Hence, it may be concluded that a period longer than 3 days prior to immunization is required for induction of the cells and/ or factors responsible for suppression of either clinical EAE or the in vitro proliferative response to MBP. Abrogation
of the MBP-SL-Induced
Suppression of EAE by Cyclophosphamide
Rats were injected ip with cyclophosphamide (Cy) 48 hr before the administration of either 1 X 10’ OA-SL or MBP-SL, which was followed 6 days later by the injection of 50 pg MBP/FCA. The results in Table 2 show that animals that received cyclophosphamide as well as MBP-SL no longer showed suppression of clinical EAE. Rats that received MBP-SL alone before injection of MBP/FCA showed a statistically significant reduction in their clinical grade. These results are taken to indicate that Cy abolished the suppressogenic effect of MBP-SL. Animals injected with Cy before pretreatment with 1 X 10’ OA-SL developed symptoms as rapidly and for the same duration as animals injected with MBP/FCA only. Hence, Cy
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ALLERGIC
255
ENCEPHALOMYELITIS
TABLE 2 Effect of Cyclophosphamide on Suppression of EAE by MBP-SL”
Cyclophosphamide b
Clinical grade score
Pretreatment’ regimen
-
OA-SL MBP-SL OA-SL MBP-SL
+
+
15.2 8.7 15.5 15.6
+ + * f
Number of rats with severe EAEd
1.7 5.9’ 3.5 2.2
718 l/8’
618 w
a Results expressed are the average of the mean results for two separate experiments. bCyclophosphamide (20 mg&) was administered ip 48 hr before either OA-SL or MBP-SL was injected. c Rats (4/group/experiment) were given I X 10’ OA-SL or MBP-SL iv and then 6 days later were immunized with 50 pg MBP/FCA. The mean amount of MBP bound to 1 X 10’ SL was 207 pg. d Severe EAE, as defined under Materials and Methods. eP < 0.05.
treatment had no effect on the induction the course of EAE. Transfer by Lymphoid by MBP-SL
of disease, i.e., Cy did not itself influence
Cells of Suppression of EAE Induced by MBP-RBC,
But Not
To establish if the suppression of EAE induced by MBP-cell conjugates in Lewis rats could be transferred by cells to naive recipients, the following experiment was performed in duplicate. Rats were pretreated with either (a) 1 X 10’ MBP-SL or OA-SL conjugate or (b) 1 X 10’ MBP-RBC or OA-RBC conjugates. These animals were then divided into two groups one of which was immunized 6 days later with 50 pg MBP/FCA to induce disease and the other group was sacrificed 14 days later; their lymph node and spleens were then removed and pooled. These donor cells (3-4 X lo*) were injected iv to recipient Lewis rats that also received 50 pg MBP/FCA 24 hr later. From the results shown in Table 3 it is clear that significant suppression of EAE was found in recipient rats when the donors had been pretreated with MBP-RBC. However, no significant suppression of EAE was brought about in the recipient rats by cells from animals pretreated with MBP-SL, though a slight reduction in the clinical grade was seen. Suppression of EAE by MBP-SL
in Histocompatible
Rat Strains
Lewis rats (RTl’, four/group) were pretreated with 1 X 10’ MBP-SL, the SL having been obtained from the MHC-compatible rat strain, F344, which is phenotypically also RTl’. These pretreated rats were then immunized with 50 pg MBP/FCA 6 days later and observed for development of EAE; their draining LNC were examined in the in vitro proliferative assay for their response to MBP. The rest&s shown in Table 4 indicate that while no significant reduction in the severity of EAE could be effected by the F344 MBP-SL, there was a reduction in the in vitro proliferative response to MBP. This suppression was deemed to be immuno-
256
MC KENNA,
CARTER, AND SEHON TABLE 3
Transfer of Lymph Node and Spleen Cells from Rats Pretreated with MBP-SL or MBP-RBC’ Pretreatment of donorsb
Disease induced in
Clinical grade score
OA-SL MBP-SL OA-SL MBP-SL OA-RBC MBP-RBC OA-RBC MBP-RBC
Donors Donors Recipients Recipients Donors Donors Recipients Recipients
20.5 15.5 23.4 20.3 20.8 10.3 18.7 12.2
+ + f f -t f + f
Number of rats with severe EAEc
1.3 0.6d 4.8 2.8 1.5 5.6d 3.7 6.2d
414 214 717 i1/12 414 214 7/8 l/gd
a Results expressed are the mean results for two separate experiments. ’ Donor rats were pretreated with either (a) I X 10’ MBP-SL or OA-SL or (b) 1 X lo* MBP-RBC or OA-RBC and either immunized 6 days later with 50 pg MBP/FCA or left for 14 days before being killed. Lymph node and spleen leukocytes (3-4 X 10’) from these sacrificed rats were then transferred iv to naive recipients syngeneic animals that were immunized with 50 pg MBP/FCA 24 hr later (4 rats/group/ experiment). The mean amount of MBP bound to 1 X 10’ SL was 45 pg and the mean amount of MBP bound to 1 X lo* RBC was 10 pg. ’ Severe EAE, as defined under Materials and Methods.
dP < 0.05.
logically specific in that LNC from animals pretreated with F344 MBP-SL showed no reduction in their proliferative response to PPD compared with control animals. Thus, MHC-histocompatible SL may substitute for syngeneic SL in suppressing the specific proliferative response to MBP. Similar experiments using MHC-histoincompatible rat strains yielded inconclusive results (data not shown).
Increasing the Suppression of EAE by Multiple Doses of MBP-SL Rats (four per group) were pretreated iv with either a single dose of 1 X lo7 MBP-SL 6 days before induction of disease or 3 doses of 1 X lo7 MBP-SL, given
TABLE 4 Effect of Pretreatment with Histocompatible MBP-SL on EAE” Source of SL
Pretreatment regimen b
Clinical grade score
Number of rata with severe EAE’
A cpm (Xl@)d MBP
PPD
F344 F344
OA-SL MBP-SL
12.6 f 3.2 11.3 k 2.8
lo/l6 9116
5.5 + 2.8 3.0 + 1.0
9.4 + 1.2 11.3 +- 2.8
LIResults shown are the average of the mean results for four different experiments. b Rats (4/group/experiment) were pretreated with either OA-SL or MBP-SL 6 days before induction of EAE with 50 +tg MBP/FCA. The mean amount of MBP bound to 1 X 10’ SL was 85 pg. c Severe EAE, as defined under Materials and Methods. d A cpm, as described in notes to Table I.
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14, 10, and 6 days before induction of EAE. Control groups received either a single dose of 1 X lo7 OA-SL or 3 doses of OA-SL, given at the same time as MBP-SL. The results of two experiments are shown in Table 5. It is clear from these results that the degree of suppression of clinical EAE as measured by the drop in the clinical grade is greater in those animals that received multiple doses of MBP-SL compared with animals receiving a single dose of MBP-SL. The effects of pretreatment of rats, with multiple doses of MBP-SL, on the in vitro proliferative response to both MBP and the unrelated antigen PPD were examined. Both single and multiple doses of MBP-SL induced a decrease of approximately 45% in the proliferative response to MBP. Thus, unlike the increase in suppression of disease shown by rats given multiple doses of MBP-SL, increasing the doses of MBP-SL did not increase the suppression of the in vitro proliferative response to MBP. By contrast there was no suppression of the in vitro proliferative response to PPD in rats given either 1 or 3 doses of MBP-SL compared with the response of lymphocytes of control rats receiving OA-SL; these results were taken to indicate that pretreatment with MBP-SL resulted in specific suppression of the in vitro response of LNC to MBP. DISCUSSION The phenotypic unresponsive state induced by syngeneic antigen-cell conjugates has been shown to be caused by two distinct mechanisms. Moody, Weksler and coworkers (12) demonstrated that tolerance to trinitrophenol (TNP) induced by TNP-syngeneic erythrocyte conjugates (TNP-RBC) was rapidly induced but the maximum suppressive activity of transferred cells required 7 days for development. Miller and Claman (13) showed by cell transfer that not only did the kinetics of phenotypic tolerance induction to a hapten differ from those of Ts induction, but that phenotypic tolerance could also exist independently of demonstrable Ts activity. TABLE Effect
Expt
of Multiple
Clinical grade score
Pretreatment regimen”
5
Doses of MBP-SL Number of rats with severe EAEb
on EAE A cpm (X10’)’ MBP
PPD
1
OA-SL MBP-SL OA-SL MBP-SL
X X X X
1 1 3 3
11.0 11.3 9.4 3.5
* + + f
5.3 4.7 3.5 2.2d
214 214 214 O/4
9.5 3.2 6.3 2.9
+ + + +
0.7 0.7 0.7 0.8
15.6 14.2 14.4 18.0
f f f f
1.4 1.3 1.1 1.9
2
OA-SL MBP-SL OA-SL MBP-SL
X X X X
1 1 3 3
15.5 7.1 11.9 2.5
+ f f +
3.2 5.7d 2.2 3.4d
314 114 214 O/4
4.6 2.2 3.0 1.7
+ f + *
0.6 0.4 0.4 0.3
8.1 6.1 5.3 5.1
+ + + +
0.4 0.6 0.4 0.3
“Rats (4/group/experiment) were pretreated with OA-SL or MBP-SL either (a) once, 6 days before immunization with 50 jog MBP/FCA, or (b) three times, 14, 10, and 6 days before immunization with MBP/FCA. The mean amount of MBP bound to 1 X 10’ SL was 114 pg. b Severe EAE, as defined under Materials and Methods. ’ A cpm, as defined in note to Table 1.
dP < 0.025.
258
MC
KENNA,
CARTER,
AND
SEHON
In a subsequent study (14), Miller et al. extended these findings to include a protein antigen and showed that, although phenotypic tolerance was evident 1 day after treatment with protein-coupled cells, active Ts cells could not be demonstrated until 4-7 days after treatment. From the results of the present study, it is evident that rats pretreated with MBP-leucocytes 3 days before MBP/FCA injection showed no evidence of disease suppression. Thus, it can be inferred that this mode of antigen presentation on leucocyte at this time does not induce either central tolerance or formation of Ts cells. However, those animals which had been injected with MBP-leucocytes 7 days prior to the MBP/FCA injection did show signs of disease suppression. Also significantly suppressed by this treatment was the in vitro proliferative response to MBP, whereas treatment on Day -3 had no apparent effect. The kinetics of unresponsiveness induced by MBP-cell conjugates thus are consistent with the interpretation that the effect was mediated by Ts cells. Unresponsiveness mediated by Ts cells can be preventing by treating donor animals with cyclophosphamide. Miller et al. (13) demonstrated that, whereas the induction of phenotypic tolerance by antigen-coupled cells was not affected by treatment with cyclophosphamide, the production of Ts cells in the treated cell donors was completely abolished. Sherr et al. (5) also showed that the phenotypic tolerance induced by administering antigen-coupled cells either 3 or 7 days before immunization was unaffected by cyclophosphamide. The conspicuous finding however, was that the production of Ts cells which, as described previously, occurred only in animals that had been injected 7 days prior to immunization, was completely abolished by the cyclophosphamide treatment. The results of these experiments support the conclusion that administering cyclophosphamide completely abolished the supressive effect of MBP-coupled leucocytes. It was evident also that cyclophosphamide treatment alone had no effect on the capacity of MBP/FCA to induce disease. Previous data (8) indicated that suppression of EAE could be induced by pretreatment of rats with conjugates of MBP and syngeneic leucocytes or erythrocytes. In our attempts to demonstrate the existence of Ts cells by cell transfer, we observed that only pretreatment with MBP and erythrocytes led to demonstrable Ts production. Although MBP-leucocyte conjugates led to direct suppression of EAE induction, we can only speculate that Ts numbers in these groups were marginal for their demonstration directly in cell-transfer experiments. In general, the level of suppression in recipients of putative supressor cells has been reported to be less than that observed in the donors of the Ts (3, 13). Claman and coworkers (15) showed previously that conjugates of hapten with H-Zcompatible leucocytes induced the activation of Ts cells in mice. We chose to examine the effect of the rat MHC (RTl) locus on the capacity of MBP-leucocytes to suppress EAE. The data showed that injection of RTl identical MBP-SL could consistently suppress the in vitro proliferative response to MBP following immunization with MBP/FCA while not significantly affecting disease. A similar discrepancy between the effects of suppression by insulin-cell conjugates on in vivo and in vitro manifestations of delayed-type hypersensitivity (DTH) was observed by Reiner and Claman (16). We have found in general (unpublished observations) that suppression of the in vitro response to MBP is more easily brought about than suppression of clinical EAE by MBP-SL.
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It is noteworthy that unresponsiveness, with respect to EAE expression, can also be induced by pretreating rats with MBP in incomplete Freund’s adjuvant (17-19). This unresponsiveness was shown to be due to a transferrable suppressor cell that acted at the inductive stage of the response. Taken collectively, the weight of the evidence supports the view that the suppression of EAE in MBP-leucocyteand MBP-erythrocyte-treated animals was caused by Ts cells. Conceivably, the reason Ts-cell activity could not be consistently demonstrated by ceil transfer is that a combination of relatively insufficient numbers of cells being transferred and the time chosen for transfer. There is no doubt however, that MBP-leucocyte and MBP-erythrocyte conjugates can be used effectively as a means for suppressing the symptoms of EAE. ACKNOWLEDGMENTS The excellent technical assistance of Ms. Eleanor Shewchuk and the cheerful cooperation of Ms. Joyce Gilmour during the typing of this paper are gratefully acknowledged.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
Scott, D. W., and Long, C. A., J. Exp. Med, 144, 1369, 1976. Battisto, J. R., and Bloom, B. R., Nature (London) 212, 156, 1966. Miller, S. D., and Claman, H. N., J. Immunol. 117, 1519, 1976. Miller, S. D., Sy, M. S., and Claman, H. N., J. Immunol. 129, 461, 1977. Sherr, D. H., Heghinian, K., Benacerraf, B., and Doti, M. E., J. Immunol. 124, 1389, 1980., Schoen, R. T., Greene, M. I., and Trentham, D. E., J. Immunol. 128,717, 1982. Braley-Mullen, H., Thompson, J. Cl., Sharp, G. C., and Hyriakos, M., Cell. Immunol. 51, 408, 1980. McKenna, R. M., Carter, B. G., Paterson, J. A., and Sehon, A. H., Ceil. Immunol. 81, 391, 1983. Sriram, S., Schwartz, G., and Steinman, L., Cell. Immunol. 75, 378, 1983. Pitts, 0. M., Barrows, A. A., and Day, E. D., Prep. Biochem. 6, 239, 1976. McConahey, P. J., and Dixon, F. J., Intl. Arch. Allergy Appl. Immunoi. 29, 185, 1966. Moody, C. E., Innes, J. B., Siskind, G. W., and Weksler, M. E., J. Immunol. 120, 844, 1978. Miller, S. D., Sy, M. S., and Claman, H. N., Eur. J. Immunol. 7, 165, 1977. Miller, S. D., Wetzig, R. P., and Claman, H. N., J. Exp. Med. 149, 758, 1979. Miller, S. D., Sy, M. S., and Claman, H. N., J. Exp. Med. 147, 788, 1978. Reiner, R. L., and Claman, H. N., Cell. Immunol. 60, 3 14, 1981. Welch, A. M., and Swanborg, R. H., Eur. J. Immunol. 6,919, 1976. Swierkosz, J. E., and Swanborg, R. H., J. Immunol. 119, 1501, 1977. Welch, A. M., Swierkosz, J. E., and Swanborg, R. H., J. Immunol. 121, 1701, 1978.
IMMUNOLOGY
88, 25 l-259 (1984)
Studies on the Mechanism of Suppression of Experimental Encephalomyelitis Induced by Myelin Basic Protein-Cell Conjugates RACHEL M. MCKENNA,’
BRIAN G. CARTER,
Allergic
AND ALEC H. SEHON
MRC Group for Allergy Research, Department of Immunology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba R3E OW3, Canada Received February 7, 1984; accepted May IO, 1984 The mechanism of suppression of experimental allergic encephalomyelitis (EAE) induced in Lewis rats by pretreatment with myelin basic protein (MBP) coupled to syngeneic spleen leukocytes (SL) was examined. Studies on the kinetics of the tolerance induction showed that pretreatment with MBP-SL suppressed EAE if given 7 but not 3 days before the diseaseinducing injection of MBP in Freund’s complete adjuvant. Treatment with cyclophosphamide 48 hr before administration of MBP-SL completely abolished the suppression of EAE. Transfer of lymph node and spleen cells from MBP-syngeneic erythrocyte conjugate (MBP-RBC) but not MBP-SL-pretreated rats resulted in suppression of disease in recipients subsequently given a disease-inducing injection of MBP. Administration of MBP coupled to SL from the histocompatible rat strain F344 resulted in suppression of the MBP-induced proliferative response of spleen cells from Lewis rats which had been given a disease-inducing injection of MBP. Taken together these results are consistent with the suppression of EAE induced by MBP-SL being mediated by suppressor T cells. o 1984 Academic RW, I”~.
INTRODUCTION It has been widely demonstrated that suppression of the humoral and cellular immune responses can be achieved by pretreatment with antigen coupled to lymphoid cells ( l-3). This suppression has been attributed to two distinct mechanisms, which may coexist (4-6), one involves T-cell tolerance (clone inhibition), the other active suppression by T suppressor (T,) cells. Antigen-lymphocyte conjugates have been used successfully to suppress the symptoms of autoimmune experimental thyroiditis (7) and collagen-induced arthritis (6). We (8) and others (9) have reported that experimental allergic encephalomyelitis (EAE) also can be suppressed using this approach, i.e., pretreatment of rats with conjugates of myelin basic protein (MBP) with lymhocytes or erythrocytes suppressed the disease. Furthermore, the suppression of both disease and perivascular cuffing in the spinal cord was found to correlate positively with the amount of MBP bound to the lymphocytes (8). In this report we provide evidence consistent with the suppression being mediated by T, cells. ’ Holder of fellowship from the Medical Research Council of Canada. 2.51 0008-8749/84 $3.00 Copyright Q 1984 by Academic Ptw, Inc. All rights of npmduction in any form reserved.
252
MC KENNA,
MATERIALS
CARTER, AND SEHON
AND
METHODS
Ruts. Female Lewis and F344 rats weighing 150-200 g were obtained from The Charles River Breeding Laboratories (Wilmington, Mass.) and were used for all experiments. Antigens. The method used to isolate MBP from whole central nervous system tissue was a modification of that described by Pitts et al. ( 10). Whole guinea pig (gp) spinal cords (Pel Freeze, Rogers, Ark.) were subjected to lipid extraction, followed by aqueous extraction, and then final acid extraction. The MBP was isolated from the acid extract by gel filtration through an ACA 54 (Ultrogel, Fisher Scientific, Canada) column (2.6 X 90 cm) equilibrated with 1 M glycine-HCl buffer (pH 2.8) containing 1 M urea. The individual fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the electrophoretie patterns were compared with those of rabbit MBP and gpMBP, appropriate fractions were pooled and tested for their encephalitogenic properties. Purified MBP gave one major band and one minor band and appeared to have a similar purity to samples of rabbit MBP and gpMBP which were kindly provided, respectively, by Dr. M. Kies, Myelin Chemistry Laboratory, NIMH (Bethesda, Md.) and Dr. C. Whitacre and Dr. P. Y. Paterson (Department of Microbiology and Immunology, Northwestern University, Evanston Ill.). The ovalbumin used was a five-times recrystallized preparation (ICN, Cleveland, Ohio). Coupling of MBP to spleen leukocytes (MBP-SL). Whole spleens were removed from healthy rats and dispersed into single-cell suspensions by gentle homogenization in a loose-fitting Teflon-glass homogenizer. Erythrocytes were removed by centrifugation through Ficoll-metrizoate (11). Leukocytes obtained from the interface were washed three times with RPM1 1640 and resuspended at a concentration of 1 X 108/ml. To this I-ethyl-(3dimethylaminopropyl)carbodiimide (EDCI; Stony Chemical Corp., Muskegon, Mich.), at a concentration of 5 mg/ml, and different amounts (0.4 to 50 mg/ml) of guinea pig MBP were added. The mixture was incubated for 30 min at room temperature on a rotator and then washed three times with RPM1 1640 to remove any unbound MBP. Cells were resuspended and the viability was established by dye exclusion. To determine the amount of MBP that was actually bound to SL in each individual experiment, a 100 ~1 sample of the cell reaction mixture was removed at the start of the incubation period and immediately a trace amount ( 10 ~1, approximately 0.5 pg) of ‘2’I-labeled MBP, which had been prepared by the chloramine-T method, was added to it (12). (Free iodine had been separated from bound iodine by passage through a Sephadex G-25 column.) This radioactive mixture was then treated in the same way as the original cell suspension and the number of counts that remained associated with the cells after extensive washing was compared to the number of counts originally added, and the percentage of cell-bound MBP was thus calculated. From the results of four different experiments in which gpMBP was coupled to SL it was evident that from 4.4 to 96.8 rg were coupled to 1 X lo7 cells. Coupling of MBP to rat erythrocytes(MBP-RBC). This reaction was carried out in essentially the same way as for coupling MBP to SL. Lewis rats were bled from the heart and the blood was collected into heparinized containers. The erythrocytes were washed three times with saline to remove the bufIy coat, counted, and adjusted to a concentration of 1 X lo9 cells/ml in RPM1 1640. To the cells EDCI, at a
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concentration of 50 mg/ml, and different amounts (0.03 to 3 mg) of gpMBP were added. The percentage of MBP bound to RBC was determined as previously described for SL. Up to 10.6 pg of protein was coupled to 1 X lo8 erythrocytes. Induction of EAE. Lewis rats were routinely immunized with a total volume of 0.1 ml of an emulsion, containing 50 pg gpMBP emulsified in a 1: 1 ratio in Freund’s complete adjuvent (FCA) supplemented with 4 mg/ml of Mycobacterium butyricum (Difco, Detroit, Mich.) and 0.05 ml of this emulsion was injected into each hind footpad. Clinical evaluation of EAE. Rats were observed daily for EAE and assigned a daily grade as follows: 1, tail tip limp plus weight loss; 2, whole tail limp; 3, unsteady gait, dragging one hind leg; 4, dragging both hind legs; and 5, paralysis with urinary and fecal incontinence. A rat that died during an experiment was assigned a grade of 5 until the end of the period of observation for the group. Each animal received a clinical grade, which was the total of all daily grades for the duration of the experiment. Clinical signs usually appeared lo-12 days after challenge with MBP in FCA, and the rats were close to recovery by Day 18. Animals were considered to have been severely ill if they had hind limb paralysis and urinary incontinence for 1 or more days. Lymphocyte proliferation assay. Popliteal lymph nodes were collected on Day 28 after injection of rats with MBP in FCA. Single-cell suspensions were made by passing the cells through coarse and fine wire-mesh screens into RPM1 1640 (Gibco Laboratories, Burlington, Ontario). The cells were washed three times and were resuspended, to a concentration of 0.5 X lo6 viable cells/ml in medium composed of RPM1 1640 supplemented with 5% fetal calf serum (Gibco Laboratories) and containing 100 kg/ml of streptomycin, 100 units/ml of penicillin (Difco), 1% glutamine, and 2 X lop5 M 2-mercaptoethanol; their viability was established using the trypan blue dye exclusion method. Cell cultures (100 ~1 each) were set up in quadruplicate in Falcon 96-well plates (Becton-Dickenson, Oxnard, Calif.) and maintained for 96 hr with an additional 100 ~1 of medium without antigen. Antigens used in proliferation assays were gpMBP, lysozyme (Miles Laboratories, South Africa), purified protein derivative of tuberculin (PPD) (Connaught, Willowdale, Ontario), and concanavalin A (Con A) (Pharmacia, Dorval, Quebec). Six hours before harvesting the cells, 1 &i of [3H]thymidine was added to each culture and the cells were then harvested onto glass-fiber filter papers. Incorporation of 3H was determined with a Beckman LS-300 liquid scintillation counter (Beckman Instruments, Irvine, Calif.) Statistics. Differences between clinical grades and A cpm were analyzed using Student’s t test. When an experiment was repeated more than twice, means for each group were compared using a paired t test. Differences between numbers of severely ill rats in control and experimental groups were analyzed using Fishers Exact Test. RESULTS Kinetic Studies on the Suppression of EAE with MBP-SL Rats (four per group) were pretreated iv with 1 X lo7 (OA)-SL either 3 or 7 days before induction of EAE, i.e., MBP/FCA. The results of three separate experiments are pretreated 3 days before induction of EAE with MBP-SL
MBP-SL or ovalbumin before administration of shown in Table 1. Rats showed no reduction in
MC KENNA,
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CARTER,
AND SEHON
TABLE 1 Kinetic Studies on the Suppression of EAE with MBP-SL” Day MBP-SL administered
Cell type! (1 x 10’)
Clinical grade score
Number of rats with severe EAEC
A cpm (X104)d MBP
PPD
-3 -3
OA-SL MBP-SL
12.3 k 2.1 12.6 + 2.4
7112 6112
4.3 f 1.7 4.2 + 1.4
8.8 * 3.5 9.5 k 3.5
-7 -7
OA-SL MBP-SL
14.5 rt 2.7 9.5 + 3.1
12112 3112’
6.2 + 1.7 3.6 + 0.9’
10.2 + 4.2 9.5 + 6.1
’ Results expressed are the mean results of three different experiments. b Rats (4/group/experiment) were pretreated with OA-SL or MBP-SL iv either 3 or 7 days Before induction of EAE with 50 pg MBP/PCA. The mean amount of MBP Bound to 1 X 10’ SL was 136 pg. c Severe EAE is, as described under Materials and Methods. d A cpm refers to the difference in cpm Between LNC (from pretreated rats) cultured with antigen plus medium and those LNC cultured with medium alone. e P < 0.025.
severity of disease compared with animals pretreated with OA-SL while rats pretreated with MBP-SL 7 days before induction of disease showed statistically significant suppression of their clinical disease compared to controls. In order to avoid the possible complication that coupling reactions performed at different times may result in varying amounts of MBP being bound to the SL, all MBP-cell conjugates were prepared and administered to the rats on the same day. In addition to the studies on disease induction the effect of administration of MBP-SL at different times before immunization with MBP/FCA on the in vitro proliferative response of lymph node cells (LNC) from pretreated rats was examined. Animals pretreated with MBP-SL 3 days before immunization with MBP showed no reduction in their proliferative response to MBP, while animals pretreated with MBP-SL 7 days before induction of disease showed a statistically significant reduction in their proliferative response to MBP. The in vitro proliferative response to PPD was not affected by pretreatment of rats with MBP-SL either 3 or 7 days before immunization with MBP/FCA. Hence, it may be concluded that a period longer than 3 days prior to immunization is required for induction of the cells and/ or factors responsible for suppression of either clinical EAE or the in vitro proliferative response to MBP. Abrogation
of the MBP-SL-Induced
Suppression of EAE by Cyclophosphamide
Rats were injected ip with cyclophosphamide (Cy) 48 hr before the administration of either 1 X 10’ OA-SL or MBP-SL, which was followed 6 days later by the injection of 50 pg MBP/FCA. The results in Table 2 show that animals that received cyclophosphamide as well as MBP-SL no longer showed suppression of clinical EAE. Rats that received MBP-SL alone before injection of MBP/FCA showed a statistically significant reduction in their clinical grade. These results are taken to indicate that Cy abolished the suppressogenic effect of MBP-SL. Animals injected with Cy before pretreatment with 1 X 10’ OA-SL developed symptoms as rapidly and for the same duration as animals injected with MBP/FCA only. Hence, Cy
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ENCEPHALOMYELITIS
TABLE 2 Effect of Cyclophosphamide on Suppression of EAE by MBP-SL”
Cyclophosphamide b
Clinical grade score
Pretreatment’ regimen
-
OA-SL MBP-SL OA-SL MBP-SL
+
+
15.2 8.7 15.5 15.6
+ + * f
Number of rats with severe EAEd
1.7 5.9’ 3.5 2.2
718 l/8’
618 w
a Results expressed are the average of the mean results for two separate experiments. bCyclophosphamide (20 mg&) was administered ip 48 hr before either OA-SL or MBP-SL was injected. c Rats (4/group/experiment) were given I X 10’ OA-SL or MBP-SL iv and then 6 days later were immunized with 50 pg MBP/FCA. The mean amount of MBP bound to 1 X 10’ SL was 207 pg. d Severe EAE, as defined under Materials and Methods. eP < 0.05.
treatment had no effect on the induction the course of EAE. Transfer by Lymphoid by MBP-SL
of disease, i.e., Cy did not itself influence
Cells of Suppression of EAE Induced by MBP-RBC,
But Not
To establish if the suppression of EAE induced by MBP-cell conjugates in Lewis rats could be transferred by cells to naive recipients, the following experiment was performed in duplicate. Rats were pretreated with either (a) 1 X 10’ MBP-SL or OA-SL conjugate or (b) 1 X 10’ MBP-RBC or OA-RBC conjugates. These animals were then divided into two groups one of which was immunized 6 days later with 50 pg MBP/FCA to induce disease and the other group was sacrificed 14 days later; their lymph node and spleens were then removed and pooled. These donor cells (3-4 X lo*) were injected iv to recipient Lewis rats that also received 50 pg MBP/FCA 24 hr later. From the results shown in Table 3 it is clear that significant suppression of EAE was found in recipient rats when the donors had been pretreated with MBP-RBC. However, no significant suppression of EAE was brought about in the recipient rats by cells from animals pretreated with MBP-SL, though a slight reduction in the clinical grade was seen. Suppression of EAE by MBP-SL
in Histocompatible
Rat Strains
Lewis rats (RTl’, four/group) were pretreated with 1 X 10’ MBP-SL, the SL having been obtained from the MHC-compatible rat strain, F344, which is phenotypically also RTl’. These pretreated rats were then immunized with 50 pg MBP/FCA 6 days later and observed for development of EAE; their draining LNC were examined in the in vitro proliferative assay for their response to MBP. The rest&s shown in Table 4 indicate that while no significant reduction in the severity of EAE could be effected by the F344 MBP-SL, there was a reduction in the in vitro proliferative response to MBP. This suppression was deemed to be immuno-
256
MC KENNA,
CARTER, AND SEHON TABLE 3
Transfer of Lymph Node and Spleen Cells from Rats Pretreated with MBP-SL or MBP-RBC’ Pretreatment of donorsb
Disease induced in
Clinical grade score
OA-SL MBP-SL OA-SL MBP-SL OA-RBC MBP-RBC OA-RBC MBP-RBC
Donors Donors Recipients Recipients Donors Donors Recipients Recipients
20.5 15.5 23.4 20.3 20.8 10.3 18.7 12.2
+ + f f -t f + f
Number of rats with severe EAEc
1.3 0.6d 4.8 2.8 1.5 5.6d 3.7 6.2d
414 214 717 i1/12 414 214 7/8 l/gd
a Results expressed are the mean results for two separate experiments. ’ Donor rats were pretreated with either (a) I X 10’ MBP-SL or OA-SL or (b) 1 X lo* MBP-RBC or OA-RBC and either immunized 6 days later with 50 pg MBP/FCA or left for 14 days before being killed. Lymph node and spleen leukocytes (3-4 X 10’) from these sacrificed rats were then transferred iv to naive recipients syngeneic animals that were immunized with 50 pg MBP/FCA 24 hr later (4 rats/group/ experiment). The mean amount of MBP bound to 1 X 10’ SL was 45 pg and the mean amount of MBP bound to 1 X lo* RBC was 10 pg. ’ Severe EAE, as defined under Materials and Methods.
dP < 0.05.
logically specific in that LNC from animals pretreated with F344 MBP-SL showed no reduction in their proliferative response to PPD compared with control animals. Thus, MHC-histocompatible SL may substitute for syngeneic SL in suppressing the specific proliferative response to MBP. Similar experiments using MHC-histoincompatible rat strains yielded inconclusive results (data not shown).
Increasing the Suppression of EAE by Multiple Doses of MBP-SL Rats (four per group) were pretreated iv with either a single dose of 1 X lo7 MBP-SL 6 days before induction of disease or 3 doses of 1 X lo7 MBP-SL, given
TABLE 4 Effect of Pretreatment with Histocompatible MBP-SL on EAE” Source of SL
Pretreatment regimen b
Clinical grade score
Number of rata with severe EAE’
A cpm (Xl@)d MBP
PPD
F344 F344
OA-SL MBP-SL
12.6 f 3.2 11.3 k 2.8
lo/l6 9116
5.5 + 2.8 3.0 + 1.0
9.4 + 1.2 11.3 +- 2.8
LIResults shown are the average of the mean results for four different experiments. b Rats (4/group/experiment) were pretreated with either OA-SL or MBP-SL 6 days before induction of EAE with 50 +tg MBP/FCA. The mean amount of MBP bound to 1 X 10’ SL was 85 pg. c Severe EAE, as defined under Materials and Methods. d A cpm, as described in notes to Table I.
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14, 10, and 6 days before induction of EAE. Control groups received either a single dose of 1 X lo7 OA-SL or 3 doses of OA-SL, given at the same time as MBP-SL. The results of two experiments are shown in Table 5. It is clear from these results that the degree of suppression of clinical EAE as measured by the drop in the clinical grade is greater in those animals that received multiple doses of MBP-SL compared with animals receiving a single dose of MBP-SL. The effects of pretreatment of rats, with multiple doses of MBP-SL, on the in vitro proliferative response to both MBP and the unrelated antigen PPD were examined. Both single and multiple doses of MBP-SL induced a decrease of approximately 45% in the proliferative response to MBP. Thus, unlike the increase in suppression of disease shown by rats given multiple doses of MBP-SL, increasing the doses of MBP-SL did not increase the suppression of the in vitro proliferative response to MBP. By contrast there was no suppression of the in vitro proliferative response to PPD in rats given either 1 or 3 doses of MBP-SL compared with the response of lymphocytes of control rats receiving OA-SL; these results were taken to indicate that pretreatment with MBP-SL resulted in specific suppression of the in vitro response of LNC to MBP. DISCUSSION The phenotypic unresponsive state induced by syngeneic antigen-cell conjugates has been shown to be caused by two distinct mechanisms. Moody, Weksler and coworkers (12) demonstrated that tolerance to trinitrophenol (TNP) induced by TNP-syngeneic erythrocyte conjugates (TNP-RBC) was rapidly induced but the maximum suppressive activity of transferred cells required 7 days for development. Miller and Claman (13) showed by cell transfer that not only did the kinetics of phenotypic tolerance induction to a hapten differ from those of Ts induction, but that phenotypic tolerance could also exist independently of demonstrable Ts activity. TABLE Effect
Expt
of Multiple
Clinical grade score
Pretreatment regimen”
5
Doses of MBP-SL Number of rats with severe EAEb
on EAE A cpm (X10’)’ MBP
PPD
1
OA-SL MBP-SL OA-SL MBP-SL
X X X X
1 1 3 3
11.0 11.3 9.4 3.5
* + + f
5.3 4.7 3.5 2.2d
214 214 214 O/4
9.5 3.2 6.3 2.9
+ + + +
0.7 0.7 0.7 0.8
15.6 14.2 14.4 18.0
f f f f
1.4 1.3 1.1 1.9
2
OA-SL MBP-SL OA-SL MBP-SL
X X X X
1 1 3 3
15.5 7.1 11.9 2.5
+ f f +
3.2 5.7d 2.2 3.4d
314 114 214 O/4
4.6 2.2 3.0 1.7
+ f + *
0.6 0.4 0.4 0.3
8.1 6.1 5.3 5.1
+ + + +
0.4 0.6 0.4 0.3
“Rats (4/group/experiment) were pretreated with OA-SL or MBP-SL either (a) once, 6 days before immunization with 50 jog MBP/FCA, or (b) three times, 14, 10, and 6 days before immunization with MBP/FCA. The mean amount of MBP bound to 1 X 10’ SL was 114 pg. b Severe EAE, as defined under Materials and Methods. ’ A cpm, as defined in note to Table 1.
dP < 0.025.
258
MC
KENNA,
CARTER,
AND
SEHON
In a subsequent study (14), Miller et al. extended these findings to include a protein antigen and showed that, although phenotypic tolerance was evident 1 day after treatment with protein-coupled cells, active Ts cells could not be demonstrated until 4-7 days after treatment. From the results of the present study, it is evident that rats pretreated with MBP-leucocytes 3 days before MBP/FCA injection showed no evidence of disease suppression. Thus, it can be inferred that this mode of antigen presentation on leucocyte at this time does not induce either central tolerance or formation of Ts cells. However, those animals which had been injected with MBP-leucocytes 7 days prior to the MBP/FCA injection did show signs of disease suppression. Also significantly suppressed by this treatment was the in vitro proliferative response to MBP, whereas treatment on Day -3 had no apparent effect. The kinetics of unresponsiveness induced by MBP-cell conjugates thus are consistent with the interpretation that the effect was mediated by Ts cells. Unresponsiveness mediated by Ts cells can be preventing by treating donor animals with cyclophosphamide. Miller et al. (13) demonstrated that, whereas the induction of phenotypic tolerance by antigen-coupled cells was not affected by treatment with cyclophosphamide, the production of Ts cells in the treated cell donors was completely abolished. Sherr et al. (5) also showed that the phenotypic tolerance induced by administering antigen-coupled cells either 3 or 7 days before immunization was unaffected by cyclophosphamide. The conspicuous finding however, was that the production of Ts cells which, as described previously, occurred only in animals that had been injected 7 days prior to immunization, was completely abolished by the cyclophosphamide treatment. The results of these experiments support the conclusion that administering cyclophosphamide completely abolished the supressive effect of MBP-coupled leucocytes. It was evident also that cyclophosphamide treatment alone had no effect on the capacity of MBP/FCA to induce disease. Previous data (8) indicated that suppression of EAE could be induced by pretreatment of rats with conjugates of MBP and syngeneic leucocytes or erythrocytes. In our attempts to demonstrate the existence of Ts cells by cell transfer, we observed that only pretreatment with MBP and erythrocytes led to demonstrable Ts production. Although MBP-leucocyte conjugates led to direct suppression of EAE induction, we can only speculate that Ts numbers in these groups were marginal for their demonstration directly in cell-transfer experiments. In general, the level of suppression in recipients of putative supressor cells has been reported to be less than that observed in the donors of the Ts (3, 13). Claman and coworkers (15) showed previously that conjugates of hapten with H-Zcompatible leucocytes induced the activation of Ts cells in mice. We chose to examine the effect of the rat MHC (RTl) locus on the capacity of MBP-leucocytes to suppress EAE. The data showed that injection of RTl identical MBP-SL could consistently suppress the in vitro proliferative response to MBP following immunization with MBP/FCA while not significantly affecting disease. A similar discrepancy between the effects of suppression by insulin-cell conjugates on in vivo and in vitro manifestations of delayed-type hypersensitivity (DTH) was observed by Reiner and Claman (16). We have found in general (unpublished observations) that suppression of the in vitro response to MBP is more easily brought about than suppression of clinical EAE by MBP-SL.
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It is noteworthy that unresponsiveness, with respect to EAE expression, can also be induced by pretreating rats with MBP in incomplete Freund’s adjuvant (17-19). This unresponsiveness was shown to be due to a transferrable suppressor cell that acted at the inductive stage of the response. Taken collectively, the weight of the evidence supports the view that the suppression of EAE in MBP-leucocyteand MBP-erythrocyte-treated animals was caused by Ts cells. Conceivably, the reason Ts-cell activity could not be consistently demonstrated by ceil transfer is that a combination of relatively insufficient numbers of cells being transferred and the time chosen for transfer. There is no doubt however, that MBP-leucocyte and MBP-erythrocyte conjugates can be used effectively as a means for suppressing the symptoms of EAE. ACKNOWLEDGMENTS The excellent technical assistance of Ms. Eleanor Shewchuk and the cheerful cooperation of Ms. Joyce Gilmour during the typing of this paper are gratefully acknowledged.
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Scott, D. W., and Long, C. A., J. Exp. Med, 144, 1369, 1976. Battisto, J. R., and Bloom, B. R., Nature (London) 212, 156, 1966. Miller, S. D., and Claman, H. N., J. Immunol. 117, 1519, 1976. Miller, S. D., Sy, M. S., and Claman, H. N., J. Immunol. 129, 461, 1977. Sherr, D. H., Heghinian, K., Benacerraf, B., and Doti, M. E., J. Immunol. 124, 1389, 1980., Schoen, R. T., Greene, M. I., and Trentham, D. E., J. Immunol. 128,717, 1982. Braley-Mullen, H., Thompson, J. Cl., Sharp, G. C., and Hyriakos, M., Cell. Immunol. 51, 408, 1980. McKenna, R. M., Carter, B. G., Paterson, J. A., and Sehon, A. H., Ceil. Immunol. 81, 391, 1983. Sriram, S., Schwartz, G., and Steinman, L., Cell. Immunol. 75, 378, 1983. Pitts, 0. M., Barrows, A. A., and Day, E. D., Prep. Biochem. 6, 239, 1976. McConahey, P. J., and Dixon, F. J., Intl. Arch. Allergy Appl. Immunoi. 29, 185, 1966. Moody, C. E., Innes, J. B., Siskind, G. W., and Weksler, M. E., J. Immunol. 120, 844, 1978. Miller, S. D., Sy, M. S., and Claman, H. N., Eur. J. Immunol. 7, 165, 1977. Miller, S. D., Wetzig, R. P., and Claman, H. N., J. Exp. Med. 149, 758, 1979. Miller, S. D., Sy, M. S., and Claman, H. N., J. Exp. Med. 147, 788, 1978. Reiner, R. L., and Claman, H. N., Cell. Immunol. 60, 3 14, 1981. Welch, A. M., and Swanborg, R. H., Eur. J. Immunol. 6,919, 1976. Swierkosz, J. E., and Swanborg, R. H., J. Immunol. 119, 1501, 1977. Welch, A. M., Swierkosz, J. E., and Swanborg, R. H., J. Immunol. 121, 1701, 1978.