Eur. J. Immunol. 1973. 3: 279-286 Synthetic copolymers cross-reactive with encephalitogen 279

14 Kibler, R.F. and Barnes, A.E., J. Exp. Med. 1962. 116: 807. 15 Levine, S., Hoenig, E.M. and Kies, M.W., Clin. Exp. Immunol.

16 Dau, P.C. and Peterson, R.D.A., Int. Arch. Allergy Appl.

17 Lennon, V.A., Whittingham, S., Carnegie, P.R., McPherson, T.A.

18 Hashim, G.A. and Eylar, E.H., Arch. Biochem. Biophys. 1969.

19 Westall, F.C., Robinson, A.B., Caccam, J., Jackson, J. and

20 Eylar, E.H. and Hashim, G.A., Arch. Biochem. Biophys. 1969.

21 Chao, L.-P. and Roboz-Einstein, E., J. Biol. Chem. 1970.

1970. 6: 503.

Immunol. 1969. 35: 353.

and Mackay, I.R., J. Immunol. 1971.107: 56.

129: 645.

Eylar, E.H., Nature 1971.229: 22.

131: 215.

245: 6397.

Cynthia Webb, Dvora Teitelbaum, Ruth Arnon and M. Sda

Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot

22 Swanborg, R.H. and Amesse, L.S., J. Immunol. 1971. 107: 281. 23 Robson, G.S.M., McPherson, T.A. and Mackay, I.R., Brit. J.

24 Paterson, P.Y. in Samter, M. (Ed.) Immunological Diseases,

25 Kersting, G. and Pette, E., Deut. Z . Nervenheilk. 1957. 176:

26 Field, E.J. and Raine, C.S. , J. Neurol. Sci. 1969. 8: 397.

27 Alvord, E.C. Jr., Shaw, C.M., Hruby, S. and Kies, M.W., Ann.

28 Roboz-Einstein, E., Csejtey, J., Davis, W.J. and Rauch, H.,

29 Rauch, H.C., Raffel, F.S. and Roboz-Einstein, E., J. Immunol.

Exp. Pathol. 1971. 52: 338.

Little Brown Inc., Boston 1965, p. 788.

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Immunochemistry 1968. 5: 567.

1969.102: 1431.

ln vivo and in vitro immunological cross-reactions between basic encephalitogen and synthetic basic polypeptides capable of suppressing experimental allergic encephalo- myelitis"

A significant extent of immunological cross-reactivity has been demonstrated between the basic encephalitogenic protein of bovine origin and several syn- thetic amino acid copolymers which have suppressive effect on experimental allergic encephalomyelitis (EAE). This cross-reactivity has been conclusively established on the cellular level, both in vivo by means of delayed hypersen- sitivity skin tests and in vitro using transformation of sensitized lymphocytes, as measured by incorporation of radioactive thymidine. The in vitro experi- ments have been conducted with lymph node cells from guinea pigs of both random bred and inbred strains, and on spleen and lymph node cells from rabbits. Definite cross-reactivity was observed between the basic encepha- litogen and all the synthetic copolymers which were previously shown effec- tive in suppression of EAE, whereas ineffective copolymers or unrelated pro- teins did not show any cross-reactivity. In the case of strain 2 guinea pigs and rabbit lymph node cells the cross-reactivity in v i t ro w.as manifested by direct cross-stimulation of the lymphocytes, whereas in random-bred or strain 13 guinea pigs and rabbit spleen cells, the cross-reaction was detected only by means of specific inhibition of the homologous stimulation by the heterolo- gous antigen.

A limited extent of cross-reactivity was observed on the humoral level as well; antibodies provoked in guinea pigs against the synthetic copolymer Cop 1 cross-reacted in the passive cutaneous anaphylaxis assay with the bovine basic encephalitogen.

1. Introduction

Previous studies from this laboratory [ 1-31 have described the suppressive effect of several related synthetic linear basic copolymers of amino acids o n experimental allergic encepha- lomyelitis (EAE). The copolymer which exerted the most

* This work was supported in part by the Freudenberg Foundation.

Correspondence: Ruth Arnon, Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot, Israel

Abbreviations: EAE: Experimental allergic encephalomyelitis BE: Basic encephalitogen Cop 1: Copolymer 1 Cop 2: Copolymer 2 Cop 3: Copolymer 3 AAspLT: A basic copolymer of alanine, aspartic acid, lysine and tyrosine AGL: A basic copolymer of alanine, glutamic acid and lysine AGT: An acidic copolymer of alanine, glutamic acid and tyrosine C F A Complete Freund's adjuvant PCA: Passive cuta- neous anaphylaxis LNC: Lymph node cells

marked suppressive effect was composed of alanine, glutamic acid, lysine and tyrosine, with a molecular weight of 2 3 000, and was designated Cop 1. This copolymer, which suppressed both clinical and histological symptoms of EAE, appeared t o be specific in its action since neither an acidic amino acid copolymer nor unrelated basic proteins had any suppressive capacity. A second batch of Cop 1 showed activity identical t o that of the first batch [ 11.

We have demonstrated that the effect of Cop 1 is not a re- stricted phenomenon, specific t o a particular species, since suppression was obtained in the case of EAE induced in rab- bits as well as in guinea pigs. Furthermore, the copolymer was equally efficient in the suppression of EAE induced in guinea pigs by encephalitogen of either human or bovine origin. It was conclusively established that Cop 1 is devoid of any im- munosuppressive activity, at least when administered under

280 C. Webb, D. Teitelbaum, R. Arnon and M. Sela Eur. J. Immunol. 1973. 3: 279-286

the conditions required for the suppression of EAE [ 3 ] . In view of the above, it seems that the suppressive effect of Cop 1 o n EAE is indeed specific, and it should be of interest t o elu- cidate the mechanism of this effect in correlation to the mech- anism of EAE. Bearing in mind that the autoimmune nature of EAE has been well established, and is believed to be a mani- festation of sensitization t o the basic encephalitogenic pro- tein of myelin (BE) [4, 51, the specific inhibition of this di- sease by Cop 1 might be explained by the possible immuno- logical cross-reaction between Cop 1 and the basic encepha- litogenic protein.

The weight of opinion at the present time is that cellular hyper- sensitivity is the primary immunological mechanism involved in the pathogenesis of EAE. One of the most significant lines of evidence in support of this view is the successful transfer of EAE by means of sensitized lymph node cells, while attempts t o passively transfer the disease by means of serum have failed [$, 61. Therefore, our first experiments, which aimed at de- monstrating immunological cross-reaction between Cop 1 and BE, involved cellular mediated immune reactions. Indeed, our preliminary results indicated the existence of cross-reaction by means of delayed hypersensitivity skin tests in guinea pigs [2].

The cellular immune response in several cases of protein sys- tems has proved more sensitive in detection of cross-reaction than the humoral immune response. Extreme cases have been reported of systems where no cross-reacting humoral antibod- ies were elicited, yet an immunological cross-reaction a t the cellular level was detected, e.g. between human and Ascaris collagens [7], between natural collagens and the synthetic polypeptide (Pro-Gly-Pro), [ 8 ] , and between native lysozyme and its reduced carboxymethylated derivative [9]. Similarly, in the system of lysozyme and a-lactalbumin, only a slight degree of cross-reactivity was observed at the humoral level, whereas at the cellular level the cross-reaction was far more pronounced [ lo].

Though many in uiuo manifestations have been associated with delayed hypersensitivity, none can be measured with the precision with which one can quantitate the correlated in vitro manifestations such as lymphocyte transformations as expressed by incorporation of radioactive thymidine [ 1 1, 121. We, therefore, proceeded to test the immunological cross-reac- tion between BE and Cop 1 at the cellular level using this quan- titative in vitro method for the assay of lymphocyte transforma- tion. In addition, we tested the existence of cross-reactivity a t the humoral level by means of the sensitive passive cutaneous anaphylaxis method.

We wish t o report in the following the existence of a signifi- cant extent of cross-reactivity, detected primarily a t the cel- lular level, between BE and the synthetic polypeptides which exert a suppressive effect on EAE.

2. Materials

2.1. Animals

Random-bred DH albino, inbred strain 2 and strain 13 guinea pigs and New Zealand albino rabbits were used in various ex- periments as specified.

2.2. Basic encephalitogenic protein

BE was prepared from bovine spinal cord, as described pre- viously [ 131, by column chromatography o n SE-Sephadex (Pharmacia, Uppsala).

2.3. Cop 1 and other synthetic polypeptides

The preparation of the various amino acid copolymers has been described in earlier publications [ 1, 31. The composition of these copolymers is summarized in Table 1.

2.4. Antigens

Hen egg-white lysozyme (2 x crystallized) was obtained from Worthington Biochemical Corp., Freehold, N.J. Phosvitin pre- pared from egg yolk was a gift from Mr. Y. Karniely of our Department. Complete Freund's adjuvant (CFA) was obtained from Difco Laboratories, Detroit, Mich.

2.5. Cell-culture media

Culture media-199 (M-199), minimum essential medium ~

for suspension (MEM-S) with added non-essential amino acids (NEAA), and glutamine, penicillin and streptomycin solutions were obtained from Microbiological Associates, Jerusalem, Israel.

Table 1. Composition of synthetic polypeptides

Residue molar ratio in copolymer Amino acid Cop 1 Cop 1 Cop 2 Cop 3 AAspLT AGL AGT

batch batch I 11

Alanine Arginine Aspartic acid Glutamic acid Glycine Histidine Leucine Lysine Phenylalanine Roline Serine Tyrosine

6.0 1.1 6.0 6.7 - 4.0 3.0 _ - - - 1.0 -

- 2.7 1.0 - - - - - -

1.9 2.1 2.0 3.2 - 2.3 1.2 - - 1.4 6.5 - - - - 0.4 1.7 - - -

4.7 4.2 2.8 3.8 2.8 4.6 -

-

- - - - - 1.0 -

- 1.2 - - - 0.4 1.8 -

1.1 3.9 - 1.0 1.0 1.0 1.0 1.0 - 1.0

- - - - - - - - - -

3. Methods

3.1. Delayed hypersensitivity reactions

Guinea pigs were sensitized [ 141 by injection into the hind footpads of 100 pg of the antigen, except in the case of BE where 10 pg were used, in CFA. They were tested 10 days later by intradermal injection of 2 0 pg or 50 p g of the test antigen in 0.1 ml physiological saline (0.15 M NaC1). The ap- pearance of erythema at the site of injection was observed 24 h later, and the extent of the delayed hypersensitivity was quantitated by measuring the diameter of the skin re- action. Only reactions above 5 mm diameter were considered positive.

3.2. Lymphocyte stimulation

Lymphocyte stimulation was studied by [ ''C]thymidine in- corporation in vitro, in part according t o the procedure de- scribed by Tarrab e t al. [ IS] . Experiments with guinea pigs were performed with lymph node cells (LNC). The animals were sensitized with 20- 100 pg of the antigen in CFA into all four footpads. Draining lymph nodes were excised twelve t o twenty days after sensitization. They were trimmed of fat

Eur. J. Immunol. 1973.3: 279-286 Synthetic copolymers cross-reactive with encephalitogen 281

and teased apart under sterile conditions in MEM-S + NEAA with added penicillin (100 pg/ml), streptomycin (100 units/ ml) and glutamine (2 mM) (see section 2.5.). Cells were washed three times in the same medium and suspended in medium supplemented with 13 % inactivated normal guinea pig serum, at a concentration of 5 x lo6 cells/ml. Experiments with rab- bits were performed with either spleen cells or LNC using a similar procedure, but replacing the above culture medium with Medium 199 supplemented with 20 % inactivated normal rabbit serum. Rabbits were sensitized with 2 mg of antigen in CFA in all four footpads and multiple intradermal sites. Their lymph nodes and spleen were removed 12-20 days later. The lymphocytes (8 x lo6 - 10 x lo6 cell/culture) were in- cubated for 24 h with varying concentrations of the test anti- gens, followed by 24 h incubation with 0.1 pCi of [2-14C]thy- midine (New England Nuclear Corp., Boston). The cells were then filtered on Whatman glass fiber paper GF/C, and washed successively with saline, trichloroacetic acid (5 %) and absolute ethanol. The filters were dried and the radioactivity measured in a Tri-Carb liquid scintillation spectrometer (Packard Instru- ment Co., Downers Grove, Ill.). The results are expressed as stimulation indices, namely the ratio of the radioactivity (cpm) in tubes containing antigen, to cpm of antigen-free tubes (con- trol), and they represent an average of values obtained in du- plicate cultures.

The percent cross-stimulation was calculated as follows:

cpm with heterologous antigen - cpm control oo. cprn with homologous antigen - cpm control

3.3. Inhibition of lymphocyte stimulation

The inhibition experiments were performed under identical conditions to those described for the stimulation experiments, except for a preincubation of the lymphocytes with the tested inhibitor [ 10, 161. Varying concentrations of heterologous antigen (inhibitor), were added in 0.1-0.25 mlof tissue cul- ture medium at time 0. Two hours later a constant amount of homologous antigen was added to these cultures as well as to cultures without inhibitor. Every concentration of inhibitor tested was also checked for possible stimulatory effect withoul added homologous antigen.

The percent inhibition was calculated as follows:

cpm without inhibitor - cpm with inhibitor cpm without inhibitor - cpm control

3.4. Preparation of antisera

Guinea pigs were immunized with 100 pg of antigen, in CFA in the four footpads and in several intradermal sites in the nuchal area. Rabbits were immunized with 2 mg of antigen in CFA in multiple intradermal sites and given a booster after 10 days. The BE injected to rabbits was complexed electro- statically with the acidic protein, phosvitin, at a ratio of 1 : 1. The animals were bled, in the case of guinea pigs by intracar- diac puncture and in the case of rabbits from the marginal ear vein, one week after the last injection.

3.5. Passive cutaneous anaphylaxis (PCA)

The PCA test was carried out in guinea pigs by the method described by Ovary [ 171, namely, intradermal injection of serial dilutions of the antiserum, followed 18 h later by in- travenous challenge with a mixture of 3 mg of the antigen and 0.5 5% Evans blue in saline.

3.6. Quantitative precipitin reactions

Increasing amounts of antigen were added to a constant amount of antiserum, the tubes were mixed, and incubated for 1 h at 37 "C and 18 h at 4 OC. The precipitates were centrifuged, washed three times in cold saline (0.15 M NaCl), dissolved in 0.1 N NaOH (1.0 ml), and quantitated by measuring the ab- sorbance at 280 nm within 10 min.

3.7. Inhibition of the precipitin reactions

Increasing amounts of inhibitor were added to a constant volume of serum, the mixtures were incubated at 37 OC for 1 h, and after that time the amount of antigen corresponding to the equivalence zone of the precipitin reaction was added to all the tubes. The reaction mixtures were kept for an ad- ditional 1 h at 37 OC and then 18 h at 4 OC. The precipitates formed were treated as described above for the precipitin re- actions.

3.8. Antigen-binding capacity: Farr technique [ 18, 191

The antigens were labeled with '''I (The Radiochemical Centre, Amersham) according to Hunter and Greenwood [20] . Increas- ing amounts of labeled antigen were incubated with a constant amount of antiserum (0.2 ml) for 1 h at 37 OC; goat antise- rum against rabbit IgG was then added, and the final mixture was maintained at 37 OC for 1 h, followed by 24 h at 4 OC. The precipitates formed were washed, and radioactivity was count- ed in an autogamma spectrometer.

4. Results

4.1. Delayed hypersensitivity reactions

Preliminary experiments have shown that random-bred DH albino guinea pigs sensitized to either BE o t Cop 1, and skin- tested 10 days later, gave positive delayed hypersensitivity reactions to both antigens [2]. We report here results of stud- ies with several synthetic copolymers as well as several strains of guinea pigs, Besides Cop 1, two additional synthetic basic polypeptides were tested, AAspLT and AGL, the composi- tion of which is listed in Table 1, They have previously been found effective in suppression of EAE, though less so than Cop 1 [3].

DH albino guinea pigs sensitized with BE, Cop 1 or lysozyme (an unrelated basic protein) were skin-tested 10 days later with various antigens as shown in Table 2. The results demon- strate clearly that two batches of Cop 1 which were prepared independently, and which possess identical activity in sup- pression of EAE [ 11, gave virtually identical skin reactions in the delayed hypersensitivity tests. AAspLT and AGL cross- reacted to a large extent with Cop 1, and all three copolymers were found to elicit cross-reactions in animals sensitized with BE. In all these experiments, lysozyme, which served as a con- trol basic antigen gave no cross-reaction with any of the above antigens. Thus lysozyme did not elicit delayed hypersensitivity reaction in guinea pigs sensitized with either Cop 1 or BE, and none of the polymers gave any reaction in animals sensi- tized with lysozyme.

Positive cross-reactivity between Cop 1 and BE was also de- monstrated in guinea pigs of the inbred strains 2 and 13 as presented in Table 3. No significant differences in the skin reactions were noted in the various strains of guinea pigs,

282 C. Webb, D. Teitelbaum, R. Arnon and M. Sefa Eur. J. Immunol. 1973.3: 279-286

Table 2. Cross-reaction between BE and synthetic polypeptides in the delayed hypersensitivity reaction

Skin test (mean diameter in mm)

Sensitization Cop 1, batch I Cop 1, batch 11 BE AAspLT

Cop 1, batch I (1 00 /lg) 12.7 ( l O / l O ) a ) 12.6 (10/10) 8.7 (7/10) 10.6 ( l O / l O )

7.4 (7/10) 7.2 (7/10) 10.8 ( l O / l O ) 7.6 (6/10)

a) The numbers in brackets represent the incidence of reacting animals,

Table 3. Cross-reaction between BE and Cop 1 in the delayed hypersensitivity reaction in the inbred strains 2 and 13

Sensitization with BE (10 f ig)

Skin test (mean diameter in mm)

BE cop 1

Strain 2 9.7 ( lO/ lO)a ) 6.3 (7/10)

Strain 13 10.2 (10/10) 7.0 (6/10)

a) The numbers in brackets represent the incidence of reacting animals.

4.2. Lymphocyte stimulation in guinea pigs

Studies were carried out using lymph node cells (LNC), main- ly from the inbred strains (strains 2 and 13) of guinea pigs. In this manner it was possible t o pool cells from several ani- mals, thereby permitting the simultaneous testing of a large number of paranieters.Using LNC from guinea pigs immu- nized with either BE or Cop 1, the extent of incorporation of labeled thymidine was followed upon exposure to various antigens. The antigens tested included those copolymers which have been found to suppress EAE, namely, the two batches of Cop 1, AAspLT and AGL, and two additional copolymers, designated Cop 2 and Cop 3, which have been found to lack such activity [ 11.

BE-sensitized lymph node cells

Cross stimulation (%)

Cop 1, batch I sensitized lymph node cells

Cross stimulation (70)

Antigen BE (pglculture)

10 5.7 100 7.6 500 5.9

1000 3.8

Results of lymphocyte stimulation experiments in strain 13 guinea pigs are summarized in Table 4. Homologous stimula- tion was recorded in both the BE and the Cop 1 systems. LNC from animals immunized with Cop 1 showed a marked degree of cross-stimulation by the two additional copolymers which have been found active in suppression of EAE, whereas no cross-stimulation was obtained by those copolymers which lack suppressive capacity, o r by an unrelated basic antigen such as lysozyme, which has also been found inactive in sup- pression [ 11. In parallel t o the findings in the delayed hyper- sensitivity reaction test, the two batches of Cop 1 are virtually indistinguishable in their stimulating'activity. However, as emerges from Table 4, in contrast t o the findings in the in vivo skin tests, no direct cross-stimulation was detected be- tween BE and Cop 1 in the in vitro test, when either material served as the sensitizing agent. LNC sensitized towards BE showed no cross-stimulation with any of the copolymers test- ed.

The cross-reactivity between BE and Cop 1 could be demon- strated only by indirect measurements of inhibition of the spe- cific stimulation of the homologous system with the hetero- logous antigen. These results, summarized in Table 5, clearly demonstrate that an appreciable extent of inhibition of the specific stimulation was brought about by addition of the heterologous antigen prior t o incubation with the sensitizing antigen. Maximum inhibition of 33-35 %was observed both in the BE and in the Cop 1 systems. Non-related basic pro- teins such as lysozyme did not cause any inhibition, nor was

Table 4. Lymphocyte stimulation by BE and synthetic copolymers in strain 13 guinea pigs

10 50

100 5 00

cop 1 batch I

0.8 0.9 1.2 1.5

0

0.9 12.2 0.9 14.2 1.2 16.4 0.9 23.1

0

AGL Lysozyme

11.2 (10/10) - (0/10)

7.1 (6/10) - (0/10)

Stimulation indices a) Cop 1 AAspLT AGL batch I1

- - - 1 .o 1.0 1.0 1.2 1.2 0.8 1.5

0 0 0

10.8 10.5 3.8 15.2 10.6 5.5 16.1 10.9 7.8 23.0 - -

98 65 44

- -

Cop 2 cop 3

- - 1.0 1.1 0.8 1.4 0.9 1.3

0 1

- 1.2 - 1.4 1.0 1.5 1.2

0 3

Lysozyme

- 0.8 0.9 1.0

0

1.0 1.1

0

a) Values represent the stimulation indices of incorporation of [ 14C]thymidine by the cells. Percent cross-stimulation was calculated at the 100 gg antigen concentration.

Em. J. Immunol. 1973.3: 279-286 Synthetic copolymers cross-reactive with encephalitogen 283

Table 5 . Inhibition of thymidine incorporation into LNC cultures from strain 13 guinea pigs

inhibitor/ Inhibition (%) stimulator (ratio) BE Cop 1 Lysozyme

BE- sensitized LNC 1 : 1 -

1 0 : 1 2 0 : 1 5 0 : 1

cop 1- sensitized LNC 1 : l

1 0 : 1 2 0 : 1 5 0 : 1

Lysozyme- sensitized LNC 1 : 1

1 0 : 1 20 : 1 5 0 : 1

-

- -

19 32 31 33

0 0 0 0

24 - 34 0 35 33 0

-

- 0 0 0 0

- - -

the specific stimulation in the non-related lysozyme system affected by BE or Cop 1 at the concentrations used.

The cross-reaction between BE and Cop 1 was also tested in the inbred strain 2 guinea pigs. In this system, the cross-reac- tions between the various synthetic copolymers were con- firmed, as shown in Table 6. As was the case in strain 13 LNC cultures, the two batches of Cop 1 were equivalent, and cross- stimulation was observed only with those copolymers active in suppression of EAE, namely, AAspLT and AGL. No signi- ficant cross-stimulation was obtained with inactive basic co- polymers nor with the acidic copolymer ACT which has n o suppressive activity either [ 11, In contradistinction to our findings in strain 13 cultures, where the cross-reaction be- tween BE and Cop 1 was demonstrable only indirectly, in strain 2 this cross-reaction was evident a t the level of direct cross-stimulation. Such cross-stimulation, t o an extent of over 30 %, was obtained with either BE-sensitized LNC or Cop 1-sensitized LNC when tested with the heterologous antigen. Moreover, the two additional copolymers AAspLT and AGL, also gave significant cross-stimulation with BE- sensitized LNC.

Since strain 2 and strain 13 cultures differed in their mani- festation of the cross-reaction between BE and Cop 1, it was of interest to investigate this point in cultures of LNC from BE-sensitized DH albino guinea pigs. As shown in Table 7, DH cultures revealed the same phenomenon as strain 13 cul- tures, namely, no direct cross-stimulation between BE and Cop 1 was detected but inhibition of specific stimulation indicated the cross-reaction between the two antigens.

Table 7. Lymphocyte stimulation in BE-sensitized DH guinea pigs

Stimulation indicesa) Inhibitor/ Inhibition ( R ) Antigen BE Cop 1 Lysozyme stimulator (P8) (ratio) Cop 1 Lysozyme

1 : l 9 4 17 1

100 7.1 1.6 0.8 4 : 1 36 0

10 6 .8 - - 50 7.6 1.2 0.8 2 : 1

200 7.2 1.8 1.6

a) The values represent the stimulation indices of incorporation of [14C]thymidine by the cells.

4.3. Lymphocyte stimulation in rabbits

As previously reported, suppression of EAE by Cop 1 can be obtained in rabbits as well as in guinea pigs [ 2 , 31. Therefore, it was of interest to test whether the cross-reaction of these antigens o n the cellular level could also be demonstrated in rabbits.

Lymphocyte cultures were prepared from both spleen and draining lymph nodes of rabbits immunized with either BE or Cop 1. In the case of BE-sensitized animals it was found that spleen cell cultures gave a poor response, whereas LNC cultures responded well in the majority of animals tested. The results summarized in Table 8 represent the average values obtained in experiments with LNC of three individual rabbits. Cross-reaction between BE and all three copolymers active in suppression of EAE was demonstrated by direct cross-stimulation, whereas copolymers devoid of suppressive activity, o r control antigens such as t h e non-related protein, lysozyme, gave n o cross-stimulation.

Table 6 . In viho immunological cross-reaction between BE and synthetic copolymers in strain 2 guinea pigs

BE-sensitized lymph node. cells

Cross stunulation (%)

COP 1, batch I- sensitized lymph node cells

Antigen BE (pg/culture)

50 4.9 100 4.8 200 4.1 500 3.4

50 4.5 100 4.1 500 3.7

1000 -

Cross stimulation (%) 31

cop 1 batch I

1.6 2.2 2.5 2.7

31.5

-

11.0 24.0 26.7 -

Stimulation indicesa) Cop 1 AAspLT AGL batch I1

1.3 2.0 1.3 2.1 2.6 2.5 2.5 3.5 3.3 3.1 3.3 3.0

29 4 2 39.5

- - -

11.7 8.6 7.4 24.5 18.1 6.1 24.3 - -

107 76 64

Cop 2 Cop 3 AGT Lysozyme

0.9 - 1.2 - 1 . 1 1.2 0.8 - 1.1 1.2 0.9 -. 1.2 1.6

5

- -

2.5 5 -

- - - - 1.6 1.9 1.5 -

2.1 2.1 2.0 - - - - -

- 6 9 5

a) The values represent the stimulation indices of incorporation of [ '%]thymidine by the cells. Percent cross-stimulation was calculated at the 100 pg cntigen concentration.

284 C. Webb, D. Teitelbaum, R. Arnon and M. Sela Em. J. Immunol. 1973.3: 279-286

Table 8. Lymph node lymphocyte stimulation in BE-sensitized rabbits

Antigen Stimulation indicesa) bg) BE Cop 1 AAspLT GAL Cop 2 ACT Lysozyme

50 5.8 1.7 2.0 1.5 0.9 0.9 1.0 100 7.2 2.2 2.6 1.7 1.1 1.2 1.1 200 8.8 2.9 2.6 3.1 1.0 1.0 0.8 500 7.0 3.6 4.6 2.2 1.2 1.0 1.2

Cross stimulation 24 21 27 0 0 0

(%I

a) The values represent the stimulation indices of incorporation of ['4C]thymidine by the cells. Only values above 2.0 were considered as resulting from specific stimulation. Percent cross stimulation was calculated at the 200 rg antigen concentration.

In the case of Cop 1-sensitized rabbits, both spleen cell cultures and LNC cultures responded well t o specific stimulation (Table 9). The response in spleen cell cultures was usually both higher and more specific then the response of LNC. The complete cross-reaction between the two batches of Cop 1 and the partial cross-reaction between Cop 1 and AAspLT and AGL was corroborated in these experiments as presented in Table 9. It is interesting t o note that direct cross-stimulation with BE was obtained only in LNC cultures and not in spleen cell cultures, despite the higher specific stimulation of the latter. However, as in the previous cases in which direct cross-stimulation was not observed, namely, in strain 13 and DH guinea pig LNC cultures, the cross-reaction could be demonstrated by means of specific inhibition of the homologous stimulation. In the case of rabbit spleen cells the specific inhibition reached the 50 % level a t inhibitor t o stimulator ratios of 1O:l to 20:l.

4.4. Cross-reaction on the humoral antibody level

In the experiments described above a definite cross-reaction between BE and suppressive synthetic copolymers was established on the cellular level. I t was, therefore, relevant to examine whether any cross-reaction on the humoral level could be detected as well. This problem was approached by

Table 9. Lymphocyte stimulation in Cop 1, batch I-sensitized rabbits

applying the precipitin reaction; Farr test and passive cutaneous anaphylaxis methods, using antisera raised in rabbits and guinea pigs.

In the precipitin reaction with anti-Cop 1 serum raised in rabbits (Fig. l ) , cross-reactivity was obtained with those synthetic copolymers possessing suppressive activity, whereas no precipitation was detected with the acidic copolymer AGT or with BE. The results of the precipitin with anti-BE serum are given in Fig. 2. High titer antisera were obtained in rabbits by using a complex of phosvitin-BE for immuni- zation. As seen in the Fig. 2 no cross-reaction could be detected with Cop 1. In the two systems cross-reaction between BE and Cop 1 could not be demonstrated by inhibi- tion of the homologous precipitin reaction with the corres- ponding heterologous antigen. No cross-reaction between BE and Cop 1 could be detected with these antisera even when the more sensitive Farr and PCA techniques were used.

A slight degree of cross-reactivity on the humoral antibody level was detected using guinea pig antisera in the PCA tech-

3.2 I

In , n 1 I n 0 200 400 600 800 1000

m Antigen added Ipg/ml serum1

Figure 1. Precipitin reactions of rabbit anti-Cop 1, batch I. Absorbance at 280 nm of solutions in 0.1 N NaOH of the precipitates obtained by addition of Cop 1, batch I (A-A), Cop 1, batch I1 (A-A),

AAspLT (@-oh AGL (0--0), ACT (m), and BE (0) to anti- Cop 1, batch I serum.

Spleen cells

Stimulation indicesa) Antigen Cop 1 cop 1 BE AAspLT (/a) batch I batch XI

10 32.4 30.1 1.8 21.0 50 34.4 33.3 1.2 22.3

100 34.6 34.8 1.7 25.0 200 29.6 27.9 1.7 28.4

Cros4-stimulation (%) 100 2 71

Lymph node cells 10 13.7 11.6 1.4 8.6 50 14.5 13.5 3.2 9.8

100 17.0 16.2 4.0 10.5 200 16.3 15.7 5.1 12.7

Cross-stimulation (%) 95 19 66

AGL

13.4 19.9 21.1 26.6

60

6.0 8.1 9.4

12.2

53

ACT

1.4 1.4 1.6 1.8

2

u.11 0.8 1.5 1.7

3

Lysozyme

1.1 1.4 1.3 1.2

1

1.2 1.2 1.6 2.0

4

a) The values represent the stimulation indices of incorporation of ['4C]thymidine by the cells. Only values above 2.0 were con- sidered as resulting from specific stimulation. Percent cross stimulation was calculated at the 100 pg antigen concentration.

Ew. J. Immunol. 1973.3: 279-286 Synthetic copolymers cross-reactive with encephalitogen 285

2.4 c

0.4 0 0 l 100 200 300 400 m Figure 2. Precipitin reactions of rabbit anti-BE. Absorbance at 280 nm of solutions in 0.1 N NaOH of the precipitates obtained by addition of BE (A-A), phosvitin (a-a) and Cop 1 (0-0) to anti-BE serum.

Antigen added l p g /ml serum I

nique. The results given in Table 10 show that guinea pig anti-Cop 1 antisera cross-react to a slight extent with BE but not vice versa.

Table 10. Cross-reaction of guinea pig antisera in passive cutaneous anaphylaxis

Antiserum used for sensitization Antigen used for challenge cop 1 BE

Anti-Cop 1 1 : 10 1 : 50 1 : 100 1 : 200 1 : 500 1 : 1000 1 : 5000 1 : 10000 1 : 50000 1 : 100000 1 : 200000 1 : 500000 1 : 2 1 : 100 1 : 500 1 : 1000 1 : 5000 1 : 10000 1 : 50000 1 : 100000 1 : 200000 1 : 500000

Normal serum 1 : 2

Anti-BE

30a) 28 25 23 22 20 19 16 13 10

19 17 14 13 -

30 30 25 24 22 18 12 9 -

a) The numbers represent the diameter of the skin reaction in mm.

5. Discussion

The present study has conclusively established the existence 7f immunologic cross-reactivity between the naturally occur- ring basic encephalitogen of myelin and synthetic polypep- tides which have been shown to exert a specific suppressive effect on EAE [ 1-31. The cross-reaction has been demonstrat- ed both.on the cellular and the humoral level, though it is far more pronounced in the former case. On the cellular level we have detected cross-r>activity both in guinea pigs, in vivo by the delayed hypersensitivity reaction (Tables 2 and 3 and

in vitro using LNC cultures (Tables 5-7), and in rabbits us- ing in vitro cultures of spleen and lymph node cells (Tables 8 and 9). On the humoral level, cross-reactivity was obtained only in the case of antisera raised in guinea pigs against the most active synthetic copolymer, Cop 1, using the sensitive passive cutaneous anaphylaxis assay (Table 10).

The incorporation of radioactive thymidine used in this study provides a quantitative and sensitive method for measuring lymphocyte transformation, resulting from specific antigenic stimulation, which is considered to be a phenomenon related to delayed hypersensitivity [ 1 1, 121. According t o the data presented here, lymphocytes sensitized to either BE or Cop 1 :an be detected in the lymph nodes of guinea pigs and in lymph nodes and spleen of rabbits. It has been reported that BE-sensitized cells can be demonstrated in homologous reac- tion by means of thymidine incorporation in spleens of guinea pigs [21] and in peripheral blood lymphocytes of rabbits [22]. However, we have found that both in guinea pigs and in rabbit: BE-sensitized cells appear preferentially in the draining lymph nodes, rather than in the spleen. The specific stimulation re- ported for peripheral blood lymphocytes in rabbits was 2-fold at most [ 2 2 ] , whereas we have obtained approximately 9-fold stimulation in LNC (Table 8). In the case of Cop 1-sensitized rabbits, though the response obtained with spleen cells was both higher and more specific than the response obtained in LNC, direct cross-stimulation between BE and Cop 1 was ob- served only in LNC (Table 9), while in spleen cells cross-re- activity was demonstrated only by means of specific inhibi- tion of homologous stimulation. In other words, the popula- tion of lymphocytes in spleen and in lymph nodes appears to differ in the sensitivity to the cross-reacting antigen. Such a phenomenon of compartmentalization of antigen-reactive lymphocytes has been reported by Schlossman et al. [23] and others [24, 251.

It is of interest to note that the cross-reaction between BE and Cop 1 manifested itself differently in LNC cultures of inbred strain 2 and strain 13 guinea pigs. In the case of strain 2 the cross-reaction was demonstrated by means of direct cross-stimulation (Table 6), whereas in the case of strain 13 the cross-reaction was detectable only by means of inhibition of specific stimulation (Table 5). These animals differ also in their susceptibility to EAE, strain 2 animals being resistant while strain 13 animals are susceptible [26]. The variance in susceptibility t o EAE is but one of the known genetic differences in the immune response of the inbred strains of guinea pigs [27]. Whether the differences in the immune response to BE in these two strains is linked to any of the identified specific immune response genes is not yet known.

We have noted significant differences in the level of the hu- moral antibody response in these two strains, whereas the level of the delayed hypersensitivity response is very similar [28]. Such differences might be interpreted as possibly involving response to different determinants of the BE r Aecule. The different manifestations of cross-reactivity betxeen BE and Cop 1 in the .two strains may, therefore, reflect t o some de- gree the genetic differences in the response to BE itself. Never- theless, the extent of in vitro cross-reaction found between BE and Cop 1 is approximately the same (ca. 33 %) in ex- periments with either strain. In a limited number of experi- ments carried out with random bred DH albino guinea pigs the typical in vitro response was found to resemble that of the strain 13 animals, and in fact the random bred animals are highly susceptible to EAE. In LNC cultures of strain 13 or DH albino guinea pigs, the cross-reaction in the cell-mediated

286 C. Webb, D. Teitelbaum, R. Arnon and M. Sela Eur. J. Immunol. 1973.3: 279-286

response was manifested not by direct cross-stimulation of lymphocytes but by specific inhibition of the homologous stimulation (Table 7). Similar phenomena were previously described in other systems [ l o , 161. A slight degree of cross-reactivity on the humoral antibody level was found using antisera raised in guinea pigs by the sensitive PCA technique (Table 10). Only anti-Cop 1 antisera gave a positive cross-reaction with BE, while the reverse re- action was not found. In view of the fact that the antibody efficiency, as measured by PCA, is similar in both systems, this cannot be due t o differences in the quantity or avidity of the antibodies to Cop 1 and t o BE, respectively. On the other hand, the experimental finding might be explained on the basis of the different nature of the two antigens. Cop 1 is a random linear copolymer of four amino acids (alanine, glutamic acid, lysine and tyrosine) and it may be possible t o postulate the occurrence of repeating sequences in such a molecule, whereas BE is a naturally occurring protein con- taining 17 different kinds of amino acids [ 131. Therefore, it is likely that common antigenic determinants might appear several times in one Cop 1 molecule, and antisera t o Cop 1 have a relatively greater chance t o contain antibodies directed towards such determinants.

Antisera raised in rabbits failed t o show any positive cross- reaction between BE and Cop 1 , using several sensitive assays. However, despite the fact that rabbit antisera gave high titers of specific antibody in the precipitin reaction, the PCA titer of these same rabbit antisera was far below that of the guinea pig antisera which gave positive cross-reactivity. The detec- tion of weak cross-reaction on the humoral level in a particular species and not in another species is not an isolated pheno- menon [ 101.

The cross-reaction between BE and synthetic copolymers in both in vivo and in vitro tests involves all those copolymers which have shown suppressive activity towards EAE [ 1-31, whereas ineffective copolymers or unrelated proteins d o not show such cross-reactivity (Tables 2, 6 and 8). This raises the interesting possibility that the cross-reactivity of these antigens with BE might be correlated with their suppressive activity. Such cross-reaction between BE and the copolymers which are capable of suppressing EAE may serve as a basis for ex- planation of the suppressive activity in terms of an immuno- logical mechanism. The exact pathogenesis of EAE has not yet been elucidated, although its autoimmune nature has been well established, and it is generally believed that cellular mediated hypersensitivity is the primary immunological mech- anism involved [4, 5, 211. Nevertheless, there are certain lines of evidence that suggest the possibility that a local release of antibody within the target organ may play a role in the patho- genesis of EAE [ 5 , 291. It is feasible that the action of Cop 1 might result in tolerization of BE-sensitized lymphoid cells o r possibly deviation of the immune response. It has been re- ported by several groups that in animals pretreated with BE to avert disease, the development of BE-sensitive cells appears t o be substantially inhibited [23, 30, 311. However, animals in which the appearance of disease was suppressed by treat- ment with BE after the EAE-inducing injection yielded sen- sitized cells in culture [3 I] . It would be interesting to try the in vitro test used in this study with cells from animals inwhich EAE has been suppressed by means of injections of Cop 1 .

An alternative explanation of the suppressive phenomenon may lie in the basicity of the copolymers either as regards net electrical charge or distribution of the charge on the molecule. Clarification of this point might be facilitated by investiga- tion of the properties of a copolymer identical in composi- tion t o that of Cop 1 but of all D configuration, where no

immunological cross-reaction is expected. Experiments are in progress t o determine the possible suppressive activity of such a copolymer. While this alternative hypothesis has not yet been eliminated, it seems that the immunological cross-reactivity of BE with the suppressive copolymers and its relationship t o the suppressive effect on EAE may con- tribute t o the further understanding of the mechanism of EAE as such.

The skillful technical assistance of Mrs. Bruria Klein is gratefully acknowledged.

Received October 20, 1972.

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