.
`(cid:14)
`Journal of Neuroimmunology 91 1998 135–146
`
`Bystander suppression of experimental autoimmune encephalomyelitis
`by T cell lines and clones of the Th2 type induced by copolymer 1
`Rina Aharoni, Dvora Teitelbaum, Michael Sela, Ruth Arnon )
`Department of Immunology, The Weizmann Institute of Science, Reho˝ot 76100, Israel
`
`Received 11 February 1998; revised 4 June 1998; accepted 23 June 1998
`
`Abstract
`The synthetic amino acid copolymer, copolymer 1 Cop 1 induces T suppressor Ts linesrclones, which are confined to the Th2
`.
`(cid:14)
`.
`(cid:14)
`.
`(cid:14)
`pathway, cross react with myelin basic protein MBP , but not with other myelin antigens on the level of Th2 cytokine secretion.
`(cid:14)
`.
`Nevertheless, Cop 1 Ts cells inhibited the IL-2 response of a proteolipid protein PLP specific line. Furthermore, Cop 1 Ts cells
`ameliorated EAE induced by two unrelated encephalitogenic epitopes of PLP: p139–151 and p178–191, that produced different forms of
`disease. This bystander suppression demonstrated by the Cop 1 Ts cells may explain the therapeutic effect of Cop 1 in EAE and MS.
`q 1998 Elsevier Science B.V. All rights reserved.
`
`.
`(cid:14)
`.
`(cid:14)
`w
`Keywords: Experimental autoimmune encephalomyelitis EAE ; Multiple sclerosis MS ; Copaxone ; Bystander suppression; Th2 cytokines; Regulatory
`T cells
`
`1. Introduction
`
`The dichotomy of the immune system into counteract-
`ing Th1 and Th2 subsets,
`that secrete distinct sets of
`cytokines, and as a result, perform distinct effector func-
`tions, has been established in both mouse and human
`(cid:14)Mosmann and Coffmann, 1989; Abbas et al., 1996; Mos-
`.
`mann and Sad, 1996 . Although this concept has been
`(cid:14)
`recently blamed as oversimplified Allen and Maizels,
`.
`1997 , it is accepted that Th1 cells which produce IL-2,
`IFN-g, and TNF, mediate pathological processes in inflam-
`matory autoimmune diseases such as multiple sclerosis
`
`(cid:14) .MS and its animal model experimental autoimmune en-
`(cid:14)
`. (cid:14)
`cephalomyelitis EAE Merrill et al., 1992; Miller and
`Karpus, 1994; Liblau et al., 1995; Nicholson and Kuchroo,
`.
`1996; Adorini and Sinigaglia, 1997 . In contrast, Th2 cells
`produce IL-4, IL-5, IL-6 and IL-10, and antagonize Th1
`(cid:14)
`cell mediated immunity Kennedy et al., 1992; Khoury et
`al., 1992; Van der Veen and Stohlman, 1993; Chen et al.,
`1994; Miller and Karpus, 1994; Liblau et al., 1995; Adorini
`.
`and Sinigaglia, 1997 . Current therapeutic approaches at-
`
`)
`
`Corresponding author. Tel.: q972 8 9344017; fax: q972 8 9469712;
`e-mail: liarnon@weizmann.weizmannac.il
`
`tempt therefore, to induce deviation from the pathological
`(cid:14)
`Th1 to the protective Th2 response Chen et al., 1994;
`Brennan and Feldmann, 1996; Nicholson et al., 1997;
`.
`Rocken et al., 1996; Adorini and Sinigaglia, 1997 .
`(cid:14)
`The synthetic random copolymer, Copolymer 1 Cop 1,
`.
`w
`Copaxone , glatiramer acetate , composed of L-Ala, L-Glu,
`(cid:14)
`.
`L-Lys and L-Tyr Teitelbaum et al., 1971, 1973 , exerts a
`marked suppressive and protective effect on EAE in vari-
`(cid:14)
`ous animal species including primates Teitelbaum et al.,
`.
`1971, 1973, 1974, 1997; Lando et al., 1979 . Cop 1
`(cid:14)
`.
`ameliorated chronic relapsing EAE Keith et al., 1979 , as
`well as EAE induced by various encephalitogens, e.g.,
`(cid:14)
`. (cid:14)
`myelin basic protein MBP
`Teitelbaum et al., 1973,
`.
`.
`(cid:14)
`(cid:14)
`1974 , proteolipid protein PLP peptides Teitelbaum et
`.
`(cid:14)
`.
`al., 1996 , and myelin oligodendrocyte glycoprotein MOG
`(cid:14)
`.
`peptides Ben-Nun et al., 1996 . Cop 1 was also shown to
`slow the progression of disability and to reduce the relapse
`(cid:14)
`.
`rate in MS patients Johnson et al., 1995, 1998 , and it was
`recently approved as a drug for MS under the trade name
`of Copaxonew. The mechanism of Cop 1 activity in EAE
`and MS involves high affinity promiscuous binding to
`(cid:14)
`.
`various class II major histocompatibility complex MHC
`(cid:14)
`.
`molecules Fridkis-Hareli et al., 1994 . This binding leads
`to both competition with myelin antigens for T cell activa-
`
`0165-5728r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved.
`(cid:14)
`.
`PII: S 0 1 6 5 - 5 7 2 8 9 8 0 0 1 6 6 - 0
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`136
`
`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`
`(cid:14)
`tion, and induction of specific suppressor T cells Teitel-
`.
`baum et al., 1997 .
`We have previously demonstrated that the unrespon-
`siveness to EAE induced by Cop 1 is regulated by T
`(cid:14)
`.
`suppressor Ts cells, since it could be adoptively trans-
`(cid:14)
`.
`ferred to normal recipients Lando et al., 1979 . Cop 1
`specific Ts cell lines and Ts hybridomas were established
`from spleens of mice that had been rendered unresponsive
`(cid:14)
`.
`to EAE by Cop 1 Aharoni et al., 1993 . These Cop 1
`induced T cells, or their supernatants, inhibited in vitro the
`response of an encephalitogenic line to MBP. Furthermore,
`they prevented the development of EAE in vivo, indicating
`that they are regulatory suppressor cells. The Cop 1 spe-
`cific T cell linesrclones expressed the CD4q phenotype.
`Recently, we have shown that Cop 1 induces T cells which
`(cid:14)
`.
`are confined to the Th2 pathway Aharoni et al., 1997 .
`Thus, the T cell lines and clones generated from Cop 1
`immunized mice and selected for Cop 1 secreted high
`amounts of IL-4, IL-6 and IL-10 in response to Cop 1, but
`not IL-2 or IFN-g. The bias towards the Th2 phenotype
`was found in Cop 1 linesrclones originating from spleens
`of mice that had been rendered unresponsive to EAE by
`injection of Cop 1 in ICFA 15–35 days earlier, a regimen
`previously shown to induce antigen specific suppressor
`(cid:14)
`.
`cells Lando et al., 1979; Aharoni et al., 1993, 1997 .
`Furthermore, even cells originating from lymph nodes of
`mice that had been immunized with Cop 1 in enriched
`CFA 10 days earlier, a procedure usually used to obtain
`(cid:14)
`.
`effector T cell lines Ben-Nun and Lando, 1983 , exhibited
`(cid:14)
`.
`this tendency to Th2 diversion Aharoni et al., 1997 .
`The Cop 1 induced T cells had never been exposed to
`the autoantigen MBP. Nevertheless,
`they responded to
`MBP by secretion of Th2 immunosuppressive cytokines
`(cid:14)
`.
`Aharoni et al., 1997 . Thus, cross reactivity between Cop
`1 and MBP on the level of Th2 cytokine secretion was
`demonstrated. We have previously shown that the cross
`reactivity of Cop 1 with the natural autoantigen MBP on
`(cid:14)
`.
`the level of both B Teitelbaum et al., 1991 , and T cell
`(cid:14)
`.
`response Webb et al., 1973 is correlated with Cop 1
`(cid:14)
`.
`suppressive activity in vivo Webb et al., 1976 . Indeed the
`Cop 1rMBP specific T cell linesrclones protected mice
`against the development of EAE induced by whole mouse
`(cid:14)
`. (cid:14)
`.
`spinal cord homogenate MSCH Aharoni et al., 1997 .
`However, MSCH contains in addition to MBP other en-
`cephalitogenic antigens that were implicated in disease
`(cid:14)
`induction Lees et al., 1991; Mendel et al., 1995; Van
`.
`Noort et al., 1995 . These findings suggested that Cop 1
`specific cells can suppress the encephalitogenic processes
`induced by other antigens and not only MBP. The phe-
`nomenon of T cells specific to one antigen which suppress
`the immunological response induced by another encephali-
`togen, has been previously described and termed bystander
`(cid:14)
`.
`suppression Chen et al., 1994; Al-Sabbagh et al., 1994 .
`In the case of EAE and MS bystander suppression must be
`due to propinquity of the antigens within the myelin
`sheath. Bystander suppression is especially important in
`
`the treatment of both MS and EAE because of the anti-
`genrepitope spreading which has been demonstrated for
`(cid:14)
`.
`these diseases McRae et al., 1995 .
`In the present study we investigated the reactivity of
`Cop 1 induced T cells with several myelin antigens. The
`ability of the Cop 1 specific Ts linesrclones to regulate
`both in vitro and in vivo, processes which are mediated by
`myelin antigens other than MBP was tested as well. In the
`following we demonstrate that even though the Cop 1
`specific cells were cross reactive only with MBP and not
`with PLP or with the other myelin antigens, they still
`inhibited IL-2 response of a PLP specific line, and pre-
`vented disease induced by PLP peptides, indicating that
`Cop 1 induced T cells that mediate bystander suppression.
`
`2. Materials and methods
`
`2.1. Mice
`SJLrJ=BALBrc F1 mice were purchased from Jack-
`.
`(cid:14)
`(cid:14)
`.
`son Laboratories Bar Harbor, ME . Female mice, 7–12
`weeks old, were used in all experiments.
`
`2.2. Antigens
`
`.
`(cid:14)
`w
`Copolymer 1 Cop 1, Copaxone , glatiramer acetate is
`a synthetic random basic polymer, prepared by polymeriza-
`tion of the N-carboxyanhydrides of L-alanine, g-benzyl-L-
`glutamate, «, N-trifluoroacetyl L-lysine, and L-tyrosine
`(cid:14)
`.
`Teitelbaum et al., 1971 followed by removal of blocking
`groups. Two Cop 1 batches obtained from Teva Pharma-
`(cid:14)
`.
`ceutical
`Industries
`Petach Tikva,
`Israel were used
`the study. Batches 02095 and 55495, with
`throughout
`average molecular weights of 6000 kDa and 5800 kDa,
`(cid:14)
`.
`respectively. Myelin basic protein MBP was isolated
`from spinal cords of mice or rats, as previously described
`(cid:14)
`.
`Hirshfeld et al., 1970 . Mouse spinal cord homogenate
`(cid:14)
`.
`MSCH was prepared by homogenizing four parts of
`mouse spinal cord and one part of saline. The homogenate
`was strained through a sieve and lyophilized. Two PLP
`(cid:14)
`.
`peptides p139–151 HSLGKWLGHPDKF and p178–191
`(cid:14)
`.
`NTWTTCQSIAFPSK , were synthesized by Merrifield
`(cid:14)
`.
`solid-phase method Merrifield, 1965 , using the peptide
`synthesizer, model 430A of Applied Biosystems, and puri-
`(cid:14)
`fied by HPLC. MOG peptide p35–56 MEVG-
`.
`WYRSPFSRVVHLYRNGKD was a gift from A. Ben-
`Nun. a B-crystallin was a gift from J.M. Van Noort.
`(cid:14)
`Lysozyme from egg-white was obtained from Sigma St.
`.
`Louis, MO .
`
`2.3. T cell lines and clones
`
`Cop 1 specific T cell lines were established from spleens
`of mice which had been rendered unresponsive to EAE by
`subcutaneous injection of Cop 1 5–10 mgrmouse , emul-
`(cid:14)
`.
`
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`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`
`137
`
`.
`(cid:14)
`sified in incomplete Freund’s adjuvant ICFA, Difco 15 to
`35 days earlier. A control T cell line was established from
`mice which had been similarly injected with lysozyme
`5–10 mgrmouse in ICFA. MBP specific line was gener-
`(cid:14)
`.
`(cid:14)
`ated from spleens of mice immunized with MBP 200
`mgrmouse in complete Freund’s adjuvant CFA 10 days
`.
`(cid:14)
`.
`earlier. PLP p139–151 specific line was established from
`(cid:14)
`spleens of mice immunized with PLP p139–151
`10
`mgrmouse in CFA 10 days earlier. Cells were cultured
`.
`(cid:14)
`and selected in vitro using the immunizing antigen 0.1–1
`mgrplate , in culture medium RPMI, 2 mM glutamine, 1
`.
`(cid:14)
`mM sodium pyruvate, non essential amino acids, 5 =10y5
`M 2-mercaptoethanol, 100 mrml penicillin, 100 mgrml
`.
`streptomycin , supplemented with 1% autologous serum.
`After 4 days, cells were transferred to culture mediumr10%
`FCS supplemented with 10% supernatant of Con A acti-
`(cid:14)
`.
`vated normal spleen cells as T cell growth factor TCGF .
`Every 14–21 days, cells were stimulated by exposures to
`(cid:14)
`Cop 1 or MBP, presented on syngeneic irradiated 3000
`rad spleen cells 50 =10 rplate for 3 days, followed by
`.
`.
`(cid:14)
`6
`propagation in TCGF medium. Cloning of T cell lines was
`performed by limiting dilution at 0.3 cellsrwell in mi-
`crotiter plate in the presence of Cop 1 10 mgrwell and
`(cid:14)
`.
`irradiated syngeneic spleen cells 5 =10 rwell . All the
`.
`(cid:14)
`Cop 1 specific T cell linesrclones expressed CD4q phe-
`notype.
`
`6
`
`2.4. Proliferation assay
`
`Cells of T lines or clones were tested for their specific
`proliferative response 10–21 days after antigenic stimula-
`4.
`(cid:14)
`tion. T Cells 1.5 =10 were cultured with 5 =10 irra-
`5
`
`diated spleen cells and with the indicated antigens in a
`final volume of 0.2 ml in 10% FCS culture medium. At the
`end of 48 h incubation, cultures were pulsed with 1 mCi
`w3
`xH -thymidine and harvested 6–12 h later. Results are
`(cid:14)
`.
`expressed as mean counts per minute cpm thymidine
`incorporation for triplicate cultures. Standard deviation
`were under 20% of the mean cpm.
`
`6
`
`2.5. Cytokine assays
`T cells from lines and clones 1 =10 rml presented
`.
`(cid:14)
`on irradiated spleen cells 5 =10 rml , were incubated
`.
`(cid:14)
`6
`with the indicated antigens in a final volume of 1 ml.
`Supernatants were collected 24 h later and assayed for
`cytokine levels. IL-2, IL-4, IL-5, IL-6, IL-10, and IFN-g
`were measured using a quantitative sandwich ELISA using
`pairs of monoclonal antibodies obtained from Pharmingen
`(cid:14)
`.
`San Diego, CA , according to the manufacturer’s instruc-
`tions. The threshold detection for all cytokines was 20–50
`pgrml. Results are expressed in nanograms as mean con-
`(cid:14)
`centration of duplicate culture supernatants standard devi-
`.
`ations were under 20% , measured in duplicate wells by
`(cid:14)
`.
`ELISA standard deviations under 10% .
`IL-2 secretion was measured also using indicator cells
`by evaluating the ability of culture supernatants to support
`the proliferation of the IL-2 dependent CTLD line. The
`tested supernatants were incubated with the indicator cells
`1 =10 rwell at a 1:1 dilution to a final volume of 0.1
`.
`(cid:14)
`4
`ml for 48 h and then labeled with 1 mCi thymidine for 16
`h. Results are expressed as mean cpm thymidine incorpora-
`tion for triplicate cultures, and the standard deviations
`were less then 20%.
`
`Fig. 1. Proliferation and cytokine secretion profile of Cop 1 specific clone S-1-4. The response to medium, Cop 1 50 mgrml , MBP 100 mgrml and
`.
`(cid:14)
`.
`(cid:14)
`ConA 5 mgrml was tested by proliferation and cytokine secretion. Proliferation was measured by thymidine incorporation for triplicate cultures.
`(cid:14)
`.
`Cytokine concentration was measured by quantitative ELISA in duplicate wells for each one of duplicate culture supernatants. Standard deviations were
`under 20% of the mean. Results represent one of four independent experiments.
`
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`138
`
`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`
`Fig. 2. Proliferation and cytokine secretion profile of lysozyme specific line Lys-1. The response to medium, lysozyme 100 mgrml , Cop 1 50 mgrml ,
`.
`(cid:14)
`.
`(cid:14)
`MBP 100 mgrml and ConA 5 mgrml was measured by proliferation and cytokine secretion as described in Fig. 1. Standard deviations were under
`(cid:14)
`.
`(cid:14)
`.
`20% of the mean. Results represent one of two independent experiments.
`
`2.6. Induction of CR-EAE by PLP peptides
`SJLrJ=BALBrc F1 7–8 weeks old female mice were
`.
`(cid:14)
`injected subcutaneously in the flank with 200 mgrmouse
`PLP peptide 139–151 or 178–191 emulsified in a 1:1 ratio
`in CFA supplemented with 3 mgrml micobacterium tuber-
`(cid:14)
`.
`culosis H37Ra. Pertussis toxin 0.25 ml, 250 ng, Sigma
`was injected i.v. immediately thereafter and 48 h later.
`Mice were examined daily from day 10 post induction for
`signs of EAE, and assessed for clinical severity from 0 to 5
`
`as follows: 0: healthy, 1: flaccid tail, 2: hind limbs para-
`lyzed, 3: hind and fore limbs paralyzed, 4: total paralysis,
`5: moribund.
`
`2.7. Inhibition of CR-EAE by Ts lines and clones
`T cells of lines and clones 0.5 =10 rml were incu-
`.
`(cid:14)
`bated with their specific antigen 50–100 mgrml and
`(cid:14)
`.
`with irradiated antigen presenting cells 5 =10 rml for 3
`.
`(cid:14)
`6
`days. Then cells were washed and injected intravenously
`
`6
`
`Fig. 3. Proliferation and cytokine secretion profile of MBP specific line. The response to medium, MBP 100 mgrml , Cop 1 50 mgrml , and ConA 5
`(cid:14)
`.
`(cid:14)
`.
`(cid:14)
`mgrml was measured by proliferation and cytokine secretion as described in Fig. 1. Standard deviations were under 20% of the mean. Results represent
`.
`one of two independent experiments.
`
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`
`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`to SJLrJ=BALBrc F1 mice, fol-
`10 =10 rmouse ,
`.
`(cid:14)
`.
`(cid:14)
`6
`lowed by disease induction.
`
`139
`
`2.8. Statistical analyses
`
`linesrclones before EAE
`Mice treated with T cell
`induction were compared to control mice induced with
`disease without any treatment. Disease incidence was ana-
`lyzed by Fisher’s exact test. Maximal score and day of
`onset were analyzed by Wilcoxon Rank Sums
`test
`(cid:14)
`.
`Mann–Whitney test . Combination statistical test was per-
`
`ConA
`
`Table 1
`Specificity of IL-4 secretion by F1-Ts-Cop 1 linesrclones
`Ts-cell
`–
`Cop 1 MBP
`PLP
`PLP
`type
`p139–151
`p178–191
`S-1-4 -0.02
`0.85 -0.02
`-0.02
`1.08
`1.87
`S-22-1 -0.02
`0.43 -0.02
`-0.02
`0.82
`1.85
`-0.02
`0.35 -0.02
`-0.02
`S-3
`1.70
`1.78
`-0.02
`0.31 -0.02
`-0.02
`S-4
`1.85
`1.98
`IL-4 secretion in response to medium, Cop 1 50 mgrml , MBP 100
`(cid:14)
`.
`(cid:14)
`mgrml , PLP p139–151 10 mgrml , PLP p178–191 10 mgrml , and
`.
`(cid:14)
`.
`(cid:14)
`.
`to ConA 5 mgrml was measured by quantitative ELISA.
`(cid:14)
`.
`Results are expressed in ngrml as mean concentration of duplicate
`culture supernatants, measured in duplicate wells.
`Standard deviations were under 20% of the mean.
`
`formed by combining several
`(cid:14)
`.
`same hypothesis Winer, 1971 .
`
`independent
`
`tests on the
`
`3. Results
`
`3.1. Specificity of Cop 1 induced Ts cells
`
`We have previously demonstrated that Cop 1 specific T
`cell lines and clones, generated from spleens of mice that
`
`(cid:14) .
`.
`(cid:14)
`Fig. 4. Specificity of Cop 1 induced S-1-4 clone. IL-4 A , IL-5 B , and
`IL-10 C , secretion in response to medium, Cop 1 50 mgrml , MBP
`.
`(cid:14) .
`(cid:14)
`100 mgrml , the following peptides 10 mgrml : PLP 139–151, PLP
`(cid:14)
`.
`(cid:14)
`.
`178–191 and MOG 35–56, a B-crystallin 100 mgrml , lysozyme 100
`(cid:14)
`.
`(cid:14)
`mgrml , and ConA 5 mgrml was measured by quantitative ELISA.
`.
`(cid:14)
`.
`Results are expressed as mean concentration of duplicate culture super-
`natants, measured in duplicate wells. Standard deviations were under 20%
`of the mean. Results represent one of two independent experiments.
`
`Fig. 5. Inhibition of IL-2 secretion of PLP p139–151 specific clone by
`supernatants of Ts lines and clones. Ts cells 1=10 rml were stimu-
`(cid:14)
`.
`6
`lated with the indicated antigens Cop 1 50 mgrml, MBP 100 mgrml,
`(cid:14)
`PLP peptides 139–151 and 178–191 10 mgrml . After 24 h, 50 ml of
`.
`culture supernatants were transferred to a PLP specific T cell clone
`stimulated with PLP p139–151 1 mgrml . The IL-2 secretion of the PLP
`(cid:14)
`.
`specific clone was measured after 24 h using the CTLD IL-2 dependent
`indicator line, as described in Section 2. Results are expressed as percent
`inhibition from the uninhibited response of the PLP clone to PLP
`p139–151—10,311 cpm. Background response of the PLP clone without
`antigen was 333 cpm. Standard deviations of thymidine incorporation for
`triplicate cultures were less then 20%.
`
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`140
`
`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`
`had been rendered unresponsive to EAE by immunization
`with Cop 1, exhibited a Th2 cytokine profile, secreting
`high amounts of IL-4, IL-6 and IL-10 but not IL-2 or
`(cid:14)
`.
`IFN-g in response to Cop 1 Aharoni et al., 1997 . We
`have now extended the study to include an additional Th2
`signature cytokine—IL-5. The Cop 1 specific linesrclones
`(cid:14)
`.
`represented here by S-1-4, Fig. 1 , secreted high amounts
`of IL-5 in response to Cop 1, similar to the amounts
`secreted in response to the mitogen Con A. Moreover, the
`cross reactivity with MBP demonstrated by the Cop 1
`specific T cells on the level of Th2 cytokines mainly by
`IL-4 secretion, was also found for IL-5. Hence, Cop 1
`specific cells stimulated by MBP secreted high amounts of
`IL-5 approaching those secreted after stimulation with Cop
`1. The cross reactivity with MBP was exhibited only by
`Cop 1 induced linesrclones. A control Th2 line, originated
`from spleens of mice that had been immunized with
`lysozyme and selected in vitro for lysozyme, secreted Th2
`cytokines in response to the immunizing antigen, but did
`(cid:14)
`.
`not cross react at all with either MBP or Cop 1 Fig. 2 .
`
`Another Th2 line specific to MBP, originated from spleens
`of mice immunized with MBP and selected in vitro with
`MBP, exhibited Th2 cytokine profile in response to MBP
`but did not cross react with Cop 1 either by proliferation or
`(cid:14)
`.
`by cytokine secretion Fig. 3 .
`The specificity of S-1-4 clone in response to various
`myelin antigens which were implicated in the pathogenesis
`of MS is depicted in Fig. 4. While the S-1-4 cells re-
`sponded to MBP by Th2 cytokine secretion, they did not
`respond to PLP p139–151, PLP p178–191, MOG p35–55,
`and a B-crystallin either by proliferation or by cytokine
`secretion. Furthermore, the Cop 1 specific cells did not
`recognize lysozyme which similarly to MBP and Cop 1 is
`positively charged. Thus, cross reactivity between Cop 1
`and the autoantigen MBP on the level of Th2 cytokine was
`not found for other myelin antigens. This was demon-
`strated for all the cytokines tested, and presented here for
`(cid:14)
`.
`(cid:14)
`.
`the Th2 cytokines IL-4 Fig. 4A , IL-5 Fig. 4B , and
`(cid:14)
`.
`IL-10 Fig. 4C . Similar results were obtained with the
`other Cop 1 specific T cell
`lines and clones. Table 1
`
`(cid:14) .
`.
`(cid:14)
`Fig. 6. Inhibition of PLP p139–151 induced chronic relapsing EAE by Cop 1 Ts lines and clones in comparison to control. A Cop 1 Ts Clone S-1-4, B
`Cop 1 Ts line S-3, C Cop 1 Ts line S-4, D Cop 1 Ts clone S-22-1, lysozyme line Lys-1, and MBP line. T cells 10=10 rmouse were injected
`(cid:14) .
`(cid:14)
`.
`(cid:14)
`.
`6
`intravenously 3 days after stimulation to SJLrJ=BALBrc F1 mice, followed by subcutaneous injection of PLP p139–151 200 mgrmouse , emulsified
`(cid:14)
`.
`(cid:14)
`.
`1:1 ratio in CFA supplemented with 3 mgrml H37Ra. Pertussis toxin 250 ngrmouse was injected intravenously, immediately after and 48 h later.
`(cid:14)
`.
`Control mice were induced with EAE without any treatment. Mice were examined daily from day 10 post induction for signs of EAE and scored as
`described in Section 2. Results are expressed as mean daily clinical score of five mice in a group. Statistical analysis is shown in Table 2.
`
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`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`presents IL-4 secretion by the different linesrclones in
`response to several antigens. Whereas all the linesrclones
`secreted IL-4 in response to Cop 1, MBP and Con A, none
`of them secreted IL-4 in response to peptides 139–151 or
`178–191 of PLP.
`
`141
`
`.
`(cid:14)
`tivity with PLP peptides Fig. 4, Table 1 . Furthermore, the
`ability of the Cop 1 specific cells to suppress the PLP
`clone was also induced by MBP, as significant inhibition
`(cid:14)
`.
`40–48% , similar to the inhibition obtained by super-
`natants from Cop 1 stimulated cells, was demonstrated. No
`such inhibition of the response to PLP p139–151 was
`obtained with supernatant from Ts cells incubated with
`PLP peptides. These results can be taken as a demonstra-
`tion of in vitro bystander suppression of the response to
`PLP by the Cop 1 specific T cell linesrclones, stimulated
`by either Cop 1 or MBP. The cross reactivity with MBP on
`the level of bystander suppression was not obtained by the
`(cid:14)
`.
`lysozyme specific line Lys-1 Fig. 5 , which produced
`inhibitory supernatant only when stimulated with the ho-
`mologous antigen lysozyme and not with Cop 1, MBP or
`PLP p139–151.
`
`3.3. In ˝i˝o bystander suppression induced by Cop 1 Ts
`cells
`The Cop 1 specific linesrclones, which had been previ-
`ously found effective in suppressing EAE induced by
`
`3.2. In ˝itro bystander suppression induced by Cop 1 Ts
`cells
`
`the Cop 1 specific T cell
`the ability of
`To test
`linesrclones to suppress the response of a T cell line of
`another specificity, cells from the various Cop 1 Ts
`linesrclones were stimulated with different antigens, and
`their culture supernatants were transferred to a PLP spe-
`cific T cell clone which presented a Th1 effector pheno-
`type. The IL-2 secretion of this line in response to its
`specific antigen—peptide 139–151 of PLP—was mea-
`(cid:14)
`.
`sured Fig. 5 . When the Cop 1 specific Ts cells were
`stimulated with Cop 1,
`their supernatants significantly
`(cid:14)
`inhibited the response to the PLP peptides, 2–3 times
`more than background inhibition by supernatant from un-
`.
`stimulated cells , even though they showed no cross reac-
`
`Table 2
`Inhibition of PLP p139–151 induced chronic relapsing EAE by Cop 1 Ts linesrclones
`Treatment
`First attack
`First relapse
`
`Second relapse
`
`Mean score
`
`Mean onset
`
`Mean score
`
`Mean onset
`
`Mean score
`
`Mean onset
`
`Ts-cell type
`a
`
`S-1-4
`Control
`bS-3
`Control
`cS-4
`Control
`d
`S-22-1
`Lys-1
`e
`MBP-1
`Control
`
`Incidence
`Incidence
`Incidence
`0r5
`1r5
`2r5
`0
`3r5
`5r5
`5r5
`1.4
`0r5
`2r5
`3r5
`0
`2r5
`4r5
`4r5
`1.2
`0r5
`0r5
`2r5
`0
`5r5
`5r5
`5r5
`3.0
`0r5
`0r5
`1r5
`0
`0r5
`2r5
`3r5
`0
`4r5
`–
`–
`–
`0r5
`2r5
`3r5
`0
`0r20
`3r20
`8r20
`m
`Ts Cop 1
`0.8
`24
`0.3
`62
`0
`–
`10r20
`16r20
`17r20
`Control
`2.0
`12
`1.9
`39
`1.4
`66
`Activated T cells of linesrclones 10 =10 cellsrmouse were injected intravenously to 7–8 weeks old female SJLrJ=BALBrc F1 mice followed by
`.
`(cid:14)
`.
`(cid:14)
`injection of 200 mgrmouse PLP peptide 139–151 emulsified in CFA supplemented with 3.5 mgrml H37Ra.
`Pertussis toxin was injected immediately after and 48 h later.
`Control group was similarly induced with disease without any treatment.
`Mice were examined daily and assessed for clinical severity.
`aPs0.05 by Wilcoxon Rank Sums compared with control.
`bPs0.07 by Wilcoxon Rank Sums compared with control.
`cPs0.02 by Wilcoxon Rank Sums compared with control.
`dPs0.08 by Wilcoxon Rank Sums compared with control.
`.
`(cid:14)
`a – d
`P -0.01 by combining several independent tests on the same hypothesis according to Winer 1971 .
`eDisease phenotype was chronic with no remissions and exacerbations.
`fPs0.004 by Fisher’s exact test compared with control.
`g Ps0.003 by Wilcoxon Rank Sums compared with control.
`hPs0.011 by Wilcoxon Rank Sums compared with control.
`iPs0.0004 by Fisher’s exact test compared with control.
`jP -0.0001 by Wilcoxon Rank Sums compared with control.
`kPs0.0072 by Wilcoxon Rank Sums compared with control.
`lPs0.0004 by Fisher’s exact test compared with control.
`m P -0.004 by Wilcoxon Rank Sums compared with control.
`
`1
`2.4
`0.8
`1.6
`1.2
`2.6
`0.2
`1.4
`2.6
`1.2
`
`27
`11
`14
`12
`41
`13
`14
`10
`12
`14
`
`6
`
`0.4
`2
`0.8
`2.0
`0
`2.6
`0
`1.6
`–
`0.8
`
`71
`45
`54
`49
`–
`29
`–
`38
`–
`33
`
`–
`71
`–
`81
`–
`48
`–
`–
`–
`–
`
`f
`
`g
`
`h
`
`i
`
`j
`
`k
`
`l
`
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`YEDA EXHIBIT NO. 2064
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`

`
`142
`
`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`
`.
`(cid:14)
`whole spinal cord homogenate Aharoni et al., 1997 , were
`tested for their ability to mediate bystander suppression in
`vivo, on EAE induced by PLP encephalitogenic peptides
`with which they showed no cross reactivity in vitro. Acti-
`vated F1-Ts-Cop 1 cells were injected to SJLrJ=
`(cid:14)
`BALBrc F1 mice, followed by disease induction. Acti-
`.
`vated lysozyme and MBP specific cells were also tested
`for their ability to inhibit PLP induced disease. In control
`mice the disease was induced with no T cell
`transfer
`treatment. Two systems of chronic EAE induced by differ-
`ent peptides of PLP, that had been found to be encephalito-
`genic in SJLrJ=BALBrc F1 mice, but produced differ-
`(cid:14)
`.
`(cid:14)
`.
`ent forms of disease Teitelbaum et al., 1996 were used.
`
`3.3.1. EAE induced by PLP p139–151
`The course of chronic EAE induced by PLP p139–151
`was relapsing–remitting with variability in the severity of
`disease between experiments. The Cop 1 specific lines and
`clones were effective in suppressing this chronic relapsing
`(cid:14)
`EAE as compered to untreated controls Fig. 6 and Table
`.
`(cid:14)
`.
`2 . The mean maximal score MMS of disease severity in
`the Cop Ts cell treated mice was always half or less of that
`seen in the control mice, although the differences demon-
`(cid:14)
`.
`strated for two lines S-3 and S-22-1 did not reach
`(cid:14)
`.
`statistical significance by Wilcoxon Rank Sums Table 2 .
`test was per-
`However, when a combination statistical
`formed, by combining several independent tests on the
`(cid:14)
`.
`same hypothesis Winer, 1971 , a statistical significance
`(cid:14)
`.
`was obtained for all the Cop 1 specific lines P -0.01 .
`Moreover, when all the Ts Cop 1 cells-treated mice were
`(cid:14)
`.
`compared to the control mice Table 2 , the suppression
`regarding disease incidence,
`obtained was
`significant
`
`severity, onset of the disease as well as in the number of
`(cid:14)
`.
`exacerbations. Most 85% of the control mice developed
`(cid:14)
`chronic disease with high frequency of relapses 80%
`.
`relapsed at least once, and 50% relapsed at least twice . On
`the other hand, mice which received Cop 1 specific T
`(cid:14)
`cells, had a reduced incidence of both the first attack only
`.
`40% developed clinical signs , and subsequent relapses
`(cid:14)only 15% displayed a single delayed relapse with no
`.
`further attacks . The MMS of each exacerbation was al-
`ways lower in the Cop 1 specific suppressor cell-treated
`mice, than in the relevant controls. This is in contrast to
`(cid:14)
`the lysozyme and MBP specific Th2 lines Fig. 6D, Table
`.2 , which did not show any inhibitory effect on the disease
`and even induced a further aggravation of the clinical
`(cid:14)
`score MMS of 1.6 and 2.6 for the lysozyme and MBP
`.
`lines, respectively in comparison to the control mice
`(cid:14)
`.
`MMS 1.2 and mice treated with the Cop 1 specific clone
`(cid:14)
`.
`S-22-1 MMS 0.2 in the same experiment.
`
`3.3.2. EAE induced by PLP p178–191
`The peptide 178–191 of PLP induced a relapsing pro-
`gressive disease, and all the untreated control mice devel-
`(cid:14)
`.
`oped a severe progressive disease with MMS 4.2 Fig. 7 .
`In contrast, only a proportion of the Cop 1 specific cell-
`treated mice developed disease, with a mild clinical score
`(cid:14)MMS 2 and 2.5 in the S-1-4 and S-22-1 treated lines,
`respectively . The Cop 1 specific linesrclones had only a
`.
`slight effect on the disease incidence and onset, but had a
`significant effect on the disease severity Ps0.005 for
`(cid:14)
`S-1-4 and Ps0.008 for S-22-1 and progression. Thus,
`.
`while all the control mice did not recover and were sick
`(cid:14)
`.
`for the entire duration of the experiment 60 days , the Cop
`
`Fig. 7. Inhibition of PLP p178–191 induced chronic progressive EAE by T cell lines and clones. Activated T Cells 10=10 rmouse were injected
`.
`(cid:14)
`intravenously to SJLrJ=BALBrc F1 mice, followed by subcutaneous injection of PLP p178–191 200 mgrmouse , emulsified 1:1 ratio in CFA
`(cid:14)
`.
`(cid:14)
`.
`supplemented with 3 mgrml H37Ra. Pertussis toxin 250 ngrmouse was injected intravenously, immediately after and 48 h later. Control mice were
`(cid:14)
`.
`induced with EAE without any treatment. Mice were examined daily for signs of EAE and scored as described Section 2. Results are expressed as mean
`daily clinical score of 5–7 mice in a group. Statistical analysis for disease severity by Wilcoxon Rank Sums test Ps0.05 for S-1-4 clone and Ps0.008
`for S-22-1 clone.
`
`6
`
`Page 8 of 12
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`YEDA EXHIBIT NO. 2064
`MYLAN PHARM. v YEDA
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`

`
`R. Aharoni et al.rJournal of Neuroimmunology 91 1998 135–146
`)
`(
`
`143
`
`1 cells-treated mice experienced only an initial episode of
`milder disease, which remitted by day 25 with no further
`exacerbations. The lysozyme specific line did not induce
`any suppressive effect on the disease in comparison to
`untreated control mice, and the MBP specific line even
`(cid:14)
`aggravated the disease, resulting in 100% mortality Fig.
`.7 .
`
`4. Discussion
`
`The cumulative data strongly suggest the protective role
`of Th2 cells in T cell-mediated organ-specific autoimmune
`(cid:14)
`diseases such as EAE and MS Kennedy et al., 1992;
`Khoury et al., 1992; Van der Veen and Stohlman, 1993;
`Chen et al., 1994; Miller and Karpus, 1994; Liblau et al.,
`.
`1995; Adorini and Sinigaglia, 1997 . Experimental results
`indicated flexibility of T cell potential
`to differentiate
`(cid:14)
`towards a certain Th phenotype Abbas et al., 1996; Rocken
`.
`et al., 1996 . In addition it was found that the degree of T
`cell polarization reflects the nature of the antigen to which
`(cid:14)
`.
`the cells have been exposed Abbas et al., 1996 . These
`findings set the basis for antigen-specific therapeutic ap-
`proaches aiming for diversion from the harmful Th1 reac-
`tions,
`towards the protective Th2 responses, which are
`currently in the focus of studies for immuno-intervention
`in organ specific