Suppression of CD4+ Th1 cell-mediated autoimmune disease via immune deviation is an attractive potential therapeutic approach. CD4+ Th2 T cells specific for myelin basic protein, induced by immunization of young adult male SJL mice, suppress or modify the progression of CNS autoimmune disease. This report demonstrates that activation of non-neuroantigen-specific Th2 cells is sufficient to suppress both clinical and histological experimental allergic encephalomyelitis (EAE). Th2 cells were obtained following immunization of male SJL mice with keyhole limpet hemocyanin. Transfer of these cells did not modify EAE, a model of human multiple sclerosis, in the absence of cognate Ag. Disease suppression was obtained following adoptive transfer and subcutaneous immunization. Suppression was not due to the deletion of myelin basic protein-specific T cells, but resulted from the presence of IL-10 as demonstrated by the inhibition of Th2-mediated EAE suppression via passive transfer with either anti-IL-10 or anti-IL-10R mAb. These data demonstrate that peripheral activation of a CD4+ Th2 population specific for an Ag not expressed in the CNS modifies CNS autoimmune disease via IL-10. These data suggest that either peripheral activation or direct administration of IL-10 may be of benefit in treating Th1-mediated autoimmune diseases.

Experimental allergic encephalomyelitis (EAE)3 is a Th1-mediated autoimmune disease of the CNS commonly used as a model for the human autoimmune disease multiple sclerosis. Regulation of self-reactive T cells via Th2 cytokines is an attractive method for ameliorating Th1-mediated autoimmune diseases. However, studies concerning the ability of either Th2 cells or their cytokines to influence CNS autoimmune disease have yielded conflicting and often confusing results. Consistent with increased Th2 cytokines in the CNS during remission (1, 2), a pre-existing Th2-like environment inhibits the active induction of EAE (3, 4). Similarly, adoptive transfer of neuroantigen-specific Th2 cells activated in vivo both suppressed clinical EAE and decreased CNS inflammation (5). By contrast, proteolipid protein (PLP)-specific Th2 cells induced in vitro by expansion in the presence of IL-4 were not only ineffective but induced clinical signs of EAE characterized by an eosinophilic CNS infiltration (6). These data contrast with the suppression of EAE mediated by rIL-4, with the secretion of IL-4 encoded by a plasmid injected directly into the CNS (7, 8) and an increased disease severity in mice deficient in IL-4 secretion (9). Similarly, administration of rIL-10 has yielded conflicting results. Intravenous injection of rIL-10 exacerbated, rather than suppressed, adoptive transfer-induced EAE (10), whereas intraperitoneal, subcutaneous, and intranasal administration partially inhibited actively induced EAE (11, 12, 13). Neither the intracranial injection of plasmids directing IL-10 secretion nor the adoptive transfer of a retrovirally transduced myelin basic protein (MBP)-specific T cell hybridoma expressing high constitutive levels of IL-10 inhibited EAE (8, 14). By contrast, Ag-inducible IL-10 secreted by PLP-specific T cells suppressed EAE (15). Similarly, secretion of IL-10 either from T cells or from APCs inhibited EAE (16, 17). The inability of IL-10 to provide consistent protection may be due in part to inhibition by IL-4, which abrogates IL-10-mediated protection from EAE (12).

The presence of a Th2 environment before Ag encounter in young adult male SJL mice (3) allowed the in vivo induction and adoptive transfer of neuroantigen-specific Th2 cells (5). Male-derived MBP-specific Th2 cells suppressed both the acute and relapse phases of passive EAE, induced by MBP-specific Th1 cells derived from female SJL mice (5). However, it is not clear whether the suppressive effects of neuroantigen-specific Th2 cells require access into the CNS. Partial inhibition of EAE by peripheral injection of Th2 cytokines suggests that cytokine secretion from a peripheral site may be sufficient to reduce an organ-specific Th1-mediated autoimmune disease. This report demonstrates that the adoptive transfer of Th2 cells specific for keyhole limpet hemocyanin (KLH) is not sufficient to suppress Th1-mediated disease in the absence of in vivo-induced activation. Th2-mediated suppression did not eliminate neuroantigen-specific Th1 cells. However, after clinical recovery, both Th1 and Th2 cytokines are secreted in response to neuroantigen. Furthermore, IL-10 appears to be the primary effector of inhibition mediated by in vivo-activated Th2 cells. These data demonstrate the efficacy of inhibiting Th1-mediated autoimmune disease via IL-10 secretion from in vivo-activated Th2 cells.

SJL mice were purchased from The Jackson Laboratory (Bar Harbor, ME), Harlan Sprague-Dawley (Indianapolis, IN), or the National Cancer Institute (Frederick, MD). No differences in responses were noted comparing mice obtained from the different vendors. Males were obtained at 4–5 wk of age and used within 2 wk of receipt. Female donors were obtained at 6 wk of age and used within 2 wk of receipt. Female recipients were obtained at 6 wk of age and used between 7 and 10 wk of age. Animals were housed and maintained in the University of Southern California vivarium.

Bovine MBP (Sigma, St. Louis, MO) was dissolved in PBS at 2 mg/ml and emulsified with an equal amount of IFA (Difco, Detroit, MI) supplemented with 20 mg/ml of heat-killed Mycobacteriumtuberculosis, strain H37Ra (Difco). Females were immunized with a total of 0.4 ml distributed at four sites over the flanks. Males were immunized i.p. with 100 μg of KLH (Calbiochem, La Jolla, CA) dissolved in 0.5 ml of PBS as previously described (3).

Inguinal, axillary, and brachial lymph nodes were removed and single-cell suspensions were prepared at 10 days post-MBP immunization or at 5 days post-KLH immunization, as previously described (3, 5). T cells from females were cultured with 50 μg/ml of MBP at 4 × 106 cells/ml. T cells from males were cultured with 50 μg/ml of KLH at 2 × 106 cells/ml. T cells from both males and females were cultured in RPMI 1640 medium supplemented with 10% prescreened FCS, 2 mM l-glutamine, 25 μg/ml gentamicin, nonessential amino acids, sodium pyruvate, and 5 × 10−5 M 2-ME and harvested after 4 days incubation at 37°C. In some experiments 3–5 × 107 cells were injected i.p. In the majority of the experiments shown, 2.5 × 107 cells were injected i.v. into naive, syngeneic female recipients. No differences were noted after either injection route. For suppression studies, equal quantities of KLH-specific cells were injected in addition to the MBP-specific Th1 cells (3–5 × 107 cells/recipient via the i.p. route or 2.5 × 107 cells/recipient via the i.v. route). Recipients were immunized subcutaneously immediately after adoptive transfer with 100 μg of KLH in IFA supplemented with 5 μg/ml H37Ra (Sigma).

Recipients were monitored daily for clinical EAE scores, graded as follows: 0, no abnormality; 1, loss of tail tone; 2, paralysis of one hind limb; 3, total paralysis of both hind limbs; 4, quadriplegia; and 5, moribund. Symptomatic mice were given ready access to food and water. Cumulative disease scores were determined from day 7 to day 15 post-adoptive transfer. Significance was determined by the Mann-Whitney U nonparametric statistical analysis. Differences were considered significant if p ≤ 0.05.

Single-cell suspensions were prepared from lymph nodes or spleens. Cells were cultured at 8 × 105 cells/ml in RPMI 1640 medium supplemented with 1% SJL serum, 2 mM l-glutamine, 25 μg/ml gentamicin, nonessential amino acids, sodium pyruvate, and 5 × 10−5 M 2-ME. A total of 2 μCi/well [3H]thymidine (ICN Radiochemicals, Irvine, CA) was added for the last 16–24 h of incubation of a 72-h incubation. [3H]Thymidine incorporation was measured by liquid scintillation spectroscopy.

Single-cell suspensions of lymph nodes or spleens were resuspended to 4 × 106 cells/ml with 50 μg/ml of Ag in RPMI 1640 medium supplemented with 10% FCS, 2 mM l-glutamine, 25 μg/ml gentamicin, nonessential amino acids, sodium pyruvate, and 5 × 10−5 M 2-ME. Cells were cultured for 60–72 h at 37°C and the supernatants were tested for secreted cytokine by ELISA as per the manufacturers instructions. Briefly, ELISA plates (Immulon II, Dynatech Laboratories, Chantilly, VA) were coated with rat anti-mouse IL-4 (11B11), anti-mouse IL-10 (JES5-2A5), or anti-mouse IFN-γ (XMG1.2) (PharMingen, San Diego, CA). Biotinylated anti-IL-4 (BVD6-24G), anti-IL-10 (SXC-1), and anti-IFN-γ (XMG-1.2) were purchased from PharMingen. Avidin peroxidase and o-phenylenediamine were obtained from Sigma.

Purified rat anti-mouse IL-10 (JES5-2A5), anti-mouse IL-10R (1B1.2), anti-mouse IL-4 (11B11), and isotype control mAb, anti-β galactosidase (GL113) mAb were prepared from serum-free culture supernatants by ion exchange chromatography and contained less than 3 IU of endotoxin/mg Ab. The mAbs were kindly provided by Robert Coffman (DNAX, Palo Alto, CA). Recipients were injected i.p. with 1 mg of anti-IL-10, anti-IL-4 mAb, or isotype control mAb. Recipients treated with anti-IL-10R received 0.5 mg i.p.

Mice were sacrificed by CO2 inhalation. Brains and spinal cords were removed and fixed by immersion in 75% ethanol and 25% glacial acetic acid and embedded in paraffin. Sections (1 mm) were stained with hematoxylin-eosin or luxol fast blue and read in a blinded fashion.

Female, but not young adult male, SJL mice are susceptible to the active induction of EAE following immunization with either MBP or mouse spinal cord homogenate (18). In contrast to the induction of Th1 cells after immunization of female SJL mice, immunization of male SJL mice results in the preferential induction of Ag-specific Th2 cells (3, 5). MBP-specific Th2 cells derived from male donors inhibit EAE induced via the adoptive transfer of MBP-specific Th1 cells derived from female donors (5). To determine whether Th2 cells reactive to an Ag not expressed in the CNS could alter EAE, T cells derived from young adult male SJL mice immunized with KLH were used as a source of non-neuroantigen-specific Th2 cells (3). T cells derived from female SJL mice immunized with MBP served as the source of Th1 cells (5). Both populations proliferated equally to their specific Ag (Fig. 1) and did not respond to the heterologous Ag (data not shown). Consistent with previous results (3, 5), T cells derived from males secreted high levels of IL-4 and IL-10 but low amounts of IFN-γ after KLH-induced activation (Table I). By contrast, no detectable IL-4, low amounts of IL-10, but high concentrations of IFN-γ were secreted following MBP-induced activation of T cells derived from females (Table I). These data are consistent with the gender-dependent differential induction of Th1 and Th2 cells in SJL mice after immunization with a wide variety of protein Ag (3, 5, 18).

FIGURE 1.

Equivalent Ag-specific proliferation of donor T cells. MBP-specific proliferation response of lymph node cells from MBP-immunized female SJL mice and KLH-specific proliferation of lymph node cells from KLH-immunized male SJL mice. Lymph node cells were prepared 10 days after immunization with MBP and 5 days after immunization with KLH.

FIGURE 1.

Equivalent Ag-specific proliferation of donor T cells. MBP-specific proliferation response of lymph node cells from MBP-immunized female SJL mice and KLH-specific proliferation of lymph node cells from KLH-immunized male SJL mice. Lymph node cells were prepared 10 days after immunization with MBP and 5 days after immunization with KLH.

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Table I.

Ag-induced cytokine secretion

GenderImmunogenIFN-γ (ng/ml)IL-4 (ng/ml)IL-10 (ng/ml)
Malea KLH 1.00 0.40 0.54 
Femaleb MBP 9.40 0.04 0.11 
GenderImmunogenIFN-γ (ng/ml)IL-4 (ng/ml)IL-10 (ng/ml)
Malea KLH 1.00 0.40 0.54 
Femaleb MBP 9.40 0.04 0.11 
a

Males were immunized with 100 μg of KLH i.p. and the lymph node cells were tested 5 days post-immunization.

b

Females were immunized with 400 μg of MBP in CFA and the lymph node cells tested 10 days of post-immunization.

Th2 cells specific for MBP suppress EAE induced by MBP-specific Th1 cells in the absence of immunization with cognate Ag (5). These data suggested that Ag presentation, T cell activation, and subsequent cytokine secretion occurred within the CNS (5). To determine whether Th2 cells specific for an Ag not expressed within the CNS could also provide protection from EAE, MBP-specific Th1 cells derived from female mice were adoptively transferred to naive female recipients with or without an equal number of KLH-specific Th2 cells derived from male mice. Neither the severity nor onset of EAE in mice receiving both KLH-specific Th2 cells and MBP-specific Th1 cells (M+K group) differed from that in controls which received MBP-specific Th1 cells only (M group; Fig. 2,A). Immunization of the MBP Th1 cell recipients with KLH (M+I group) also did not alter the clinical course of EAE (Fig. 2 A). These data suggest that in the absence of cognate Ag, Th2 cells are unable to influence MBP-specific Th1 cell-induced EAE.

FIGURE 2.

In vivo activation suppresses EAE. A, Female SJL mice received 2.5 × 107 MBP-specific Th1 cells i.v. and were either left untreated (M), immunized subcutaneously with 100 μg of KLH subcutaneously (M+I), or received MBP-specific Th1 cells (2.5 × 107) plus 2.5 × 107 KLH-specific Th2 cells i.v. (M+K). B, Female SJL mice received 2 × 107 MBP-specific Th1 cells i.v. and were either left untreated (M), or received MPB-specific T cells plus 2.5 × 107 KLH-specific Th2 cells, then were subcutaneously immunized with 100 μg of KLH (M+K+I). Data present are representative of three experiments, n = 3–4.

FIGURE 2.

In vivo activation suppresses EAE. A, Female SJL mice received 2.5 × 107 MBP-specific Th1 cells i.v. and were either left untreated (M), immunized subcutaneously with 100 μg of KLH subcutaneously (M+I), or received MBP-specific Th1 cells (2.5 × 107) plus 2.5 × 107 KLH-specific Th2 cells i.v. (M+K). B, Female SJL mice received 2 × 107 MBP-specific Th1 cells i.v. and were either left untreated (M), or received MPB-specific T cells plus 2.5 × 107 KLH-specific Th2 cells, then were subcutaneously immunized with 100 μg of KLH (M+K+I). Data present are representative of three experiments, n = 3–4.

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To determine whether Ag deposition at a peripheral site could induce sufficient activation to suppress CNS disease, recipients were injected subcutaneously with KLH (M+K+I group) after adoptive transfer of both cell populations. Recipients of both Th2 and Th1 cells immunized with KLH were protected from EAE compared with mice which received MBP-specific Th1 cells only (Fig. 2,B). To examine the extent of suppression, mice were sacrificed at day 16 post-adoptive transfer for histological analysis following resolution of the acute phase in recipients of MBP-specific T cells only. CNS immunopathology associated with KLH-mediated suppression demonstrated reduced mononuclear cell infiltrates in both the brain and spinal cord compared with recipients of MBP-specific Th1 cells only (Fig. 3). Although demyelination was prominent in the spinal cords of the mice which received MBP-specific Th1 cells only (Fig. 3), no demyelination was observed within the spinal cords of the KLH-protected group. These findings are consistent with the reduced clinical scores (Fig. 2 B) and with the ability of MBP-specific Th2 cells derived from young adult male SJL mice to suppress EAE (5).

FIGURE 3.

Histology of spinal cords associated with KLH-mediated suppression of EAE. Mice were sacrificed at 16 days post-transfer of MBP-specific T cells. The average clinical score of the group which received 2.5 × 107/recipient of MPB-specific T cells was 1.2 ± 0.4. The average clinical scores of the mice which had received MBP-specific T cells and 2.5 × 107/recipient KLH-specific T cells and then were subcutaneously immunized with 100 μg of KLH, was 0.7 ± 0.5. Tissues were fixed in 75% ethanol and 25% glacial acetic acid and embedded in paraffin. Sections were stained with hematoxylin-eosin (A and C) or luxol fast blue for myelin (B). Spinal cords of the recipients of MBP-specific T cells only show prominent plagues of inflammation (A, outlined by arrowheads) associated with demyelination (B, outlined by arrowheads). Spinal cords of the recipients of both MBP- and KLH-specific T cells which were immunized with KLH show a marked decrease in the amount of inflammation (C, compare with A) and no demyelination (data not shown). Magnification, ×120.

FIGURE 3.

Histology of spinal cords associated with KLH-mediated suppression of EAE. Mice were sacrificed at 16 days post-transfer of MBP-specific T cells. The average clinical score of the group which received 2.5 × 107/recipient of MPB-specific T cells was 1.2 ± 0.4. The average clinical scores of the mice which had received MBP-specific T cells and 2.5 × 107/recipient KLH-specific T cells and then were subcutaneously immunized with 100 μg of KLH, was 0.7 ± 0.5. Tissues were fixed in 75% ethanol and 25% glacial acetic acid and embedded in paraffin. Sections were stained with hematoxylin-eosin (A and C) or luxol fast blue for myelin (B). Spinal cords of the recipients of MBP-specific T cells only show prominent plagues of inflammation (A, outlined by arrowheads) associated with demyelination (B, outlined by arrowheads). Spinal cords of the recipients of both MBP- and KLH-specific T cells which were immunized with KLH show a marked decrease in the amount of inflammation (C, compare with A) and no demyelination (data not shown). Magnification, ×120.

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To determine whether KLH-induced suppression was due to elimination or altered activation of MBP-reactive T cells, proliferative T cell responses in recipients of either MBP-specific T cells only or MBP- plus KLH-specific T cells immunized with KLH were compared. No difference in MBP-specific T cell proliferation responses were detected comparing these two groups of recipients (Fig. 4). These data suggest that the ability of KLH-specific Th2 cells to suppress EAE was not due to elimination of MPB-reactive T cells. Although T cells derived from the two groups proliferated equally in response to MBP (Fig. 4), T cells derived from the KLH-immunized recipients, receiving both Th1 and Th2 cells, secreted high levels of IL-4, IL-10, and IFN-γ following MBP stimulation (Table II). By contrast, T cells derived from the recipients of MBP-specific T cells only secreted high levels of IFN-γ and only minimal amounts of IL-4 and IL-10 (Table II). Ag-specific IFN-γ secretion was not diminished in the T cells derived from MBP-only recipients compared with immunized recipients of both Th1 and Th2 cells, which is consistent with equivalent proliferative responses (Fig. 4). By contrast, the MBP-induced secretion of both IL-4 and IL-10 increased 3- to 4-fold in immunized recipients of Th1 and Th2 cells compared with mice that received MBP-specific T cells only. These data suggest that in vivo activation of MBP-specific T cells was influenced by the presence of a Th2 response, and that it contrasts with the absence of IL-4 secretion from T cells derived from mice protected from EAE via the secretion of IL-10 (17).

FIGURE 4.

Th2-mediated suppression of EAE does not eliminate MBP-reactive T cells. Splenocytes from groups of mice which either received MBP-specific T cells (M) or were immunized with KLH after receiving MBP-specific T cells plus KLH-specific T cells (M+K+I) were tested for MBP-specific proliferation.

FIGURE 4.

Th2-mediated suppression of EAE does not eliminate MBP-reactive T cells. Splenocytes from groups of mice which either received MBP-specific T cells (M) or were immunized with KLH after receiving MBP-specific T cells plus KLH-specific T cells (M+K+I) were tested for MBP-specific proliferation.

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Table II.

Th2 activation during EAE influences neuroantigen-specific T cells

Recipient GroupaMBP-Specific Cytokine Secretion (ng/ml)b
IFN-γIL-4IL-10
MBP 15.6 0.37 0.30 
MBP+KLH 12.0 1.43 0.99 
Recipient GroupaMBP-Specific Cytokine Secretion (ng/ml)b
IFN-γIL-4IL-10
MBP 15.6 0.37 0.30 
MBP+KLH 12.0 1.43 0.99 
a

Mice were sacrificed 18 days post-adoptive transfer.

b

Splenic T cells (4 × 106/ml) were stimulated with 50 μg/ml MBP. Supernatants were tested for cytokines by ELISA following 72-h incubation.

Secretion of Th2 cytokines by MBP-specific T cells following recovery from acute disease (Table II) suggested that Ag-induced cytokine secretion from the KLH-specific cells was responsible for EAE suppression (Fig. 2). To determine whether cytokines were indeed responsible for EAE suppression, immunized recipients of both cell populations were treated with anti-IL-4, anti-IL-10, or an isotype control mAb. A single injection of anti-IL-10 mAb on the day of adoptive transfer partially reversed the protective effects (Fig. 5,A). This group exhibited an onset of disease similar to that of the recipients of MBP cells only. The clinical scores for this group exceeded those of the group treated with the isotype control mAb. Maximum clinical scores were significantly different from both the KLH-protected group (M+K+I) and the group which received MBP cells only (M group) (p ≤ 0.05), suggesting partial amelioration of suppression. By contrast, treatment with anti-IL-4 mAb resulted in only a small increase in clinical score (Fig. 5,A). To confirm that in vivo activation-mediated EAE suppression via IL-10 was not due to a generalized increase in clinical severity following IL-10 inhibition, groups of MBP-specific T cell recipients were treated with either anti-IL-10 or the isotype control mAb on the day of adoptive transfer. Neither mAb altered the course of EAE induced by the adoptive transfer of MBP-specific Th1 cells (Fig. 5 B).

FIGURE 5.

Inhibition of IL-10 reverses Th2-mediated protection. Clinical scores of groups of mice which received 2.5 × 107 MBP-specific T cells i.v. (M), or groups immunized with 100 μg KLH following transfer of both MBP- and KLH-specific T cells (2.5 × 107) (M+K+I) are shown. Immunized recipients of both T cell populations were treated on the day of transfer with 1 mg/recipient anti-IL-4, anti-IL-10, or an isotype control mAb (A). The dates represent the average clinical scores for two experiments with a total of seven mice per group. Mice which received MBP-specific T cells were only treated with either anti-IL-10 or an isotype control mAb on the day of adoptive transfer (B). The dates represent the average clinical scores for two experiments with a total of seven mice per group. Mice received either MBP-specific T cells only or were immunized with KLH following transfer of equal numbers of both MBP- and KLH-specific T cells. Immunized recipients were treated with either anti-IL-10 (1 mg/recipient) or anti-IL-10R (0.5 mg/recipient) on the day of adoptive transfer and also 5 days later. Clinical scores were determined daily in a blinded fashion for 7 days post-transfer (C).

FIGURE 5.

Inhibition of IL-10 reverses Th2-mediated protection. Clinical scores of groups of mice which received 2.5 × 107 MBP-specific T cells i.v. (M), or groups immunized with 100 μg KLH following transfer of both MBP- and KLH-specific T cells (2.5 × 107) (M+K+I) are shown. Immunized recipients of both T cell populations were treated on the day of transfer with 1 mg/recipient anti-IL-4, anti-IL-10, or an isotype control mAb (A). The dates represent the average clinical scores for two experiments with a total of seven mice per group. Mice which received MBP-specific T cells were only treated with either anti-IL-10 or an isotype control mAb on the day of adoptive transfer (B). The dates represent the average clinical scores for two experiments with a total of seven mice per group. Mice received either MBP-specific T cells only or were immunized with KLH following transfer of equal numbers of both MBP- and KLH-specific T cells. Immunized recipients were treated with either anti-IL-10 (1 mg/recipient) or anti-IL-10R (0.5 mg/recipient) on the day of adoptive transfer and also 5 days later. Clinical scores were determined daily in a blinded fashion for 7 days post-transfer (C).

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To determine whether partial inhibition mediated by the passive transfer of the anti-IL-10 mAb was due to incomplete IL-10 neutralization or to the participation of another mechanism(s) (19), immunized recipients of both cell populations were treated with either anti-IL-10 or anti-IL-10R mAb both on the day of adoptive transfer and again at 5 days post-transfer. In contrast to the partial protection afforded by a single anti-IL-10 injection (Fig. 5,A), two injections of either anti-IL-10 or anti-IL-10R mAb completely reversed the protective effect (Fig. 5,C; Table III). Although the mean day of onset of clinical disease in the protected group was delayed 2 days relative to the group which received MBP-specific T cells only, disease onset in mice treated with anti-IL-10 or anti-IL-10R was identical to that in mice which received only the MBP-specific T cells (Fig. 5,C; Table III). Similarly, the maximum clinical scores and cumulative disease scores in protected recipients treated with either mAb regime were similar to those recipients of MBP-specific Th1 cells only (Fig. 5,C; Table III). In contrast to a single injection of anti-IL-10, preliminary experiments suggest that a single injection of 500 μg of anti-IL-10R on the day of adoptive transfer is sufficient to reverse protection. These data indicate that peripheral activation results in IL-10 secretion sufficient to inhibit the progression of EAE. In contrast to immunized recipients (Fig. 3), histological analysis of the CNS of immunized recipients treated twice with either anti-IL-10 or anti-IL-10R mAb revealed significant inflammation and demyelination at day 16 post-transfer, a condition comparable to that in control recipients receiving MBP T cells only (data not shown).

Table III.

Summary of the effects of IL-10 inhibition

GroupamAbbNumberClinical Scores
Day of onsetMaximumcCumulatived
Control 2.9 ± 0.4 16.6 
M+K+I Control 1.0 ± 0.4 4.6 
M+K+I Anti-IL-10 10 3.3 ± 0.2 17.4 
M+K+I Anti-IL-10R 3.2 ± 0.2 20.1 
GroupamAbbNumberClinical Scores
Day of onsetMaximumcCumulatived
Control 2.9 ± 0.4 16.6 
M+K+I Control 1.0 ± 0.4 4.6 
M+K+I Anti-IL-10 10 3.3 ± 0.2 17.4 
M+K+I Anti-IL-10R 3.2 ± 0.2 20.1 
a

Mice received MBP-specific Th1 cells only (M); or MBP-specific cells plus KLH-specific Th2 cells, then immunization with 100 μg KLH (M+K+I).

b

Control mAb (GL113), anti-IL-10, or anti-IL-10R mAb was injected on day 0 and on day 5 post-adoptive transfer.

c

Maximum clinical scores per group ± SEM.

d

Cumulative clinical scores were calculated from day 7 until day 15 post-adoptive transfer.

Suppression of Th1 cell-mediated autoimmune disease via in vivo immune deviation is an attractive potential therapeutic approach. Immune deviation is currently understood as distinct patterns of cytokine secretion by CD4+ Th1 and Th2 cells which mutually inhibit each others development and function (20, 21). Activation of Th1 and Th2 cells is dependent upon the cytokine environment present during priming (20, 21). The presence of IL-12 during priming preferentially induces Th1 cells, whereas the presence of IL-4 results in the preferential activation of Th2 cells. The presence of IL-4 and IL-10, which is due to concomitant parasitic infections (22, 23, 24), altered sex hormones (18), or genetic predisposition (25), preferentially lead to the induction of a Th2 response. However, the interactions between the immune system and the target organ, in this case the CNS, have made the analysis of Th2 cytokine-mediated inhibition of a Th1-induced autoimmune disease complex. Mice which preferentially activate Th2 cells due to an alteration in APC activity are resistant to active, but not to passive, EAE (18). Consistent with these data, the induction of a Th2 response specific for a non-neuroantigen inhibits actively induced EAE (4). These examples are similar to the ability of preexisting parasitic infections to facilitate Th2 responses to heterologous Ag (22, 23, 24). In addition, comparison of relapsing EAE and a progressive CNS infection with Theiler’s virus suggested that IL-4 is associated with EAE remissions (2). Treatment with rIL-4 suppresses the induction of EAE (7), and mice unable to secrete IL-4 due to a targeted mutation exhibit increased clinical signs of EAE (9). These data suggest that the ability of Th2 responses to modify the induction of Th1 CD4+ T cells in vivo is via an IL-4-dependent mechanism (22, 23, 24). However, cotransfer of encephalitogenic Th1 cells along with highly polarized PLP-specific Th2 cells, induced by in vitro culture with rIL-4, did not prevent clinical symptoms of EAE and resulted in a CNS disease characterized by eosinophil infiltration (6). This result is not surprising in light of the recent demonstration that adoptive transfer of an MBP-specific Th2 cell line induced eosinophil-mediated CNS destruction in Rag−/− mice (26). These results suggest that both Th1 and Th2 cells are potentially pathogenic in an environment lacking appropriate immune regulation and that high levels of IL-4 may have adverse effects on CNS inflammation.

The adoptive transfer of IL-4- and IL-10-secreting lymph node cells from young male SJL mice primed in vivo with MBP resulted in a reduction of both clinical and histological EAE (5). In this model, inhibition of EAE is associated with increased expression of IL-10 mRNA in the CNS of protected mice (5). The passive transfer of anti-IL-10 mAb and the analysis of mice deficient in IL-10 secretion suggested that this cytokine is critical in the regulation of EAE (16). Furthermore, recent analysis of two transgenic models, one in which IL-10 secretion was controlled by the CD2 promoter resulting in IL-10 secretion by T cells, and one controlled by the MHC class II promoter resulting in IL-10 secretion by APC, finds that both support previous data demonstrating that rIL-10 has a protective effect on the progression of EAE (16, 17).

In an attempt to resolve the relative roles of IL-4 and IL-10 in inhibition of EAE, as well as to determine whether the activation of Th2 cells specific for a non-neuroantigen at a peripheral site could alter the course of EAE, Th2 cells specific for KLH were induced in young adult male SJL mice (3, 5, 18). These T cells were cultured in vitro and cotransferred with similarly activated EAE-inducing neuroantigen-specific Th1 cells. In the absence of activation by cognate Ag, Th2 cells were unable to influence the induction, progression, or severity of EAE. These results suggested that without in vivo activation, Th2 cells could not antagonize the function of adoptively transferred encephalitogenic Th1 cells, even though it has been suggested that Th1 and Th2 cells with different Ag specificities can interact in vivo (5, 27, 28). For example, activation of Th2 cells during parasitic infection alters the Th1 response to heterologous Ag such as mycobacteria and viruses (23, 24). Consistent with these data, activation of the Th2 cells by immunization with the cognate Ag was efficient in suppressing the Th1-mediated EAE.

The possibility of clonal deletion of MBP-specific Th1 cells was excluded by demonstrating the retention of Ag-specific proliferation in the KLH-protected and control groups after remission. However, examination of the Ag-specific cytokine secretion following MBP-induced activation showed that the MBP-specific T cells derived from the KLH-immunized groups secreted both Th1- and Th2-type cytokines, i.e., high IL-4, IL-10, and IFN-γ. These data contrast with the MBP-induced secretion of the T cells derived from the control group which was predominantly a Th1 type of cytokine profile. These data suggest that the cytokine secretion profile by MBP-specific T cells recovered following KLH-induced protection resulted from the in vivo activation of KLH-specific Th2 cells, most likely via secretion of IL-4 (21). The data cannot exclude a primary effect of IL-10; however, analysis of transgenic mice demonstrated that IL-10 can prevent EAE in the absence of a preferential induction of Th2 cells (17). Effector CD4+ T cells appear to be irreversibly committed to one given type (29); therefore, the altered cytokine secretion profile exhibited by the MBP-specific cells in a KLH-induced Th2 cytokine environment may reflect cytokine secretion by a mixture of the adoptively transferred Th1 cells and in vivo-activated MBP-specific T cells derived from naive T cells of the recipients.

Partial inhibition of the protective effect induced by transfer and in vivo activation of the KLH-specific Th2 cells by a single injection of anti-IL-10 suggested that activation induced a significant amount of IL-10 or that an additional mediator was participating in EAE suppression. However, two injections of either anti-IL-10 or anti-IL-10R reversed protection. The possibility that another mediator also participates in EAE suppression induced by the peripheral activation of Th2 cells cannot be ruled out. However, the data support the previous reports suggesting that IL-10 plays a dominant role in suppression of EAE (17, 18). Protection required in vivo activation of the transferred population and, interestingly, treatment with a neutralizing anti-IL-4 mAb did not alter protection. The inability of anti-IL-4 to prevent protection suggests that Th2 cell proliferation is not required, implying that secretion of IL-10 is not dependent upon continued proliferation. In addition, a number of studies have demonstrated that IL-10 can be induced in the absence of ongoing Th2 responses (30, 31). For example, IL-4-deficient mice recovering from EAE had high levels of IL-10 mRNA in the CNS (9), which correlates with the reduction of both EAE severity (5) and disease remission (1). Recent studies have shown that in vitro-generated regulatory T cells (21, 32, 33) are capable of secreting large amounts of IL-10 and/or TGF-β, but not IL-4. This suggests the possibility that a regulatory T cell population indeed may be the source of IL-10. Although the mechanism of IL-10-mediated inhibition is unclear, the loss of blood brain barrier integrity during EAE may allow IL-10 to access the CNS. Alternatively, IL-10 may modify or prevent EAE by inhibiting the loss of blood brain barrier integrity (34). Both of these possibilities are consistent with the reduced CNS inflammation associated with protection. These data provide evidence that peripheral activation of IL-10-secreting cells can suppress a Th1-mediated autoimmune disease via the secretion of IL-10. Therefore, these data support the concepts that either activation of a preexisting Th2 regulatory population or direct peripheral administration of IL-10 may be viable approaches to ameliorate or modify human autoimmune disease.

1

This work was supported by Grant NS 35058 from the National Institutes of Health and Grant RG 2431 from the National Multiple Sclerosis Society. DNAX Research Institute of Molecular and Cellular Biology is supported by Schering Plough Corporation.

3

Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; PLP, proteolipid protein; MBP, myelin basic protein; KLH, keyhole limpet hemocyanin.

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