Abstract
We examined the role of B7-1 and B7-2, costimulatory molecules critical to full activation of T cells, in the development of Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to B7-1 resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of TNF-α and IFN-γ in the spleen cells was decreased. The delayed-type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. In contrast, treatment with Abs to B7-2, resulted in no effect on TMEV-IDD. These data suggest that B7-1 is critically involved in the pathogenesis of TMEV-IDD and that Abs to B7-1 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.
Multiple sclerosis (MS)3 is an immune-mediated chronic inflammatory demyelinating disease of the CNS in humans (1). Although the etiology of MS is unknown, epidemiological studies and investigations with experimental animal models have supported a potential role for viruses as the environmental trigger in disease induction (2). Thus, a similar demyelinating disease induced by a virus could be one of the most attractive animal models in the study of the pathogenesis of MS. Theiler’s murine encephalomyelitis viruses (TMEV), members of the genus Cardiovirus in the family Picornaviridae, are natural enteric pathogens that cause CNS disease in mice (3). Intracerebral (i.c.) injection of TMEV into susceptible strains of mice results in a chronic, progressive demyelinating disease characterized histologically by perivascular inflammatory cell infiltrates and primary demyelination of the CNS (4). The clinical signs of TMEV-induced demyelinating disease (TMEV-IDD) include a spastic waddling gait, extensor spasm, and incontinence (5). TMEV-IDD is considered an infectious mouse model for MS because the disease displays similar histopathologic, genetic, and clinical similarities to human MS (6, 7, 8, 9). The demyelination is linked to persistent TMEV infection in the CNS (10), and histological findings are characterized by perivascular inflammatory cell infiltrates and primary demyelination in CNS (5, 11). TMEV persists in the white matter of the spinal cord, mainly in macrophages, but also in astrocytes and oligodendrocytes throughout the life of a mouse (11, 12).
Persistent CNS virus infection in susceptible mouse strains triggers clonal expansion and differentiation of TMEV-specific MHC class II-restricted effector delayed-type hypersensitivity (DTH) (Th1) cells that are poorly controlled by normal immunoregulatory mechanism. Activated Th1 cells, specific for viral capsid protein, release proinflammatory cytokines in the CNS, resulting in the subsequent recruitment and activation of mononuclear phagocytes that initiate myelin destruction via both direct and bystander mechanisms (13, 14).
Full activation of T cells requires two signals from APCs. The first signal, the binding of the TCR to its Ag-MHC ligand, provides specificity. The second signal is provided by costimulatory molecules expressed on APCs. Of the known costimulatory molecules, the family of proteins termed B7 appears to be the most potent. The B7 costimulatory pathway involves at least two molecules, B7-1 (CD80) (15) and B7-2 (CD86) (16). The B7 molecules on the APC, B7-1 and B7-2, are the counterreceptors for CD28 and CTLA-4 (17). This costimulatory signal controls T cell activation and suppression and involves the induction of Th1/Th2 cell from Th0 cell (18). Blocking B7-1 interactions during T cell activation induces functional inactivation in Th1 cells, leading to a state of hyporesponsiveness or anergy (19). In experimental autoimmune encephalomyelitis (EAE), another model of MS, encephalitogenic CD4+ Th1 cells play an important role in pathogenesis. In the mouse EAE system, several researchers examined the effect of anti-B7 Abs. The suppressive effect of anti-B7-1 mAb treatment and the aggravated effect of anti-B7-2 mAb treatment have been reported (18, 20, 21, 22, 23). Pope et al. showed that B7-1 and B7-2 were also expressed on the surface of macrophages/microglia and CD4+ T cells in the CNS of TMEV-infected mice (24). These findings suggest that the B7 costimulatory molecule may play an important role in the pathogenesis of TMEV-IDD.
As far as we know, the effect of anti-B-7 mAbs on TMEV-IDD has not been studied yet. We recently generated mAbs to mouse CD80 (RM80) and CD86 (PO3) (25). By utilizing these mAbs, we examined the separate roles of the different costimulatory molecules B7-1 (CD80) and B7-2 (CD86) in TMEV-IDD.
Materials and Methods
Animals
Female pathogen-free SJL/J mice from The Jackson Laboratory (Bar Harbor, ME) were housed and cared for in an approved facility, in accordance with the National Institutes of Health guidelines.
Virus
The BeAn 8386 strain of TMEV was propagated in baby hamster kidney (BHK)-21 cells grown in DMEM supplemented with 7.5% donor calf serum and purified by isopyknic centrifugation on Cs2SO4 gradients as previously described (26).
mAbs
A rat IgG2a mAb to mouse CD80 (RM80) and a rat IgG2b to mouse CD86(PO3) were obtained from PharMingen (San Diego, CA) (25). Hybridoma cells that produce these mAbs were cultured in RPMI 1640 supplemented with 10% FBS and 0.1% gentamicin. These cells were injected into nude mice; mAbs harvested as ascites were purified with the use of a protein G affinity column. All of these mAbs were prepared in PBS at 1 mg/ml.
Injection of mice with TMEV
For i.c. inoculation of virus, 1.3 × 106 PFU of virus in 30 μl were administered into the right cerebral hemisphere of mice anesthetized with methoxyflurane. This inoculum consistently induces neurological signs in susceptible mouse strains (27).
Treatment with mAbs
Six- to eight-wk-old female SJL/J mice were separated into groups (A-E). Group A mice (control) were treated with PBS. In each experiment, TMEV was injected into SJL/J mice i.c. at day 0. mAbs (RM80, PO3, M18/2(nonspecific rat IgG2a mAb)) were injected i.p. into mice on days 4 and 14 after i.c. infection with TMEV at a dose of 500 μg at a volume of 100 μl/mouse per injection. Details of the experimental design are given in Table I⇓. We performed this mAb treatment experiment three times. In one experiment, five groups of mice were under investigation (n = 20 for each group). Before experiments, five mice were blindly selected from each group for histological study, and another five mice were also blindly selected from each group for immunological studies such as TMEV-specific DTH, TMEV-specific T cell proliferation assay, and enumeration of cytokine-producing cells assay. Other mice were clinically observed until 80 days post i.c. infection (n = 10 for each group).
Experimental design
Assessment of demyelinating disease
TMEV-infected mice were examined daily for clinical symptoms of demyelination. Mice were allowed to walk on a polyethylene (Dynalab, Rochester, NY) walking board and observed for exhibition of symptoms including a waddling gait, extensor spasms, paralysis, loss of righting reflex, incontinence, and hunched posture. Neurological signs were recorded using the following grading system: normal = 0, slight waddling gait = 1, waddling gait = 2, spastic hind limb paralysis = 3, and severe hind limb paralysis accompanied by incontinence = 4 (28). These clinical scores have been shown to be indicative of demyelination (29). A clinical score was recorded daily for each mouse in each experiment using this grading system. The mean clinical score for each group of mice on each day was calculated by dividing the sum of all clinical scores of the mice in a given group by the number of mice in that group.
Histology
In each experiment, mice were blindly selected from each group (n = 5 from each group) beforehand for histological examination and sacrificed on day 40 post i.c. infection. Since we had repeated this experiment three times, 15 mice from each group were histologically examined. Mice were perfused under anesthesia by the intraventricular route with 4% paraformaldehyde in PBS, pH 7.4. Spinal cords were removed, fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in PBS, and embedded in epoxy resin. These epoxy-embedded (1 μm thick) sections were stained with toluidine blue. These sections from 12 segments/mouse were read under light microscopy, and grading was done in a blinded fashion by two independent investigators who were unaware of the treatment each animal had received. The score for inflammation was determined according to the following criteria: 0, none; 1, a few inflammatory cells; 2, numerous scattered cells with an occasional perivascular cuff; 3, many perivascular cuffs; and 4 and 5, increasing perivascular infiltration and subarachnoid inflammation. The extent of demyelination was determined according to the following scoring system: 0, no demyelination; 1, a few scattered naked axons; 2, small groups of naked axons; and 3, large groups of demyelinated axons with confluent plaques of demyelination (30).
Ag-specific DTH
A 24-h ear swelling assay was used to quantitate DTH (31). Before experiments, five mice were also blindly selected from each group for immunological studies. At 36 days post i.c. infection, prechallenge ear thickness of these mice was determined using a Mitutoyo digimatic micrometer (Mitutoyo, Tokyo, Japan). Subsequently, 5 μg of purified TMEV in 10 μl of saline was injected into the dorsal surface of the ear using a Hamilton syringe fitted with a 30-gauge needle. Twenty-four hours later, ear thickness was again measured, and the increase in thickness was expressed in units of 10−4 inches. Ear swelling reactions were due to mononuclear cell infiltration and showed typical DTH kinetics (i.e., minimal swelling at 4 h and maximal swelling at 24 to 48 h).
T cell proliferation assay
After DTH measurement, the same mice were sacrificed. Spleen cells were harvested from three animals in each group and pooled. Cells (5 × 105) were cultured in 96-well flat-bottom microculture plates in RPMI 1640 containing 0.5% syngeneic mouse serum, 5 × 10−5 M 2-ME and antibiotics. Triplicate cultures were stimulated with three different concentrations of UV-inactivated TMEV (0.5, 5, and 10 μg) and were incubated for 72 h in a humidified atmosphere of 5% CO2 and 95% air. Cultures were then pulsed with 1.0 μCi of [3H] dThd and harvested 24 h later. Measurement of [3H]dThd incorporation was determined using a scintillation counter and expressed as cpm. Background proliferation was less than 1/7 of TMEV-specific proliferation.
Anti-TMEV Ab and anti-TMEV subclass titration
TMEV-specific Ab titers were determined using ELISA as described earlier (32) utilizing sera from individual mice. Briefly, 0.3 μg of purified virus was used to coat microtiter plates. A BSA solution (0.3 μg) was also used to coat the plates, to serve as a negative control. Unless otherwise stated, 2-fold serial diluents of sera starting from a 1:100 (20 × 100) dilution were reacted with the Ags on the microtiter plates and then with goat anti-mouse secondary Ab conjugated with alkaline phosphatase (KPL, Gaithersburg, MD). For anti-TMEV subclass Ab titration, sera were reacted with the Ag on the microplates and then with biotinylated rat monoclonal anti-mouse IgG subclass Ab (Zymed, San Francisco CA). After the plates were washed, streptavidin-alkaline phosphatase was added to each well, and the plates were incubated. After the plates were again washed, substrate (p-nitrophenyl phosphate) for the enzyme was added, and the enzyme reaction was colorimetrically measured by an ELISA reader (Bio-Rad, Hercules, CA) at 410 nm. The Ab titers of ELISA represent log2 × 100.
Cytokine assay by ELISA
The concentration of circulating cytokines, such as TNF-α, IFN-γ, IL-4, or IL-10, were measured using commercially available ELISA kits (Genzyme, Cambridge, MA).
Enumeration of cytokine-producing cells
On day 40 post i.c. infection, spleen cells from the same mice used for Ag-specific DTH and T cell proliferation assay (n = 5 from each group) were harvested from animals in each group. The levels of TNF-α, IFN-γ, IL-4, or IL-10 producing spleen cells were examined using an ELISPOT assay. The original reverse ELISPOT assay (33) was modified by using nitrocellulose membrane (Bio-Rad). Wells were filled (50 μl/well) with monoclonal hamster anti-murine TNF-α, IFN-γ, IL-4, or IL-10 mAbs (Genzyme) at a concentration of 10 μg/ml in 0.5% BSA in PBS overnight at 4°C. Unabsorbed Abs were removed, and wells were washed with PBS. The plates were then blocked with 1% BLOTTO (nonfat dry milk) for 2 h at 37°C. The outer surface of the nitrocellulose membrane was carefully dried. Spleen cells (1 × 105/well) in the culture medium (RPMI 1640 supplemented with 10% FBS and 0.1% gentamicin) were dispensed among individual wells (100 μl/well). Plates were then incubated for 48 h at 37°C in a humidified, 5% CO2 atmosphere and were washed three times with Tris-buffered saline with Tween 20 (TBST). Fifty μl of a 1:250 dilution of polyclonal rabbit anti-murine TNF-α, IFN-γ, IL-4, or IL-10 Abs (Genzyme) was added to each well followed by incubation for 2 h at 37°C. Plates were washed with TBST again and were treated with 50 μl of 1 μg/ml alkaline phosphatase-conjugated goat anti-rabbit IgG (KPL) for 2 h at 37°C. After another washing with TBST, cytokines secreted by single cells were visualized by adding a mixture of nitro-blue tetrazolium and 5-bromo-4-chloro-3-indole phosphate (Life Technologies, Grand Island, NY). The color reaction of the enzyme was halted after 30 min by washing with water, and spots were enumerated under ×40 magnification.
Statistical analysis
Clinical scores were analyzed using the Mann-Whitney U test; other results were statistically evaluated using the Student t test (StatView program, Abacus Concepts, Berkeley CA). A p value of <0.05 was considered statistically significant.
Results
Protection from demyelinating disease after administration of mAbs specific to B7-1.
The results of experiments are summarized in Fig. 1⇓ and Table II⇓. Control animals (Group A, nontreated; group B, treated with control mAb M18/2) showed the typical disease course of TMEV-IDD. On day 40, about 35% of mice showed clinical signs such as waddling gait, extensor spasm, and hind leg paralysis; mean clinical scores were 2.8 in group A and 2.7 in group B, respectively. On day 80, all mice from groups A, B, and D (treated with anti-B7-2 mAb) and with E (treated with both anti-B7-1 mAb and anti-B7-2 mAb) developed TMEV-IDD; the mean clinical scores were 3.6 in group A and 3.5 in group B. In contrast, only 6.7% (1/15) of mice treated with anti-B7-1 mAb (group C) had clinical signs on day 40; the mean clinical score was 1.3. On day 80, 40% (4/10) of mice treated with anti-B7-1 mAb had clinical signs with a mean clinical score of 1.9. These results demonstrate that clinical signs of demyelinating disease are significantly suppressed (p < 0.01) in animals treated with anti-B7-1 mAb (group C), as compared with those in control groups (groups A and B). In addition, we could not detect any significant differences in other treatment groups (groups D and E) that received either anti-B7-2 alone or together with anti-B7-1 mAb. Representative mice were blindly selected from each group (n = 5 from each group) for histological examination and sacrificed on day 40 post i.c. infection. The characteristic perivascular and parenchymal mononuclear cell infiltration and extensive demyelination in the white matter of spinal cord were observed in mice from groups A, B, D, and E (Fig. 2⇓A). The inflammation scores and demyelination scores were significantly lower in mice from group C (p < 0.01) than those from control groups (Fig. 2⇓B, Table II⇓).
Effect of treatment with mAbs specific for B7-1/B7-2 on the clinical course of TMEV-IDD. Mice were infected i.c. at day 0. Monoclonal Abs (group A, none; group B, nonspecific IgG mAb (M18/2); group C, anti-B7-1 mAb; group D, anti-B7-2 mAb; group E, anti-B7-1 mAb + anti-B7-2 mAb) were injected i.p. into mice on days 4 and 14 after i.c. infection of TMEV at a dose of 500 μg in a volume of 100 μl/mouse each time. On days 40 and 80 post i.c. infection, clinical signs of demyelinating disease were significantly suppressed (∗, p < 0.01) in animals treated with anti-B7-1 mAb (group C), as compared with those in control groups (groups A and B). No significant differences were observed in other treatment groups (groups D and E).
Histological findings. Epon-embedded section, 1 μm thick, stained with toluidine blue (×150). In each experiment, mice were blindly selected from each group (n = 5 from each group) beforehand for histological examination and sacrificed on day 40 post i.c. infection. Since we repeated this experiment three times, 15 mice from each group were histologically examined. A, Cross-section of a spinal cord from a mouse treated with nonspecific IgG mAbs (M18/2). This mouse showed severe neurological signs. Extensive demyelinative lesion with parenchymal mononuclear cell infiltrates in white matter is observed. B, Cross-section of a spinal cord from a mouse treated with anti-B7-1 mAb. This mouse showed no clinical sign. This section shows almost normal myelinated spinal cord white matter.
Clinical and pathological evaluation of anti-B7-1/B7-2 mAb-treated and control mice
TMEV-specific DTH
Virus-specific DTH, as measured by the ear-swelling assay, is known to correlate strongly with susceptibility to TMEV (31). DTH has been shown to be mediated by the Th1 lymphocyte subset (34). To compare the clinical signs and the level of TMEV-specific DTH, we assessed the level of DTH 36 days after viral infection in mice selected for immunological examination (n = 5 from each group). The levels of DTH at 36 days postinfection in mice of control groups (groups A and B) were increased at almost the same levels previously reported (35). Conversely, they were significantly lower (p < 0.01) in mice treated with neutralizing anti-B7-1 Ab (group C). These results show that administration of anti-B7-1 mAb inhibits the level of TMEV-specific DTH. The levels of DTH at 36 days postinfection in mice treated with anti-B7-2 mAb, and mice treated with both anti-B7-1 mAb and anti-B7-2 mAb, were increased to similar levels of control groups (Fig. 3⇓A).
Before experiments, five mice were blindly selected from each group for TMEV-specific DTH and TMEV-specific T cell proliferation assay. Since these studies were examined three times, the initial data points are representative of 15 mice per group. A, The level of TMEV-specific DTH in mice treated with neutralizing mAb specific for B7-1 is low. On day 36 post viral infection, five SJL/J mice from groups A, B, C, D, and E and a group mock infected with baby hamster kidney cell lysate were examined for ear-swelling response to UV-inactivated purified TMEV. Responses from this experiment are expressed as mean Δ ear swelling in units of 10 −4 inchesd ± SEM. Average background ear-swelling responses (from mock-infected mice) were subtracted from the individual specific ear-swelling responses. The average background ear-swelling was 3.2 ± 1.2. The responses of group B were significantly lower (∗, p < 0.01) than those of other groups. B, The level of TMEV-specific T cell proliferative response was significantly lower in mice treated with mAbs to B7-1 than control groups treated with either PBS (none) or a nonspecific IgG mAb (∗p < 0.01). On day 40 post viral infection, the same mice from the DTH study were sacrificed and examined for TMEV-specific T cell proliferative response. Spleen cells (5 × 105) were cultured with 10 μg of intact UV-inactivated TMEV for 72 h and then pulsed with 1 μCi of [3H]TdR for the final 24 h. Data represent specific proliferation minus nonspecific proliferation of spleen cells. Results are expressed as mean cpm ± SEM from triplicate cultures. Group A, none (PBS) treated; group B, nonspecific IgG mAb treated; group C, anti-B7-1 mAb treated; group D, anti-B7-2 mAb treated; group E, anti-B7-1 mAb + anti-B7-2 mAb treated.
TMEV-specific T cell proliferation
T cell proliferative responses have been used frequently to assess the level of response of CD4+ T helper cells to the virus (36). After DTH measurement, same mice were sacrificed on day 40 post i.c. infection. Spleen cells were taken out and used for examination for TMEV-specific T cell proliferation and ELISPOT assay. When the TMEV-specific T cell proliferative responses of mice treated with neutralizing anti-B7-1 mAb were compared with those of control groups, we observed a distinct difference (Fig. 3⇑B). These results may indicate that administration of anti-B7-1 mAb inhibits the ability of TMEV-specific T cell proliferation of TMEV-specific mouse spleen cells or inhibits the generation of TMEV-specific T cells at the precursor level. TMEV-specific T cell proliferative responses of mice treated with anti-B7-2 mAb, and mice treated with both anti-B7-1 mAb and anti-B7-2 mAb, were increased to almost the same levels as those of control groups.
TMEV-specific Ab responses
We examined the Ab responses in the experimental groups of mice to determine a possible effect of anti-B7-1 and anti-B7-2 Abs on the production of TMEV-specific Abs. Sera were taken from all mice of each group (n = 10 from each group) on day 56 and day 80 post i.c. infection. On day 56, there was no significant difference in the TMEV-specific Ab levels among the six groups. We also explored whether Ab isotypes were affected. We detected IgG1 and IgG2b Abs in all groups, with no significant difference among the five groups. We also detected IgG2a Ab in control groups (Groups A, B, D, and E), but not in the anti-B7-1 mAb neutralizing Ab-treated group (group C) (Fig. 4⇓). These results indicate that, despite an unaltered level of total Abs, there is a lack of IgG2a component in animals treated with anti-B7-1 neutralizing Abs. This result is consistent with the suppression of Th1 response since IgG2a production is dependent on the presence of Th1 population.
Analysis of TMEV-specific Abs. Since small amounts of sera were enough for Ab titration and we did not have to sacrifice mice, we took out sera from all mice from the clinical course observation group (n = 10 for each group). Since these studies were examined three times, the initial data points are representative of 30 mice per group. SDs represent values obtained from individual mice from different groups of mice, i.e., sera from each individual were assayed separately. We detected TMEV-specific IgG Ab in all groups. We could detect IgG2a Ab in control-treated groups (group A and B), but not in anti-B7-1 neutralizing mAb-treated groups (group C).
Assessments of cytokine production
After DTH measurement, the same mice (n = 5 from each group) were sacrificed on day 40 post i.c. infection. Spleen cells were taken out and used for ELISPOT assay. We could not detect any TNF-α, IFN-γ, IL-4, or IL-10 in the sera from animals of any groups. The levels of cytokine-producing cells in the spleens of animals were also assessed using the ELISPOT method. TNF-α production by spleen cells from mice treated with neutralizing Abs to B7-1 was significantly suppressed (p < 0.01) compared with other groups (Fig. 5⇓A). IFN-γ production by spleen cells from mice treated with anti-B7-1 neutralizing Abs was also significantly suppressed (p < 0.01) compared with other groups (Fig. 5⇓B). No significant differences in the production of Th2-derived cytokines such as IL-4 and IL-10 were observed among all groups (Fig. 5⇓, C and D). No cytokine production by spleen cells was detected in uninfected mice. These results suggest that production of proinflammatory cytokines such as TNF-α and Th1 cell-derived inflammatory cytokines such as IFN-γ was down-regulated in TMEV-infected mice treated with anti-B7-1 neutralizing Abs.
ELISPOT assays for cytokine production. The same mice used for TMEV-specific DTH and for the TMEV-specific T cell proliferation assay were examined in this assay. Since these studies were examined three times, the initial data points are representative of 15 mice per group. SDs represent values obtained from individual mice from different groups of mice, i.e., splenocytes from each individual were assayed separately. A, Results of TNF-α assay by ELISPOT. TNF-α production of spleen cells was significantly suppressed in the mice of anti-B7-1 neutralizing mAb-treated group (group C) (∗, p < 0.01) compared with control-treated groups (groups A and B). B, Results of IFN-γ assay. The number of IFN-γ-producing spleen cells was significantly decreased in the mice of the anti-B7-1 neutralizing mAb-treated group (group C) (∗, p < 0.01) compared with control-treated groups (groups A and B). C, Results of IL-4 assay. The number of IL-4-producing spleen cells was significantly increased in the mice of the anti-B7-1 neutralizing mAb-treated group (group C) (∗, p < 0.01) compared with control-treated groups (groups A and B). D, Results of IL-10 assay. The number of IL-10-producing spleen cells was significantly increased in the mice of the anti-B7-1 neutralizing mAb-treated group (group C) (∗, p < 0.01) compared with control-treated groups (group A and B)
Discussion
In this study, we examined the role of the members of the B7 family, B7-1 (CD80) and B7-2 (CD86), in TMEV-IDD. We showed that administration of Abs to B7-1 suppressed the clinical signs of TMEV-IDD. Furthermore, infiltration of the CNS with mononuclear cells and demyelination were dramatically reduced by treatment with this mAb. Insofar as we are aware, the effects of anti-B7-1 neutralizing Abs have not been studied in TMEV-IDD, one of a few available experimental models of MS. After i.c. inoculation with TMEV, an initial viremia is followed by a persistent, low-level CNS infection. TMEV infects neurons, glial cells, and macrophages in the spinal cords (37, 38). Ag presentation is mediated by either macrophages or glial cells, which contain TMEV Ags. As a consequence of the immunological process, MHC class II-restricted TMEV-specific DTH T cells are activated, which in turn participate in immune-mediated inflammatory responses. It has been proposed that virus-specific DTH results in the recruitment and accumulation of macrophages in the CNS, producing demyelination via a nonspecific “bystander” mechanism (39). Myelin damage induced following TMEV infection is an immune-mediated process. In susceptible mice, the persistence of viral Ags within the CNS may result in continuous activation of T cells that can mediate pathogenesis through locally produced cytokines or direct cytolytic mechanisms. The role of T lymphocytes in immune-mediated demyelination has been confirmed by the fact that thymectomized, irradiated, and bone marrow-restored SJL/J mice fail to develop any clinical signs of TMEV-IDD (40). It has been shown that treatment of susceptible mice with Abs to the CD4 molecule or its restricting MHC class II molecules can significantly reduce the development of inflammation and demyelination after infection with TMEV (40, 41, 42). In contrast, depletion of the CD8+ T cells has no inhibitory effect on demyelination induced by the BeAn strain of TMEV (40, 43). Demyelination correlates well with the development of a chronic, virus-specific DTH response (39). In addition, TMEV-IDD is inhibited after induction of virus-specific tolerance, which down-regulates Th1-type responses (28, 44), and recent T cell epitope study showed that Th1-type cells specific for VP1233–250 and VP274–86 were involved in the pathogenesis of viral demyelination induced by TMEV (45). These studies strongly suggest that virus-specific CD4+, class II-restricted Th1 cells are a major component in myelin destruction by TMEV infection. The activation and differentiation of T cells require both Ag/MHC recognition and costimulatory signals. The costimulatory signals are provided by binding of specific receptors on T cells with their ligands on APC and appears to be necessary for functional T cell activation (46). Among these signals, those involving interactions between CD28 and B7-1 (CD80) and/or B7-2 (CD86) molecules appear to be the most critical (47). Blockade of this pathway results in the induction of Ag-specific unresponsiveness in vitro and in vivo. For example, treatment with CTLA4-Ig, that binds to both human and mouse B7-1 and B7-2 molecules, suppresses rejection of xenogenic islets. The cells from mice treated with CTLA4Ig are specifically unresponsive to the xenoantigens expressed on the islet graft during CTLA4Ig therapy (48). Treatment with Abs to CD80 and CD86 also decreases the inflammatory cell infiltration and prolongs the survival of rat cardiac xenografts in mice (49). EAE, another animal model of multiple sclerosis, is induced by CD4+ MHC class II-restricted encephalitogenic T cells of the Th1 phenotype that predominantly secrete the cytokines IL-2 and IFN-γ (50). CD80 (B7-1) is preferentially up-regulated on T and B cells and macrophages of the CNS during EAE and in acute CNS plaques from patients with multiple sclerosis (22, 51). The effects of anti-B7 mAbs in mouse EAE have been reported. In myelin basic protein (MBP)-induced mouse EAE system, the proliferation and IL-2 production of MBP-specific lymph node cells (LNC) were inhibited, and the clinical signs were reduced by using CTLA4Ig (52). They also showed that anti-CD80 (B7-1) mAb blocked development of the first disease episode and anti- CD86 (B7-2) mAb had no effect. Injection of CTLA4Ig on day 2 postimmunization protected the disease, but combined anti-CD80 (B7-1) mAb and anti-CD86 (B7-2) mAb treatment on day 2 resulted in marked exacerbation of disease (20). The same group also showed that administration of anti-CD80 (B7-1) in actively induced EAE resulted in almost complete suppression of clinical disease. Administration of both anti-CD80 (B7-1) mAb and anti-CD86 (B7-2) mAb on day 2 resulted in a delay in disease onset and reduced disease severity, but disease suppression was not as complete as with anti-CD80 (B7-1) alone. Conversely, anti-CD86 (B7-2) mAb treatment resulted in modest disease exacerbation. In vitro activation of MBP-specific lymph node cells was inhibited by the combination of anti-B7-1 mAb plus anti-B7-2 mAb, but not individually (21). The opposite results reported in Refs. 20 and 21 with the injection of both anti-B7-1 mAb and anti-B7-2 mAb may be due to the different protocols for disease in induction. In Ref. 20 , the authors used a pertussis toxin-dependent model of actively induced EAE. In Ref. 21 , the authors used a pertussis toxin-independent model of active EAE (involving two injections of MBP) and adoptively transferred EAE.
Kuchroo et al. showed that anti-B7-1 mAb drove naive MBP-specific helper T cells along a Th2 pathway whereas anti-B7-2 mAb favored Th1 development. Administration of anti-B7-1 mAb inhibited the generation of Th1 cells and ameliorated the disease, whereas injection of anti-B7-2 mAb inhibited the generation of Th2 cells and worsened the disease. Blocking B7 molecules in vivo did not inhibit the generation of Ag-specific T cells, consistent with the in vitro differentiation results, but affected the cytokine profiles of the responding T cells (18). Miller’s group showed preferential up-regulation of B7-1 during the course of relapsing EAE and a selective increase in its functional costimulatory activity relative to B7-2 in relapsing EAE. When these Abs were administered during the first remission after the acute phase of the disease, anti-B7-2 treatment had no effect, whereas blockade of B7-1/CD28 interactions using the F(ab) fragments of anti-B7-1 resulted in the blockade of disease relapses (22). Taken together, in the mouse EAE system, administration of anti-B7-1 mAb ameliorates and that of anti-B7-2 mAb worsens the disease, and the various EAE results suggest a requirement for B7-1 in the pathogenesis. These results are similar to our study. In TMEV-IDD, anti-B7-1 mAb treatment suppressed the disease. Our study showed this treatment also suppressed TMEV-specific T cell proliferation and DTH, and down-regulated proinflammatory and Th1 cell-derived inflammatory cytokines. These result suggest that inhibition of encephalitogenic CD4+ Th1 cell activation caused by this treatment may lead to the suppression of TMEV-IDD. Though anti- B7-2 mAb treatment did not change the clinical course of demyelination, this treatment may suppress Th2 cell function. It is interesting to note that treatment with both anti-B7-1 mAb and anti-B7-2 mAb had no effect on TMEV-IDD. Suppression of both Th1 and Th2 cells may not change the Th1-Th2 balance and eventually lead to demyelination. However, the effect of both anti-B7-1 mAb and anti-B7-2 mAb treatment is controversial in EAE. In another autoimmune disease model, autoimmune diabetes of nonobese diabetic (NOD) mouse, anti-B7-2 mAb treatment prevented, whereas anti-B7-1 exacerbated, the disease (53, 54). Since Th1-type cytokines should mediate disease in nonobese diabetic mice (55), these results may suggest that the concept B7-1 costimulation preferentially results in development of Th1 cells whereas B7-2 results in the development of Th2 cells may be overly simplified. The conflicting results between immune-mediated demyelinating disease and autoimmune diabetes may be due to the difference in the nature of APCs on the side of the autoimmune disease, or the presence of the blood-brain barrier. Although the importance of the role of CD28/B7 costimulatory signals in the regulation TMEV-IDD has been shown in this study, further investigation should be needed in the pathogenesis of demyelinating disease. Finally, since methods of clinical therapy of MS are still incomplete, it is important to examine the possibility of anti-cytokine therapy, including mAb to B7-1.
Footnotes
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1 This study was supported by Grants 07670707, 07457155, 09670649, and 11670619 from the Ministry of Education and Culture, by a research Grant for neuroscience, and by a research grant for allergy and immunity from the Ministry of Health and Welfare, Japan.
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↵2 Address correspondence and reprint requests to Dr. Chang-Sung Koh, Third Department of Medicine (Neurology), Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. E-mail address: kshosei{at}hsp.md.shinshu-u.ac.jp
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↵3 Abbreviations used in this paper: MS, multiple sclerosis; TMEV, Theiler’s murine encephalomyelitis virus; TMEV-IDD, TMEV-induced demyelinating disease; EAE, experimental autoimmune encephalomyelitis; DTH, delayed-type hypersensitivity; i.c., intracerebral.
- Received July 8, 1999.
- Accepted September 17, 1999.
- Copyright © 1999 by The American Association of Immunologists
References
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