Endogenous presentation of self myelin epitopes by CNS-resident APCs in Theiler’s virus–infected mice

The mechanisms underlying the initiation of virus-induced autoimmune disease are not well understood. Theiler’s murine encephalomyelitis virus–induced demyelinating disease (TMEV-IDD), a mouse model of multiple sclerosis, is initiated by TMEV-specific CD4⁺ T cells targeting virally infected central nervous system–resident (CNS-resident) antigen-presenting cells (APCs), leading to chronic activation of myelin epitope–specific CD4⁺ T cells via epitope spreading. Here we show that F4/80⁺, I-A^s+, CD45⁺ macrophages/microglia isolated from the CNS of TMEV-infected SJL mice have the ability to endogenously process and present virus epitopes at both acute and chronic stages of the disease. Relevant to the initiation of virus-induced autoimmune disease, only CNS APCs isolated from TMEV-infected mice with preexisting myelin damage, not those isolated from naive mice or mice with acute disease, were able to endogenously present a variety of proteolipid protein epitopes to specific Th1 lines. These results offer a mechanism by which localized virus-induced, T cell–mediated inflammatory myelin destruction leads to the recruitment/activation of CNS-resident APCs that can process and present endogenous self epitopes to autoantigen-specific T cells, and thus provide a mechanistic basis by which epitope spreading occurs.

The mechanisms underlying the initiation of autoimmunity are not well understood, but studies in a number of human autoimmune diseases strongly suggest that infections often precipitate clinical episodes in genetically predisposed individuals. This has perhaps been best documented in multiple sclerosis (MS), a human autoimmune disease characterized by CD4⁺ T cell–mediated demyelination of the central nervous system (CNS) and by autoimmune responses to myelin proteins such as myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG) (1–4). In MS, epidemiological evidence strongly supports the hypothesis that the disease may be initiated by a virus infection (5), although no single virus has been consistently found in MS lesions. It is therefore of great interest to explore mechanisms by which infectious agents trigger autoimmune disease. Potential mechanisms include direct activation of autoreactive T cells that are cross-reactive with a viral epitope(s) (molecular mimicry; ref. 6); nonspecific activation of autoreactive T cells by virus-encoded superantigens (7); and de novo activation of autoreactive T cells by autoepitopes released secondary to virus-specific T cell–mediated bystander damage to self tissues (epitope spreading). We have recently shown that in SJL mice, epitope spreading plays a major role in the progression of 2 clinically distinct T cell–mediated demyelinating diseases that resemble the 2 distinct clinical presentations of MS: relapsing experimental autoimmune encephalomyelitis (R-EAE) and chronic-progressive Theiler’s murine encephalomyelitis virus–induced demyelinating disease (TMEV-IDD) (8–11).

R-EAE in the SJL mouse is a CD4⁺ T cell–mediated autoimmune demyelinating disease that can be induced with a variety of myelin proteins/peptides. The disease presents with a relapsing-remitting paralytic course, characterized clinically by flaccid hindlimb paralysis and histologically by mononuclear cell infiltration and demyelination (12). In PLP139-151–induced R-EAE, the initial clinical relapse is mediated primarily by T cells specific for the secondary, non–cross-reactive PLP178-191 epitope that are activated after acute myelin destruction mediated by CD4⁺ T cells specific for the initiating PLP139-151 determinant (13).

More relevant to initiation of virus-induced autoimmune diseases, we have also recently shown that the chronic stages of TMEV-IDD involve the activation of autoreactive CD4⁺ T cells specific for a variety of myelin epitopes (14). TMEV-IDD is the most relevant of the available virus-induced animal models of immune-mediated demyelination (8), and serves as an excellent system in which to assess the potential contribution of antimyelin autoimmune responses to initiation and progression of clinical disease. TMEV, a picornavirus and natural mouse pathogen, induces a lifelong persistent infection of central nervous system–resident (CNS-resident) antigen-presenting cells (APCs) (15–17), and results in a chronic immune-mediated CNS demyelinating disease when inoculated intracerebrally into susceptible strains of mice. Infected SJL mice develop progressive symptoms of gait disturbance, spastic hindlimb paralysis, and urinary incontinence (18), histologically related to perivascular and parenchymal mononuclear cell infiltration and demyelination of white matter tracts within the spinal cord (19–21). In the highly susceptible SJL mouse strain, current evidence indicates that the myelin damage is initiated by TMEV-specific CD4⁺ T cells targeting persistent virus antigen (22–26), whereas the chronic stage of the disease also involves the activity of CD4⁺ myelin epitope–specific T cells primed by epitope spreading (14).

These results illustrate that autoimmune responses are not static, as T-cell responses to endogenous self epitopes emerge during the chronic course of these 2 T cell–mediated CNS inflammatory diseases. As importantly, they indicate that bystander myelin damage initiated by virus-specific immune responses can lead to the activation of naive autoreactive T cells, demonstrating that epitope spreading is an important alternate mechanism to molecular mimicry for explaining the etiology of certain virus-induced, organ-specific autoimmune diseases. Here we show that F4/80⁺, I-A^s+ macrophages/microglia isolated from the CNS of TMEV-infected SJL mice have the ability to endogenously process and present virus epitopes at both acute and chronic stages of the disease. Relevant to the initiation of virus-induced autoimmune disease, CNS APCs isolated from the spinal cords of TMEV-infected mice with preexisting myelin damage, but not CNS APCs isolated from mice at disease initiation or microglia from naive brain, were able to endogenously present a variety of PLP epitopes to specific Th1 lines. To our knowledge, this is the first demonstration of presentation of endogenous autoepitopes in the target organ of a virus-induced disease. These results offer a mechanism by which a localized virus-induced inflammatory myelin destruction results in processing and presentation of endogenous self epitopes by CNS-resident APCs that can activate autoantigen-specific T cells.

Distinct clinical disease patterns in SJL mice with TMEV-IDD and PLP139-151–induced R-EAE. Groups of 15–20 SJL mice were monitored for development of clinical signs of demyelination after infection with TMEV and active immunization with PLP139-151/CFA. The TMEV-IDD and R-EAE models demonstrate 2 distinct clinical disease courses that resemble the chronic-progressive and relapsing-remitting forms of human MS. Figure 1a shows a typical disease course of SJL/J mice, inoculated intracerebrally with the BeAn strain of TMEV. Initial clinical symptoms of a mild waddling gait first appear approximately 30 days after infection, and follow a chronic-progressive disease course, with most animals progressing to total hindlimb paralysis by 150–200 days after infection. The relapsing-remitting nature of R-EAE is demonstrated in Figure 1b, which shows that the acute clinical disease signs appear around 12–13 days after disease induction and peak on day 14. This is followed by a remission and a number of subsequent relapses peaking on days 20, 29, and 37.

Figure 1

TMEV-IDD and PLP139-151–induced R-EAE have distinct clinical disease courses in SJL mice. TMEV-IDD (a) and R-EAE (b) were induced in 2 groups of 20 SJL/J mice, and all animals were graded for clinical signs of demyelination as described in the Methods. Results are expressed as mean clinical score of affected animals versus days after immunization/infection. Disease incidence in both groups was 100%.

Endogenous presentation of myelin epitopes by APCs isolated from the spinal cords of SJL mice with ongoing TMEV-IDD and PLP139-151–induced R-EAE. Because the progression of both TMEV-IDD and R-EAE are characterized by the activation of CD4⁺ T cell–mediated immune responses to endogenous myelin epitopes (13, 14), we asked whether CNS APCs isolated from the spinal cords of mice undergoing either or both of the demyelinating diseases could endogenously present self myelin epitopes. We recently reported that endogenously acquired TMEV epitopes could be presented by CNS-resident microglia/macrophages isolated from the spinal cords of TMEV-infected mice around the time of onset of clinical disease (40 days after infection) (31). To test the possibility of endogenous antigen presentation in the CNS of affected mice, plastic-adherent CNS-resident mononuclear cells were purified from the spinal cords of mice undergoing both TMEV-IDD and R-EAE, irradiated, and used as APCs to stimulate a panel of T-cell lines and hybridomas specific for various viral and myelin epitopes.

Plastic adherent CNS-resident mononuclear cells isolated from mice 88–95 days after infection with TMEV (about 60 days after initiation of clinical symptoms) were irradiated and cultured with a panel of SJL-derived Th1 lines specific for the immunodominant TMEV epitope (VP2 70-86) and for a number of encephalitogenic epitopes on PLP (PLP56-70, PLP104-117, PLP139-151, and PLP178-191). As seen in Figure 2a, 3 × 10⁴ to 4 × 10⁴ irradiated APCs from the CNS of the TMEV-infected mice were able to activate significant proliferative responses of 3 × 10⁴ to 4 × 10⁴ Th1 line cells specific for both the virus and each of the PLP epitopes in the absence of addition of exogenous antigen (Figure 2, filled bars). In general, the level of activation was not as great as that achieved by irradiated splenic APCs pulsed with the relevant peptides (Figure 2, hatched bars), but it was nevertheless highly significant in comparison with naive splenocytes in the absence of antigen (open bars) and was reproducible in multiple experiments. Endogenous activation of the Th1 lines depended on the number of CNS APCs added to the culture, and these APCs induced the secretion of IL-2 from the Th1 lines (data not shown). We have recently reported (31) that CNS APCs from TMEV-infected mice do not trigger an I-A^s–restricted horse myoglobin–specific Th1 line, indicating that activation is specific to TMEV and endogenous myelin epitopes. The current results indicate that there is a significant amount of processed, MHC class II–associated VP2 70-86 peptide endogenously present on mononuclear APCs from the spinal cords of mice infected with TMEV almost 3 months previously. This supports the documented ability of TMEV to persist long-term in macrophages/microglia in the CNS target organ (15). Most significantly, APCs resident in the inflammatory CNS environment of TMEV-infected mice were able to process and present a number of endogenous PLP epitopes that have been associated with chronic disease (14).

Figure 2

Endogenous presentation of myelin epitopes by CNS-resident APCs from the spinal cords of mice with ongoing TMEV-IDD and PLP139-151–induced R-EAE. (a) Plastic-adherent spinal cord mononuclear cells were prepared from SJL mice 88–95 days after intracerebral infection with TMEV. A total of 3 × 10⁴ to 4 × 10⁴ irradiated (30 Gy) CNS APCs were cultured with 4 × 10⁴ of the indicated T-cell lines for 60 hours, and pulsed with 1 μCi of [³H]TdR for the final 16 hours of culture. T-cell lines cultured with 4 × 10⁴ irradiated splenocytes + 50 μM of the appropriate peptide served as the positive control. (b) Plastic-adherent spinal cord mononuclear cells were prepared from SJL mice during the recovery period after the acute phase of PLP139-151–induced R-EAE (18 days after immunization). A total of 4 × 10⁴ of the indicated T-cell lines were cultured with either 4 × 10⁴ irradiated naive splenocytes or 2 × 10⁴ irradiated CNS APCs, without the addition of antigen, with 25 μg/mL of the MP-4 MBP/PLP fusion protein, or with 50 μM of the appropriate peptide. Cultures were pulsed with 1 μCi of [³H]TdR at 48 hours and harvested 24 hours thereafter. Values for both groups represent the mean cpm ± SEM of duplicate or triplicate cultures. Stimulation indices are indicated above each bar, and were calculated using the cpm of the T-cell line cultured with naive splenic APCs in the absence of peptide.

Plastic-adherent CNS-resident mononuclear cells harvested from mice recovering from acute R-EAE were also able to activate myelin-specific Th1 lines in the absence of added antigen. As seen in Figure 2b, CNS APCs from mice with PLP139-151–induced R-EAE triggered proliferation of a Th1 line specific for the initiating PLP139-151 epitope, as we have shown previously (32). More significantly, these APCs activated Th1 lines specific for relapse-associated, non–cross-reactive epitopes on both PLP (PLP178-191) and MBP (MBP84-104) (filled bars). However, at this point in disease, the CNS APCs did not endogenously activate proliferation of a Th1 line specific for the less dominant PLP104-117 epitope. It is significant that these CNS APCs could both present exogenous peptide (hatched bars) and process and present the MP-4 fusion protein (gray bars), containing the 21.5-kDa isoform of MBP and a recombinant variant of human PLP (27, 28), to all of the different T cells. Thus, a variety of immunodominant epitopes within the major myelin proteins PLP and MBP are endogenously displayed by CNS APCs isolated from mice during the acute phase of PLP139-151–induced R-EAE, and these cells are highly efficient at processing encephalitogenic myelin determinants.

Analysis of specificity of presentation of endogenous myelin epitopes by CNS-resident APCs. To determine the specificity of endogenous antigen presentation, we compared the ability of CNS APCs from mice with TMEV-IDD (90 days after infection) to APCs from mice with PLP139-151–induced R-EAE to activate a Th1 line (sTV1) specific for the immunodominant TMEV VP2 70-86 epitope. As shown in Figure 3, irradiated splenocytes from naive mice were able to process and present ultraviolet-inactivated TMEV virions and present the VP2 70-86 peptide to the sTV1 line. CNS APCs from mice with both TMEV-IDD and R-EAE were also able to process efficiently intact virions and present peptide to the VP2 70-86–specific T-cell line. However, only CNS APCs from TMEV-infected mice were able to endogenously activate the virus-specific Th1 line. As expected, CNS APCs from mice with R-EAE, which had never been exposed to TMEV, did not induce proliferation of the virus-specific cells, indicating the specificity of the endogenous presentation.

Figure 3

Specificity of endogenous antigen presentation by CNS-resident APCs from the spinal cords of mice with ongoing TMEV-IDD and PLP139-151–induced R-EAE. Plastic-adherent spinal cord mononuclear cells were prepared from groups of SJL mice both 90 days after intracerebral infection with TMEV and 17 days after PLP139-151 immunization. A total of 10⁴ T cells from the sTV1 T-cell line, specific for TMEV VP2 70-86 epitope, were cultured with either 5 × 10⁴ irradiated (30 Gy) naive SJL splenocytes or 5 × 10⁴ irradiated CNS APCs, without the addition of antigen, with 5 μg of purified ultraviolet-inactivated TMEV virions, or with 25 μM of VP2 70-86 peptide. Cultures were pulsed with 1 μCi of [³H]TdR at 48 hours, and were harvested 24 hours thereafter. Values represent the mean cpm ± SEM of triplicate cultures. Stimulation indices are indicated above each bar. The background for both CNS-derived APC groups was the value obtained from the culture of the sTV1 T-cell line with CNS APCs from R-EAE mice in the absence of added antigen.

To ensure that the endogenous activation of virus- and myelin-specific T cells was due to preexisting peptide/I-A^s complexes on the cell surface of CNS APCs and not due to the uptake of myelin and/or viral fragments during cell isolation, we performed several control experiments. First, we dissociated spinal cords from naive mice in parallel with spinal cords from TMEV-infected donors. Before Percoll gradient separation, either naive splenocytes or splenocytes preactivated with LPS for 3 days in vitro were added to the naive spinal cord suspension. Figure 4 demonstrates that both peptide-pulsed splenocytes and CNS APCs isolated from TMEV-infected donors (85 days after infection) could activate a Th1 line specific for the PLP56-70 epitope. However, neither naive nor LPS-preactivated splenocytes mixed with dissociated spinal cords from normal mice and later reisolated on Percoll gradients were capable of activating the myelin peptide–specific T-cell line. Similar results were obtained with T cells specific for the immunodominant PLP139-151 epitope (data not shown). Thus, uptake of myelin proteins during APC isolation appears to be minimal. We also showed the endogenous presentation of myelin epitopes by CNS APCs from TMEV-infected mice was not diminished by the addition of the antigen-processing inhibitor leupeptin during the entire isolation procedure (data not shown).

Figure 4

Myelin epitopes are not generated during the isolation of CNS-resident APCs. Spinal cords from naive SJL mice and a group of SJL mice infected with TMEV 85 days previously were dissociated and treated with collagenase. The spinal cord suspension from the naive mice was split and mixed with either naive splenocytes or LPS-preactivated splenocytes at the ratio of 1 spleen equivalent per spinal cord. All 3 suspensions were separated on discontinuous Percoll gradients. The cells were incubated in plastic dishes for 1.5 hours, and the plastic-adherent cells were irradiated (30 Gy). A total of 2.8 × 10⁴ cells from a PLP56-70–specific T-cell line were cultured with the following: 3 × 10⁴ irradiated naive SJL splenocytes in the absence of added peptide (group A); 3 × 10⁴ irradiated naive SJL splenocytes + 50 μM PLP56-70 (group B–positive control); 3 × 10⁴ irradiated, plastic-adherent CNS APCs from TMEV-infected mice (group C); 3 × 10⁴ irradiated naive splenocytes recovered from Percoll gradients after admixture with the naive spinal cord suspension (group D); 3 × 10⁴ irradiated, LPS-preactivated splenocytes (group E); and 3 × 10⁴ irradiated, LPS-preactivated splenocytes recovered from Percoll gradients after admixture with the naive spinal cord suspension (group F). Cultures were pulsed with 1 μCi of [³H]TdR at 48 hours, and were harvested 16 hours thereafter. Values represent the mean cpm ± SEM of triplicate cultures. Stimulation indices are indicated above each bar, and were calculated using the cpm of the PLP56-70–specific T-cell line cultured with irradiated naive splenic APCs in the absence of peptide. *Naive and LPS-preactivated splenocytes in these groups were processed similarly to the CNS APCs.

Efficient endogenous presentation of myelin epitopes by CNS APCs from TMEV-infected mice requires preexistent myelin damage. It is logical to assume that loading of myelin peptides on APCs in the CNS of TMEV-infected mice would require prior myelin damage, whereas endogenous presentation of virus epitopes should be independent of the level of myelin destruction. We thus isolated CNS APCs from TMEV-infected mice that displayed minimal clinical and histological signs of disease (days 40–42 after infection). Unlike APCs derived from mice with severe clinical disease at 88–95 days after infection (Figures 2a, 3, and 4), APCs isolated from mice at onset of TMEV-induced disease (Figure 5a) stimulated vigorous proliferation (SI = 41) and IL-2 secretion (data not shown) of the sTV1 VP2 70-86–specific T-cell line but were incapable of activating a T-cell line specific for the immunodominant PLP139-151 epitope to either proliferate (SI = 1.5) or secrete IL-2. CNS APCs purified from TMEV-infected mice at 45–50 days after infection also failed to activate a PLP56-70–specific T cells (data not shown). This result indicates that a threshold level of myelin destruction must occur before CNS APCs can efficiently activate myelin-specific T cells. In addition, this result again indicates that there is little or no exogenous uptake of myelin proteins during isolation of the CNS APCs. These conclusions are supported by the fact that microglia isolated from the brains of naive mice (it was not possible to isolate sufficient cells from naive spinal cords) failed to endogenously activate either a PLP139-151–specific T-cell line (Figure 5b) or a VP2 70-86–specific T-cell hybridoma (Figure 5c). In contrast, naive microglia could present myelin and virus peptides in a dose-dependent manner, and were capable of processing the MP-4 fusion protein for presentation of PLP139-151.

Figure 5

Viral, but not myelin, epitopes are endogenously presented by CNS-resident APCs isolated from SJL mice at the onset of TMEV-IDD. (a) A total of 1.8 × 10⁴ plastic-adherent spinal cord mononuclear cells were prepared from SJL mice 42 days after intracerebral infection with TMEV. The CNS APCs were irradiated and cultured with either 3 × 10⁴ sTV1 T cells (specific for VP2 70-86) or the same number of T cells from a long-term PLP139-151–specific line. T-cell lines cultured with 1.8 × 10⁴ irradiated splenocytes + 50 μM of the appropriate peptide served as the positive control. (b) A total of 10⁴ to 10⁵ irradiated microglia isolated from the brains of naive SJL mice or irradiated naive splenocytes were cultured with 3 × 10⁴ T cells from a long-term PLP139-151–specific line in the presence or absence of 50 μM of PLP139-151 or 25 μg/mL of the MP-4 MBP/PLP fusion protein. (c) A total of 10⁴ to 10⁵ irradiated microglia isolated from the brains of naive SJL mice or irradiated naive splenocytes were cultured with 3 × 10⁴ cloned T-cell hybridoma cells specific for TMEV VP2 70-86 in the presence or absence of the peptide. For a and b, cultures were pulsed with 1 μCi of [³H]TdR at 48 hours, and were harvested 16–24 hours thereafter. For c, culture supernates were harvested after 48 hours and tested for their ability to support the proliferation of the IL-2–dependent CTLL-2 cell line. Values represent the mean cpm ± SEM of triplicate cultures. Stimulation indices are indicated above each bar.

Endogenous presentation of myelin epitopes by CNS APCs from TMEV-infected mice is B7 dependent and MHC class II restricted. The CD28/B7 costimulatory pathway is critical for T-cell activation, particularly activation of naive T cells (33). To characterize further the in vivo potential of APCs resident in the CNS of mice with ongoing TMEV-IDD to activate naive T cells specific for endogenous myelin epitopes, we examined the B7 dependency and MHC restriction of the CNS APCs. As seen in Figure 6a, endogenous presentation of myelin epitopes by CNS APCs from TMEV-infected mice is MHC class II restricted, as activation of a PLP139-151–specific T-cell line was specifically inhibited by a monoclonal anti–I-A^s antibody. The B7 dependence of the presentation is illustrated by data showing that T-cell activation is significantly inhibited both by the combination of anti–B7-1 and anti–B7-2 mAb’s (Figure 6b) and by murine CTLA4-Ig (Figure 6a), which blocks B7-1 and B7-2. A predominance of B7-2–mediated costimulation on the CNS APCs is suggested by the enhanced ability of anti–B7-2 (72% inhibition), compared with anti–B7-1 (44% inhibition), to block activation of the PLP139-151–specific Th1 line (Figure 6b).

Figure 6

Endogenous presentation of myelin epitopes by CNS-resident APCs isolated from SJL mice with ongoing TMEV-IDD is B7 dependent and MHC class II restricted. (a) A total of 5 × 10⁴ irradiated, plastic-adherent CNS APCs isolated from mice infected 115 days previously with TMEV were cultured with 3 × 10⁴ PLP139-151–specific T cells for 72 hours and pulsed with 1 μCi of [³H]TdR for the final 24 hours of culture. CNS APCs were incubated with 10 μg/mL of mouse control Ig, mCTLA-4 Ig, anti–I-A^d, or anti–I-A^s for 30 minutes before the addition of the T-cell line. (b) A total of 3 × 10⁴ irradiated, plastic-adherent CNS APCs isolated from mice infected 125 days previously with TMEV were cultured with 3 × 10⁴ PLP139-151–specific T cells for 72 hours and pulsed with 1 μCi of [³H]TdR for the final 24 hours of culture. CNS APCs were incubated with 10 μg/mL of hamster control Ig, anti–B7-1, anti–B7-2, or a combination of anti–B7-1 and anti–B7-2 for 30 minutes before the addition of the T-cell line. Values for both experiments represent the mean cpm ± SEM of triplicate cultures. Figures in parentheses above each bar indicate the percentage inhibition in comparison to the addition of control Ig. *Inhibition is statistically significant (P < 0.01).

Phenotypic characterization of CNS-resident mononuclear APCs. Flow cytometric analysis of the CNS-resident mononuclear cells isolated from the spinal cords of TMEV-infected SJL mice 90 days after infection on discontinuous Percoll gradients was carried out. In general, these preparations yielded between 5 × 10⁴ and 5 × 10⁵ total mononuclear cells per spinal cord. Approximately 50% of these cells bore the F4/80 marker (Figure 7a) and Mac1 (data not shown), which are specific macrophage/microglia lineage markers (34). The percentage of F4/80⁺/Mac1⁺ cells is enriched to 70–80% of the total lymphoid cells following the plastic-adherence step, with a usual yield of 1.25 × 10⁴ to 2.5 × 10⁴ cells per spinal cord (data not shown). These cells are large and vacuolated under the light microscope. The majority of CNS-resident F4/80⁺ cells (70–85%) express I-A^s (Figure 7b), B7-1 (Figure 7c), and B7-2 (Figure 7d). Based on mean fluorescence intensity, relatively more B7-2 than B7-1 molecules are expressed on the cell surface of the F4/80⁺ population. This correlates with the enhanced ability of anti–B7-2, compared with anti–B7-1, to inhibit endogenous activation of the PLP56-70–specific Th1 line by CNS APCs from TMEV-infected mice (Figure 6b). Our previous analyses (31) have shown that only 30% of F4/80⁺ cells from the spleen of TMEV-infected SJL mice express MHC class II, and that F4/80⁺ cells account for only 4% of the I-A^s+ cells.

Figure 7

Phenotypic characterization of CNS APCs isolated from SJL mice with ongoing TMEV-IDD. Spinal cord CNS mononuclear cells were isolated on discontinuous Percoll gradients from 25 SJL mice infected 102 days previously with TMEV. The cells were stained with anti–F4/80-FITC and anti–I-A^s-biotin, anti–B7-1-PE, anti–B7-2-PE, or anti–CD45-PE followed by A-PE (anti–I-A^s group only). Approximately 50% of the CNS mononuclear cells expressed F4/80 (a), and the remainder were CD3⁺ (data not shown). By electronic gating on the F4/80⁺ cells, it can be seen that 70–85% of the CNS-resident macrophage/microglial cells expressed I-A^s (b), B7-1 (c), and B7-2 (d). Approximately 90% of the F4/80⁺ cells were CD45⁺, with 60% bearing intermediate levels and 30% bearing high levels of this marker (e).

The CNS F4/80⁺ population could be separated into 2 subpopulations based on surface expression of the leukocyte common antigen CD45. Approximately 60% of these cells bear intermediate levels of CD45, whereas around 30% express high levels of CD45 (Figure 7e). Gating on the CD45⁺ subpopulations revealed that expression of both B7-1 and B7-2 was higher on the CD45^hi population than on the CD45^lo population. This is significant in that Ford et al. (35) have previously shown that the CD45^hi population represents perivascular macrophages in brain tissue that are functionally capable of presenting MBP to a specific T-cell line, whereas the CD45^lo population represents microglia that are relatively poor APCs. It is therefore of interest to compare the relative ability of sorted CD45^hi and CD45^lo populations to present endogenous myelin epitopes.

The initiation and progression of a number of autoimmune diseases, including MS (5, 36), are strongly suspected to arise secondary to virus infections. Therefore, determining the mechanisms by which infections lead to autoimmune sequelae is an area of great interest, and may provide important clues about the pathogenesis and regulation of autoimmune diseases. Molecular mimicry has been postulated to be a prime mechanism whereby infections can lead to the initiation of T-cell autoreactivity (6, 37–39), but in general there has been a paucity of direct evidence supporting this hypothesis. Alternatively, epitope spreading — the process whereby epitopes distinct from, and non–cross-reactive with, an inducing epitope become major targets of an ongoing immune response — has been implicated in the pathogenesis of both human autoimmune diseases and their experimental models. In animal models of autoimmunity, there is mounting evidence that chronic inflammatory-mediated tissue damage can lead to de novo activation of autoreactivity via epitope spreading. The primary examples of epitope spreading have been in T cell–mediated autoimmune models, such as R-EAE (13, 40, 41) and diabetes in nonobese diabetic mice (42–44). Epitope spreading has also been demonstrated after infection with the picornaviruses, TMEV (14), and Coxsackie virus (45). Recent evidence has suggested that local expression of proinflammatory cytokines such as TNF-α promotes autoimmunity by enhancing presentation of autoantigens (46). Although epitope spreading has generally been described as a Th1 phenomenon, recent evidence shows that Th2 epitope spreading may serve as an intrinsic negative feedback mechanism to regulate autoimmune pathology (47). Epitope spreading has also been described at the B-cell level in systemic lupus erythematosis, which is mediated by pathogenic autoantibodies (48, 49). Although there is yet no conclusive proof that epitope spreading is a general feature in human autoimmune diseases, recent evidence suggests that it may play a role in autoimmune skin diseases (50) and in the chronic-progressive course of MS (51).

Our laboratory has used the R-EAE and TMEV-IDD models of CD4⁺ T cell–mediated CNS demyelination to study the functional significance of T-cell responses specific for endogenous myelin epitopes that arise during the chronic course of CNS damage in these 2 diseases. R-EAE in the SJL mouse is a Th1-mediated autoimmune demyelinating disease, useful in dissecting the immune response to chronic tissue damage because disease can be induced in a peptide-specific manner, and the identity and relative dominance of encephalitogenic epitopes on a variety of myelin proteins, including MBP, PLP, and MOG, have been well defined. Both intra- and intermolecular epitope spreading play important pathologic roles in the progression of ongoing autoimmune disease in R-EAE, because blockade of T-cell reactivity to the relapse-associated myelin epitopes, either by costimulatory antagonists (52, 53) or by antigen-specific tolerance (13, 41, 54) (C.L. Vanderlugt et al., unpublished study), results in inhibition of clinical relapses. Myelin damage in TMEV-infected SJL mice is initiated by TMEV-specific CD4⁺ T cells targeting virus persisting in CNS-resident APCs, leading to upregulation of proinflammatory cytokines in the CNS (22–26, 55). Similar to R-EAE, the chronic stage of TMEV-IDD is associated with the activation of CD4⁺ myelin epitope–specific T cells primed by epitope spreading, as there are no apparent virus epitopes that are shared with the encephalitogenic myelin epitopes on PLP, MBP, or MOG (14).

There are a variety of cells within the normal CNS with antigen presentation potential, including astrocytes, microglia, and macrophages. IFN-γ–treated primary astrocytes (56, 57) and microglia (58, 59) cultured from neonatal mouse brain upregulate MHC class II and can present antigens to T cells in vitro. It is important to realize that antigen presentation by neonatal cells in long-term culture may not faithfully reproduce the in vivo state in adult animals. Microglia directly isolated from adult rats can more efficiently present MBP to T-cell lines in vitro than can neonatally derived microglia (35), but they are inefficient in endogenously activating myelin-specific T cells (Figure 5b). Studies using allogeneic bone marrow chimeras have supported the idea that cells of hematopoietic origin (i.e., microglia and macrophages) are the principal APCs active in the CNS during the initiation of EAE (60–62). Although they are much more abundant than microglia, astrocytes are significantly less potent when inducing EAE in chimeras (62).

How and where do T cells specific for endogenous myelin epitopes become activated during the initiation and progression of R-EAE and TMEV-IDD? It is possible that T cells specific for relapse-associated epitopes are activated in the peripheral immune organs. After inflammatory disruption of the blood-brain barrier, myelin debris and/or macrophages/microglia that have ingested myelin proteins within the CNS may gain access to the cervical lymph nodes, which drain the cerebrospinal fluid (63), or to the spleen, which concentrates blood-borne material. In support of the idea that APCs can traffic from the CNS to peripheral lymphoid organs, it has been reported that donor cells from alloantigen-disparate solid CNS grafts placed intracerebrally can be later identified in the host spleen and lymph nodes (64). It is also possible that T cells specific for endogenous myelin epitopes are activated in the local inflammatory environment within the CNS. In both R-EAE and TMEV-IDD, the lymphocytic infiltrate is composed of numerous I-A^s+, B7⁺-activated microglia, and macrophages, derived from both the CNS-resident pool and macrophages or monocytes infiltrating from the peripheral blood (31, 32, 52). Macrophages/microglia within the demyelinated areas also contain phagocytized myelin debris (65). Considered together with the fact that the majority of T cells infiltrating the CNS during immune-mediated demyelinating diseases are thought to be naive “bystander” cells that are not specific for the inducing antigen, the CNS theoretically provides an ideal environment for the activation of naive autoreactive CD4⁺ T cells.

In the present report, we examined the potential presentation of viral and myelin epitopes within the CNS of mice with ongoing T cell–mediated demyelinating disease. After a recent report in which we demonstrated endogenous presentation of an immunodominant viral epitope by CNS APCs from TMEV-infected mice (31), we asked whether myelin destruction initiated by TMEV-specific CD4⁺ T cells would also lead to the uptake, processing, and presentation of myelin epitopes. The current results clearly indicate that a variety of self myelin epitopes are endogenously processed and displayed in the context of MHC class II molecules on the surface of plastic-adherent, F4/80⁺, I-A^s+ mononuclear APCs isolated from the CNS of SJL mice with preexisting myelin damage (Figure 2a), but not from naive mice (Figure 5, b and c) or mice in the initial stages of TMEV-IDD (Figure 5a). In contrast, TMEV epitopes were associated with CNS-resident mononuclear APCs both before the onset of clinical disease and in mice with extensive myelin damage (85–120 days after TMEV infection) (Figure 2a), supporting previous findings that TMEV persist long term in macrophages/microglia in the CNS (15). The temporal difference in the availability of TMEV versus myelin epitopes indicates a lack of cross-reactivity between viral and self myelin epitopes. This lack of cross-reactivity is supported by the failure of APCs from mice with R-EAE to activate a TMEV VP2-specific T-cell line (Figure 3), and by our previous report showing that T-cell clones and lymph node T cells specific for immunodominant TMEV and myelin epitopes do not cross-react (14).

The phenotypic characteristics of the cells presenting endogenous myelin epitopes (i.e., F4/80⁺, MHC class II⁺, B7-1⁺, B7-2⁺; Figure 7) are characteristic of activated macrophages and microglia. This conclusion is also supported by the bimodal distribution of the leukocyte common antigen CD45, expressed on the APCs (Figure 7). Previous studies have indicated that naive CNS-resident microglia express low levels of CD45, whereas activated microglia express intermediate levels of this marker. Both naive and activated parenchymal microglia are relatively poor APCs, as determined by induction of T-cell proliferation, but they are more efficient in stimulating Th1 differentiation, as assessed by IFN-γ production. In contrast, CD45^hi perivascular microglia and infiltrating macrophages have been shown to be highly efficient APCs (35, 66, 67). Our finding that the CNS APCs capable of efficient presentation of endogenous myelin epitopes were of the macrophage/microglia lineage is consistent with previous studies. Using immunohistochemistry, we showed that MHC class II was predominantly expressed on macrophages/microglia in spinal cord demyelinating lesions in TMEV-infected mice, and that the majority of cells in these lesions were sialoadhesin-bearing infiltrating macrophages (31). Macrophages/microglia are the cells that predominantly harbor persistent TMEV infection (15–17), and the number of F4/80⁺ cells increases to 2- to 3-fold the number of CD4⁺ T cells in spinal cords 50–60 days or more after infection (55), around the time when endogenous myelin epitopes are functionally expressed (Figure 1a). In addition, these MHC class II–bearing cells expressed B7-1 and B7-2 at levels exceeding those in the spleens of infected mice (31). B7-1 was expressed predominantly on infiltrating macrophages, whereas B7-2 was expressed on both F4/80⁺ macrophages/microglia and a subpopulation of CD4⁺ T cells. Thus, CNS-resident mononuclear APCs present virus and myelin epitopes, and express the requisite costimulatory molecules required for activation of naive myelin epitope–specific CD4⁺ T cells.

Comparison of MHC class II and B7 expression of CNS-resident APCs between TMEV-IDD and R-EAE is important in relation to our data on the manipulation of B7 molecules in vivo during R-EAE in the SJL mouse (52). The current functional (Figure 6) and FACS (Figure 7) analyses indicate that both B7-1– and B7-2–mediated costimulatory processes are important on CNS-resident F4/80⁺ cells isolated from SJL mice with ongoing TMEV-IDD. In contrast, our recent studies have shown that that B7-1 becomes the dominant costimulatory molecule in SJL mice with ongoing R-EAE, as its surface expression level and functional role in T-cell activation are significantly increased relative to B7-2 both in the CNS and in the peripheral lymphoid tissues (32, 52). Moreover, blockade of B7-1 by treatment of SJL mice with anti–B7-1 F(ab) fragments during R-EAE remission inhibited subsequent disease relapses by preventing activation of T cells specific for endogenous myelin epitopes (13, 52). Thus, the costimulatory dependence of epitope spreading differs in these 2 models of T cell–mediated demyelination.

For multiple reasons, the finding that CNS mononuclear APCs from TMEV-infected mice contain and present viral and myelin antigens to T cells ex vivo is significant to understanding the pathogenesis of MS. First, MHC class II–bearing macrophages, astrocytes, and endothelial cells have been observed in lesions from patients with MS (68–70). Expression of B7 costimulatory molecules has also been demonstrated in MS lesions (71–73). Therefore, multiple cells in MS lesions are equipped to activate fully both naive and memory T cells within the CNS. Second, IL-2R–bearing cells have been observed in the lesions (68), as have products of activated T cells, including IFN-γ (74) and IL-2 (68), further suggesting local antigen presentation. Third, the epidemiology of MS strongly suggests a role for an infectious agent, perhaps a virus, that is widespread, chronic, and usually subclinical (5). Presentation within the CNS of viral antigens (leading to bystander demyelination), of neuroantigens cross-reactive with viral antigens (molecular mimicry), or of neuroantigens liberated by virus-induced CNS damage (epitope spreading) are all possible mechanisms by which pathogenic immune reactions could be initiated by viruses within the CNS. Our results showing the temporal availability of first, virus epitopes, and later, both virus and myelin epitopes, on CNS APCs support a chronic CNS infection, such as that observed in SJL mice infected with TMEV, as a plausible initiating event in the autoimmune pathogenesis of MS.

This work was supported by the United States Public Health Service/National Institutes of Health (grants NS23349, NS26543, and NS34819) and a by National Multiple Sclerosis Society Postdoctoral Fellowship (to Y. Katz-Levy).

Yael Katz-Levy and Katherine L. Neville contributed equally to this work.

1. Wekerle, H. Experimental autoimmune encephalomyelitis as a model of immune-mediated CNS disease. Curr Opin Neurobiol 1993. 3:779-784.
   View this article via: PubMed CrossRef Google Scholar
2. Ota, K, et al. T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature 1990. 346:183-187.
   View this article via: PubMed CrossRef Google Scholar
3. Bernard, CC, de Rosbo, NK. Immunopathological recognition of autoantigens in multiple sclerosis. Acta Neurol (Napoli) 1991. 13:171-178.
   View this article via: PubMed Google Scholar
4. de Rosbo, NK, et al. Predominance of the autoimmune response to myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis: reactivity to the extracellular domain of MOG is directed against three main regions. Eur J Immunol 1997. 27:3059-3069.
   View this article via: PubMed CrossRef Google Scholar
5. Kurtzke, JF. Epidemiologic evidence for multiple sclerosis as an infection. Clin Microbiol Rev 1993. 6:382-427.
   View this article via: PubMed Google Scholar
6. Fujinami, RS, Oldstone, MB. Amino acid homology between the encephalitogenic site of myelin basic protein and virus: mechanism for autoimmunity. Science 1985. 230:1043-1045.
   View this article via: PubMed CrossRef Google Scholar
7. Scherer, MT, Ignatowicz, L, Winslow, GM, Kappler, JW, Marrack, P. Superantigens: bacterial and viral proteins that manipulate the immune system. Annu Rev Cell Biol 1993. 9:101-128.
   View this article via: PubMed CrossRef Google Scholar
8. Miller, SD, Gerety, SJ. Immunologic aspects of Theiler’s murine encephalomyelitis virus (TMEV)–induced demyelinating disease. Semin Virol 1990. 1:263-272.
9. Miller, SD, et al. Evolution of the T cell repertoire during the course of experimental autoimmune encephalomyelitis. Immunol Rev 1995. 144:225-244.
   View this article via: PubMed CrossRef Google Scholar
10. Vanderlugt, CL, Miller, SD. Epitope spreading. Curr Opin Immunol 1996. 8:831-836.
    View this article via: PubMed CrossRef Google Scholar
11. Vanderlugt, CL, Karandikar, NJ, Bluestone, JA, Miller, SD. The functional significance of epitope spreading and its regulation by costimulatory interactions. Immunol Rev 1998. 164:63-72.
    View this article via: PubMed CrossRef Google Scholar
12. McRae, BL, Kennedy, MK, Tan, LJ, Dal Canto, MC, Miller, SD. Induction of active and adoptive chronic-relapsing experimental autoimmune encephalomyelitis (EAE) using an encephalitogenic epitope of proteolipid protein. J Neuroimmunol 1992. 38:229-240.
    View this article via: PubMed CrossRef Google Scholar
13. McRae, BL, Vanderlugt, CL, Dal Canto, MC, Miller, SD. Functional evidence for epitope spreading in the relapsing pathology of experimental autoimmune encephalomyelitis. J Exp Med 1995. 182:75-85.
    View this article via: PubMed CrossRef Google Scholar
14. Miller, SD, et al. Persistent infection with Theiler’s virus leads to CNS autoimmunity via epitope spreading. Nat Med 1997. 3:1133-1136.
    View this article via: PubMed CrossRef Google Scholar
15. Lipton, HL, Kratochvil, J, Sethi, P, Dal Canto, MC. Theiler’s virus antigen detected in mouse spinal cord 2 1/2 years after infection. Neurology 1984. 34:1117-1119.
    View this article via: PubMed Google Scholar
16. Clatch, RJ, Miller, SD, Metzner, R, Dal Canto, MC, Lipton, HL. Monocytes/macrophages isolated from the mouse central nervous system contain infectious Theiler’s murine encephalomyelitis virus (TMEV). Virology 1990. 176:244-254.
    View this article via: PubMed CrossRef Google Scholar
17. Lipton, HL, Twaddle, G, Jelachich, ML. The predominant virus antigen burden is present in macrophages in Theiler’s murine encephalomyelitis virus-induced demyelinating disease. J Virol 1995. 69:2525-2533.
    View this article via: PubMed Google Scholar
18. Lipton, HL, Gonzalez-Scarano, F. Central nervous system immunity in mice infected with Theiler’s virus. I. Local neutralizing antibody response. J Infect Dis 1978. 137:145-151.
    View this article via: PubMed Google Scholar
19. Dal Canto, MC, Lipton, HL. Ultrastructural immunohistochemical localization of virus in acute and chronic demyelinating Theiler’s virus infection. Am J Pathol 1982. 106:20-29.
    View this article via: PubMed Google Scholar
20. Dal Canto, MC, Barbano, RL. Immunocytochemical localization of MAG, MBP and P0 protein in acute and relapsing demyelinating lesions of Theiler’s virus infection. J Neuroimmunol 1985. 10:129-140.
    View this article via: PubMed CrossRef Google Scholar
21. Lipton, HL, Dal Canto, MC. Susceptibility of inbred mice to chronic central nervous system infection by Theiler’s murine encephalomyelitis virus. Infect Immun 1979. 26:369-374.
    View this article via: PubMed Google Scholar
22. Miller, SD, et al. Class II–restricted T cell responses in Theiler’s murine encephalomyelitis virus (TMEV)–induced demyelinating disease. III. Failure of neuroantigen-specific immune tolerance to affect the clinical course of demyelination. J Neuroimmunol 1990. 26:9-23.
    View this article via: PubMed CrossRef Google Scholar
23. Miller, SD, Clatch, RJ, Pevear, DC, Trotter, JL, Lipton, HL. Class II–restricted T cell responses in Theiler’s murine encephalomyelitis virus (TMEV)–induced demyelinating disease. I. Cross-specificity among TMEV substrains and related picornaviruses, but not myelin proteins. J Immunol 1987. 138:3776-3784.
    View this article via: PubMed Google Scholar
24. Gerety, SJ, Rundell, MK, Dal Canto, MC, Miller, SD. Class II–restricted T cell responses in Theiler’s murine encephalomyelitis virus (TMEV)–induced demyelinating disease. VI. Potentiation of demyelination with and characterization of an immunopathologic CD4⁺ T cell line specific for an immunodominant VP2 epitope. J Immunol 1994. 152:919-929.
    View this article via: PubMed Google Scholar
25. Karpus, WJ, Pope, JG, Peterson, JD, Dal Canto, MC, Miller, SD. Inhibition of Theiler’s virus–mediated demyelination by peripheral immune tolerance induction. J Immunol 1995. 155:947-957.
    View this article via: PubMed Google Scholar
26. Pope, JG, Karpus, WJ, Vanderlugt, CL, Miller, SD. Flow cytometric and functional analyses of CNS-infiltrating cells in SJL/J mice with Theiler’s virus–induced demyelinating disease: evidence for a CD4⁺ T cell–mediated pathology. J Immunol 1996. 156:4050-4058.
    View this article via: PubMed Google Scholar
27. Elliott, EA, et al. Immune tolerance mediated by recombinant proteolipid protein prevents experimental autoimmune enceph-alomyelitis. J Neuroimmunol 1997. 79:1-11.
    View this article via: PubMed CrossRef Google Scholar
28. Elliott, EA, et al. Treatment of experimental encephalomyelitis with a novel chimeric fusion protein of myelin basic protein and proteolipid protein. J Clin Invest 1996. 98:1602-1612.
    View this article via: JCI PubMed Google Scholar
29. Lipton, HL, Melvold, R. Genetic analysis of susceptibility to Theiler’s virus–induced demyelinating disease in mice. J Immunol 1984. 132:1821-1825.
    View this article via: PubMed Google Scholar
30. Lipton, HL, Dal Canto, MC. Chronic neurologic disease in Theiler’s virus infection of SJL/J mice. J Neurol Sci 1976. 30:201-207.
    View this article via: PubMed CrossRef Google Scholar
31. Pope, JG, Vanderlugt, CL, Lipton, HL, Rahbe, SM, Miller, SD. Characterization of and functional antigen presentation by central nervous system mononuclear cells from mice infected with Theiler’s murine encephalomyelitis virus. J Virol 1998. 72:7762-7771.
    View this article via: PubMed Google Scholar
32. Karandikar, NJ, et al. Tissue-specific up-regulation of B7-1 expression and function during the course of murine relapsing experimental autoimmune encephalomyelitis. J Immunol 1998. 161:192-199.
    View this article via: PubMed Google Scholar
33. Lenschow, DJ, Walunas, TL, Bluestone, JA. CD28/B7 system of T cell costimulation. Annu Rev Immunol 1996. 14:233-258.
    View this article via: PubMed CrossRef Google Scholar
34. Perry, VH, Hume, DA, Gordon, S. Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain. Neuroscience 1985. 15:313-326.
    View this article via: PubMed CrossRef Google Scholar
35. Ford, AL, Goodsall, AL, Hickey, WF, Sedgwick, JD. Normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting. Phenotypic differences defined and direct ex vivo antigen presentation to myelin basic protein-reactive CD4+ T cells compared. J Immunol 1995. 154:4309-4321.
    View this article via: PubMed Google Scholar
36. Ebers, GC, Sadovnick, AD. The geographic distribution of multiple sclerosis: a review. Neuroepidemiology 1993. 12:1-5.
    View this article via: PubMed CrossRef Google Scholar
37. Wucherpfennig, KW, Strominger, JL. Molecular mimicry in T cell-mediated autoimmunity: viral peptides activate human T cell clones specific for myelin basic protein. Cell 1995. 80:695-705.
    View this article via: PubMed CrossRef Google Scholar
38. Zhao, Z-S, Granucci, F, Yeh, L, Schaffer, PA, Cantor, H. Molecular mimicry by herpes simplex virus-type 1: autoimmune disease after viral infection. Science 1998. 279:1344-1347.
    View this article via: PubMed CrossRef Google Scholar
39. Ufret-Vincenty, RL, et al. In vivo survival of viral antigen-specific T cells that induce experimental autoimmune encephalomyelitis. J Exp Med 1998. 188:1725-1738.
    View this article via: PubMed CrossRef Google Scholar
40. Lehmann, PV, Forsthuber, T, Miller, A, Sercarz, EE. Spreading of T-cell autoimmunity to cryptic determinants of an autoantigen. Nature 1992. 358:155-157.
    View this article via: PubMed CrossRef Google Scholar
41. Yu, M, Johnson, JM, Tuohy, VK. A predictable sequential determinant spreading cascade invariably accompanies progression of experimental autoimmune encephalomyelitis: a basis for peptide-specific therapy after onset of clinical disease. J Exp Med 1996. 183:1777-1788.
    View this article via: PubMed CrossRef Google Scholar
42. Tisch, R, et al. Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature 1993. 366:72-75.
    View this article via: PubMed CrossRef Google Scholar
43. Kaufman, DL, et al. Spontaneous loss of T cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature 1993. 366:69-72.
    View this article via: PubMed CrossRef Google Scholar
44. Tian, J, et al. Nasal administration of glutamate decarboxylase (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. J Exp Med 1996. 183:1561-1567.
    View this article via: PubMed CrossRef Google Scholar
45. Horwitz, MS, et al. Diabetes induced by Coxsackie virus: initiation by bystander damage and not molecular mimicry. Nat Med 1998. 4:781-786.
    View this article via: PubMed CrossRef Google Scholar
46. Green, EA, Eynon, EE, Flavell, RA. Local expression of TNFα in neonatal NOD mice promotes diabetes by enhancing presentation of islet antigens. Immunity 1998. 9:733-743.
    View this article via: PubMed CrossRef Google Scholar
47. Tian, J, Lehmann, PV, Kaufman, DL. Determinant spreading of T helper cell 2 (Th2) responses to pancreatic islet autoantigens. J Exp Med 1997. 186:2039-2043.
    View this article via: PubMed CrossRef Google Scholar
48. James, JA, Gross, T, Scofield, RH, Harley, JB. Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B′-derived PGMRPP and PGIRGP induce spliceosome autoimmunity. J Exp Med 1995. 181:453-461.
    View this article via: PubMed CrossRef Google Scholar
49. Topfer, F, Gordon, T, McCluskey, J. Intra- and intermolecular spreading of autoimmunity involving the nuclear self-antigens La (SS-B) and Ro (SS-A). Proc Natl Acad Sci USA 1995. 92:875-879.
    View this article via: PubMed CrossRef Google Scholar
50. Chan, LS, et al. Epitope spreading: lessons from autoimmune skin diseases. J Invest Dermatol 1998. 110:103-109.
    View this article via: PubMed CrossRef Google Scholar
51. Tuohy, VK, Yu, M, Weinstock-Guttman, B, Kinkel, RP. Diversity and plasticity of self recognition during the development of multiple sclerosis. J Clin Invest 1997. 99:1682-1690.
    View this article via: JCI PubMed Google Scholar
52. Miller, SD, et al. Blockade of CD28/B7-1 interaction prevents epitope spreading and clinical relapses of murine EAE. Immunity 1995. 3:739-745.
    View this article via: PubMed CrossRef Google Scholar
53. Howard, LM, et al. Mechanisms of immunotherapeutic intervention by anti-CD40L (CD154) antibody in an animal model of multiple sclerosis. J Clin Invest 1999. 103:281-290.
    View this article via: JCI PubMed Google Scholar
54. Tan, LJ, Kennedy, MK, Miller, SD. Regulation of the effector stages of experimental autoimmune encephalomyelitis via neuroantigen-specific tolerance induction. II. Fine specificity of effector T cell inhibition. J Immunol 1992. 148:2748-2755.
    View this article via: PubMed Google Scholar
55. Smith Begolka, W., Vanderlugt, C.L., Rahbe, S.M., and Miller, S.D. 1998. Differential expression of inflammatory cytokines parallels progression of CNS pathology in two clinically distinct models of MS. J. Immunol. 4437–4446.
    View this article via: PubMed Google Scholar
56. Fontana, A, Fierz, W, Wekerle, H. Astrocytes present myelin basic protein to encephalitogenic T-cell lines. Nature 1984. 307:273-276.
    View this article via: PubMed CrossRef Google Scholar
57. Tan, LJ, Gordon, KB, Mueller, JP, Matis, LA, Miller, SD. Presentation of proteolipid protein epitopes and B7-1-dependent activation of encephalitogenic T cells by IFN-gamma-activated SJL/J astrocytes. J Immunol 1998. 160:4271-4279.
    View this article via: PubMed Google Scholar
58. Frei, K, et al. Antigen presentation and tumor cytotoxicity by interferon-gamma–treated microglial cells. Eur J Immunol 1987. 17:1271-1278.
    View this article via: PubMed CrossRef Google Scholar
59. Cash, E, Rott, O. Microglial cells qualify as the stimulators of unprimed CD4⁺ and CD8⁺ T lymphocytes in the central nervous system. Clin Exp Immunol 1994. 98:313-318.
    View this article via: PubMed Google Scholar
60. Hinrichs, DJ, Wegmann, KW, Dietsch, GN. Transfer of experimental allergic encephalomyelitis to bone marrow chimeras. Endothelial cells are not a restricting element. J Exp Med 1987. 166:1906-1911.
    View this article via: PubMed CrossRef Google Scholar
61. Hickey, WF, Kimura, H. Perivascular microglial cells of the CNS are bone marrow–derived and present antigen in vivo. Science 1988. 239:290-292.
    View this article via: PubMed CrossRef Google Scholar
62. Myers, KJ, Dougherty, JP, Ron, Y. In vivo antigen presentation by both brain parenchymal cells and hematopoietically derived cells during the induction of experimental autoimmune encephalomyelitis. J Immunol 1993. 151:2252-2260.
    View this article via: PubMed Google Scholar
63. Cserr, HF, Knopf, PM. Cervical lymphatics, the blood-brain barrier and the immunoreactivity of the brain: a new view. Immunol Today 1992. 13:507-512.
    View this article via: PubMed CrossRef Google Scholar
64. Broadwell, RD, Baker, BJ, Ebert, PS, Hickey, WF. Allografts of CNS tissue possess a blood-brain barrier. III. Neuropathological, methodological, and immunological considerations. Microsc Res Tech 1994. 27:471-494.
    View this article via: PubMed CrossRef Google Scholar
65. Dal Canto, MC, Melvold, RW, Kim, BS, Miller, SD. Two models of multiple sclerosis: experimental allergic encephalomyelitis (EAE) and Theiler’s murine encephalomyelitis virus (TMEV) infection: a pathological and immunological comparison. Microsc Res Tech 1995. 32:215-229.
    View this article via: PubMed CrossRef Google Scholar
66. Carson, M.J., Sutcliffe, J.G., and Campbell, I.L. 1999. Microglia stimulate naive T-cell differentiation without stimulating T-cell proliferation. J. Neurosci. Res. In press.
    View this article via: PubMed Google Scholar
67. Aloisi, F, Ria, F, Penna, G, Adorini, L. Microglia are more efficient than astrocytes in antigen processing and in Th1 but not Th2 activation. J Immunol 1998. 160:4671-4680.
    View this article via: PubMed Google Scholar
68. Hofman, FM, et al. Immunoregulatory molecules and IL 2 receptors identified in multiple sclerosis brain. J Immunol 1986. 136:3239-3245.
    View this article via: PubMed Google Scholar
69. Traugott, U, Reinherz, EL, Raine, CS. Multiple sclerosis: distribution of T cell subsets within active chronic lesions. Science 1983. 219:308-310.
    View this article via: PubMed CrossRef Google Scholar
70. Traugott, U. Multiple sclerosis: relevance of class I and class II MHC-expressing cells to lesion development. J Neuroimmunol 1987. 16:283-302.
    View this article via: PubMed CrossRef Google Scholar
71. Windhagen, A, et al. Expression of costimulatory molecules B7-1 (CD80), B7-2 (CD80), and interleukin 12 cytokine in multiple sclerosis lesions. J Exp Med 1995. 182:1985-1996.
    View this article via: PubMed CrossRef Google Scholar
72. De Simone, R, et al. The costimulatory molecule B7 is expressed on human microglia in culture and in multiple sclerosis acute lesions. J Neuropathol Exp Neurol 1995. 54:175-187.
    View this article via: PubMed Google Scholar
73. Williams, K, Ulvestad, E, Antel, JP. B7/BB-1 antigen expression on adult human microglia studied in vitro and in situ. Eur J Immunol 1994. 24:3031-3037.
    View this article via: PubMed CrossRef Google Scholar
74. Traugott, U, Lebon, P. Demonstration of alpha, beta, and gamma interferon in active chronic multiple sclerosis lesions. Ann NY Acad Sci 1988. 540:309-311.
    View this article via: PubMed CrossRef Google Scholar