Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.     

Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis.

Relapsing experimental autoimmune encephalomyelitis (R-EAE) can be induced in SJL/J mice by immunization with spinal cord homogenate and adjuvant. The specific Ag(s) responsible for acute disease and subsequent relapses in this model is unknown. Myelin basic protein (BP), an encephalitogenic peptide of BP (BP 87-99), and proteolipid protein (PLP) can each induce R-EAE in SJL/J mice, and a peptide of PLP (PLP 139-151) has been reported to induce acute EAE. To determine the encephalitogens in cord-immunized mice with R-EAE, the in vitro proliferative responses of lymph node cells (LNC) and central nervous system mononuclear cells to BP, BP peptides, and PLP peptides were examined during acute EAE and during relapses. LNC responded only to PLP peptides 139-151 and 141-151 and did not respond to BP or its peptides during acute or chronic disease. Central nervous system mononuclear cells also preferentially responded to PLP 139-151 and 141-151 during acute and relapsing disease. A PLP 139-151 peptide-specific Th cell line was selected from LNC of cord-immunized donors. Five million peptide-specific line cells transferred severe relapsing demyelinating EAE to naive recipients. We conclude that PLP peptide 139-151 is the major encephalitogen for R-EAE in cord-immunized SJL/J mice. We demonstrate for the first time that Th cells specific for this peptide are sufficient to transfer relapsing demyelinating EAE. The predominance of a PLP immune response rather than a BP response in SJL/J mice suggests that genetic background may determine the predominant myelin Ag response in human demyelinating diseases such as multiple sclerosis.     

Identification and characterization of a second encephalitogenic determinant of myelin proteolipid protein (residues 178-191) for SJL mice.

We previously described a synthetic peptide of myelin proteolipid protein (PLP), peptide 139-151, which induces experimental allergic encephalomyelitis in SJL/J (H-2s) mice. We have now identified an additional determinant, PLP residues 178-191, that is also a potent encephalitogen in this strain. When PLP peptide 178-191 was compared with peptide 139-151 on an equimolar basis, the day of onset of disease induced by PLP 178-191 was earlier, but the incidence, severity, and histologic features were indistinguishable. Lymph node cells from animals immunized with the whole PLP molecule responded to both PLP 178-191 and 139-151, suggesting immunologic codominance of the two epitopes. PLP 178-191 elicited stronger proliferative responses and this may relate to the earlier onset of disease induced with this peptide. Two CD4+, peptide-specific, I-A(s)-restricted T cell lines, selected by stimulation of lymph node cells with either PLP 178-191 or 139-151, were each encephalitogenic in naive syngeneic mice. The presence of multiple encephalitogenic codominant PLP epitopes within a single mouse strain emphasizes the complexity of the immune response to PLP and its potential as a target Ag in autoimmune demyelinating diseases.     

Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis

Chronic progression of two T cell-mediated central nervous system (CNS) demyelinating models of multiple sclerosis, relapsing EAE (R-EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is dependent on the activation of T cells to endogenous myelin epitopes (epitope spreading). Using transfer of carboxyfluorescein succinyl ester (CFSE)-labeled T-cell receptor (TCR)-transgenic T cells and mixed bone marrow chimeras, we show that activation of naive proteolipid protein (PLP)139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE or TMEV-IDD occurs directly in the CNS and not in the cervical lymph nodes or other peripheral lymphoid organs. Examination of the antigen-presentation capacity of antigen-presenting cell (APC) populations purified from the CNS of mice with PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi dendritic cells (DCs) efficiently present endogenous antigen to activate naive PLP139-151-specific T cells in vitro. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia activate a PLP139-151-specific helper T cell line. The data suggest that naive T cells enter the inflamed CNS and are activated by local APCs, possibly DCs, to initiate epitope spreading.     

Frequencies of neuroantigen-specific T cells in the central nervous system versus the immune periphery during the course of experimental allergic encephalomyelitis

Direct measurements of the frequency and the cytokine signature of the neuroantigen-specific effector cells in experimental allergic encephalomyelitis (EAE) are a continuing challenge. This is true for lymphoid tissues, and more importantly, for the CNS itself. Using enzyme-linked immunospot analysis (ELISPOT) assays, we followed proteolipid protein (PLP) 139--151-specific T cells engaged by active immunization of SJL mice. The total numbers of PLP(139--151)-specific CD4 cells were highest before disease onset. At this time, these cells resided in lymphoid and nonlymphoid tissues, but were not detected in the CNS. While the PLP(139--151)-specific cells reached high frequencies in the CNS during clinical EAE, in absolute numbers, less than 20% of them were present in the target organ, with the majority residing in the periphery throughout all stages of the disease. The numbers of PLP(139--151)-specific cells gradually declined in both compartments with time. While eventually this first wave of effector cells completely disappeared from the CNS, PLP(178--191)-specific cells became engaged, being detected first in the CNS. These data suggest that throughout all stages of EAE, the effector cells in the CNS are recruited from a vast peripheral reservoir, and that the second wave of effector cells is engaged while the first wave undergoes exhaustion.     

Amelioration of established experimental autoimmune encephalomyelitis by an MHC anchor-substituted variant of proteolipid protein 139-151

Murine experimental autoimmune encephalomyelitis (EAE) is a CD4+ T cell-mediated autoimmune disorder directed against myelin proteins within the CNS. We propose that variant peptides containing amino acid substitutions at MHC anchor residues will provide a unique means to controlling the polyclonal autoimmune T cell response. In this study, we have identified an MHC variant of proteolipid protein (PLP) 139-151 (145D) that renders PLP(139-151)-specific T cell lines anergic in vitro, as defined by a significant reduction in proliferation and IL-2 production following challenge with wild-type peptide. In vivo administration of 145D before challenge with PLP(139-151) results in a significant reduction in disease severity and incidence. Importantly, we demonstrate the ability of an MHC variant peptide to ameliorate established EAE. An advantage to this treatment is that the MHC variant peptide does not induce an acute hypersensitivity reaction. This is in contrast to previous work in the PLP(139-151) model demonstrating that anaphylactic shock resulting in death occurs upon rechallenge with the encephalitogenic peptide. Taken together, these data demonstrate the effectiveness of MHC anchor-substituted peptides in the treatment of EAE and suggest their utility in the treatment of other autoimmune disorders.     

Kinetics and organ distribution of IL-17-producing CD4 cells in proteolipid protein 139-151 peptide-induced experimental autoimmune encephalomyelitis of SJL mice

In experimental autoimmune encephalomyelitis (EAE), the production of proinflammatory cytokines by neuroantigen-specific T cells is thought to initiate and maintain the inflammatory autoimmune pathology. Because gene knockout strategies have shown that IFN-gamma and TNF are not essential for EAE development, there is increasing interest in establishing the role of other proinflammatory cytokines, primarily IL-17 in EAE. We used an IL-17 ELISPOT assay to track the neuroantigen-specific IL-17-producing T cells at single-cell resolution in various organs of SJL mice undergoing PLP 139-151-induced EAE. Overall, the migration patterns and population kinetics of the PLP 139-151-specific IL-17-producing CD4 cells were reminiscent of the IFN-gamma-producing cells, with the exception of IL-17 producers far outnumbering the IFN-gamma and IL-2 producers in the inflamed CNS. The selective enrichment of IL-17-producing CD4 cells in the CNS is suggestive of the pathogenic role of an independent (non-Th1) IL-17-producing proinflammatory effector T cell class in EAE.     

Pathologic role and temporal appearance of newly emerging autoepitopes in relapsing experimental autoimmune encephalomyelitis

Relapsing experimental autoimmune encephalomyelitis (R-EAE) is a CD4+ T cell-mediated demyelinating disease model for multiple sclerosis. Myelin destruction during the initial relapsing phase of R-EAE in SJL mice initiated by immunization with the proteolipid protein (PLP) epitope PLP139-151 is associated with activation of T cells specific for the endogenous, non-cross-reactive PLP178-191 epitope (intramolecular epitope spreading), while relapses in R-EAE induced with the myelin basic protein (MBP) epitope MBP84-104 are associated with PLP139-151-specific responses (intermolecular epitope spreading). Here, we demonstrate that T cells specific for endogenous myelin epitopes play the major pathologic role in mediating clinical relapses. T cells specific for relapse-associated epitopes can serially transfer disease to naive recipients and are demonstrable in the CNS of mice with chronic R-EAE. More importantly, induction of myelin-specific tolerance to relapse-associated epitopes, by i.v. injection of ethylene carbodiimide-fixed peptide-pulsed APCs, either before disease initiation or during remission from acute disease effectively blocks the expression of the initial disease relapse. Further, blockade of B7-1-mediated costimulation with anti-B7-1 F(ab) during disease remission from acute PLP139-151-induced disease prevents clinical relapses by inhibiting activation of PLP178-191-specific T cells. The protective effects of anti-B7-1 F(ab) treatment are long-lasting and highly effective even when administered following the initial relapsing episode wherein spreading to a MBP epitope (MBP84-104) is inhibited. Collectively, these data indicate that epitope spreading is B7-1 dependent, plays a major pathologic role in disease progression, and follows a hierarchical order associated with the relative encephalitogenic dominance of the myelin epitopes (PLP139-151 > PLP178-191 > MBP84-104).     

Location of a new encephalitogenic epitope (residues 43 to 64) in proteolipid protein that induces relapsing experimental autoimmune encephalomyelitis in PL/J and (SJL x PL)F1 mice.

Synthetic peptides of proteolipid protein (PLP) were screened for their ability to induce experimental autoimmune encephalomyelitis (EAE) in SJL/J, PL/J, and (SJL x PL)F1 mice, and T cell lines were selected by stimulation of lymph node cells with PLP peptides. PLP 141-151 was found to be less encephalitogenic in SJL/J mice than PLP 139-151, due to deletion of two amino acids from the amino-terminal end. PLP 139-151 immunization induced relapsing EAE in SJL/J and F1 mice but not PL/J mice. In contrast, PLP 43-64 induced relapsing EAE in PL/J and F1 mice but not SJL/J mice. F1 T cell lines specific for either PLP 43-64 or PLP 139-151 adoptively transferred demyelinating EAE to naive F1 recipients. Haplotypes H-2s and H-2u appear to be immunologically co-dominant in F1 mice in the PLP EAE system, which differs from the H-2u dominance in F1 mice in the myelin basic protein EAE system. The identification of a PLP peptide that is encephalitogenic in PL/J mice, in addition to the previous demonstration of PLP peptides that are encephalitogenic for SWR mice (PLP 103-116) and SJL/J mice (PLP 139-151), lends support to a role for PLP as a target Ag in autoimmune demyelinating diseases.     

Detection of autoreactive myelin proteolipid protein 139-151-specific T cells by using MHC II (IAs) tetramers

Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IA(s) class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP(139-151)) and used it to analyze myelin PLP(139-151)-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IA(s) tetramers stimulated and stained the PLP(139-151)-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP(139-151), but not a control T cell line specific for PLP(178-191). We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH ( approximately 8.0) and neuraminidase treatment enhances the staining capacity of PLP(139-151) tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP(139-151)-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP(139-151)-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.     

Cutting edge: CD4+CD25+ regulatory T cells contribute to gender differences in susceptibility to experimental autoimmune encephalomyelitis

Female B10.S mice are highly resistant to proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) and depletion of PLP 139-151-reactive CD4+CD25+ regulatory T (Treg) cells can slightly increase their EAE susceptibility. Although male B10.S mice are moderately susceptible to EAE, we report that depletion of Treg cells in male B10.S mice before immunization with PLP 139-151 renders them highly susceptible to severe EAE with more CNS neutrophil infiltrates than nondepleted controls. Increased susceptibility is associated with an enhanced PLP 139-151-specific T cell response and greater production of IFN-gamma, IL-6, and IL-17. Male CD4+CD25- effector cells depleted of Treg cells proliferate to a greater degree than those from females in response to either anti-CD3 or PLP 139-151. These data suggest that because of their capacity to regulate potent autoaggressive effector cells, Treg cells partly contribute to the resistance to autoimmunity in the male mice.     

Identification of an encephalitogenic determinant of myelin proteolipid protein for SJL mice

PLP is the major protein constituent of central nervous system myelin. We have previously shown that SJL/J (H-2s) mice develop an acute form of EAE after immunization with PLP. The purpose of the present study was to identify an encephalitogenic determinant of PLP for SJL mice. We immunized SJL/J mice with a synthetic peptide identical to residues 130-147 QAHSLERVCHCLGKWLGH of murine PLP, a sequence having an amphipathic alpha-helical conformation. Although it did not induce disease, an overlapping peptide containing residues 139-154 HCLGKWLGHPDKFVGI was encephalitogenic. Immunization with this peptide induced severe clinical and histologic EAE in 3 of 20 mice. T cell enriched ILN cells from these mice responded specifically (3H-thymidine incorporation) to this peptide as well as to shorter analogues of this domain containing serine in place of cysteine at residues 138 and 140. Immunization with the serine-substituted PLP peptides 137-151 VSHSLGKWLGHPDKF and 139-151 HSLGKWLGHPDKF induced severe, acute EAE in 4 of 9 and 15 of 15 SJL mice, respectively, and their T cell enriched ILN cells responded not only to the analogues, but also to the native PLP sequence 139-154. These results indicate that residues 139-151 of murine PLP is an encephalitogenic determinant for SJL mice. Furthermore, like the PLP encephalitogenic domain for SWR (H-2q) mice, this determinant is also a T cell epitope with a coding sequence at the end of an exon.     

CD4+CD25+ regulatory T cells specific for a thymus-expressed antigen prevent the development of anaphylaxis to self

A role for CD4(+)CD25(+) regulatory T cells (Tregs) in the control of allergic diseases has been postulated. We developed a mouse model in which anaphylaxis is induced in SJL mice by immunization and challenge with the fragment of self myelin proteolipid protein (PLP)(139-151), that is not expressed in the thymus, but not with fragment 178-191 of the same protein, that is expressed in the thymus. In this study, we show that resistance to anaphylaxis is associated with naturally occurring CD4(+)CD25(+) Tregs specific for the self peptide expressed in the thymus. These cells increase Foxp3 expression upon Ag stimulation and suppress peptide-induced proliferation of CD4(+)CD25(-) effector T cells. Depletion of Tregs with anti-CD25 in vivo significantly diminished resistance to anaphylaxis to PLP(178-191), suggesting an important role for CD4(+)CD25(+) Tregs in preventing the development of allergic responses to this thymus-expressed peptide. These data indicate that naturally occurring CD4(+)CD25(+) Tregs specific for a peptide expressed under physiological conditions in the thymus are able to suppress the development of a systemic allergic reaction to self.     

Differential role of programmed death-ligand 1 [corrected] and programmed death-ligand 2 [corrected] in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis

Programmed death-1 (PD-1) is a negative costimulatory molecule, and blocking the interaction of PD-1 with its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), enhances autoimmune disease in several animal models. We have studied the role of PD-1 ligands in disease susceptibility and chronic progression in experimental autoimmune encephalomyelitis (EAE). In BALB/c mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, PD-L1 but not PD-L2 blockade significantly increased EAE incidence. In B10.S mice immunized with myelin proteolipid protein (PLP) peptide 139-151, both PD-L1 and PD-L2 blockade markedly enhanced EAE severity. In prediabetic NOD mice immunized with PLP48-70, PD-L2 blockade worsened EAE but did not induce diabetes, whereas PD-L1 blockade precipitated diabetes but did not worsen EAE, suggesting different regulatory roles of these two ligands in EAE and diabetes. B6 mice immunized with MOG35-55 developed chronic persistent EAE, and PD-L2 blockade in the chronic phase exacerbated EAE, whereas PD-L1 blockade did not. In contrast, SJL/J mice immunized with PLP139-151 developed chronic relapsing-remitting EAE, and only PD-L1 blockade during remission precipitated EAE relapse. The strain-specific effects of PD-1 ligand blockade did not correlate with the expression of PD-L1 and PD-L2 on dendritic cells and macrophages in lymphoid tissue, or on inflammatory cells in the CNS. However, EAE enhancement is correlated with less prominent Th2 cytokine induction after specific PD-1 ligand blockade. In conclusion, PD-L1 and PD-L2 differentially regulate the susceptibility and chronic progression of EAE in a strain-specific manner.     

IL-10 plays an important role in the homeostatic regulation of the autoreactive repertoire in naive mice

We have previously shown that naive SJL (H-2(s)) mice, which are highly susceptible to myelin proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE), have a very high frequency (1/20,000 CD4 T cells) of PLP(139-151)-reactive T cells in the naive repertoire. In this study, we examine the function of this endogenous PLP(139-151)-reactive repertoire in vivo and find that this repertoire encompasses the precursors of pathogenic T cells. Because SJL mice do not develop spontaneous EAE, we have explored the mechanisms that keep this autopathogenic repertoire in check and prevent the development of spontaneous autoimmunity. We crossed IL-4 and IL-10 deficiency onto the SJL background and analyzed the roles of these two immunoregulatory cytokines in regulating the size and effector function of the endogenous PLP(139-151)-reactive repertoire and development of autoimmune disease. We find that IL-10 is important in the homeostatic regulation of the endogenous PLP(139-151)-reactive repertoire in that it both limits the size of the repertoire and prevents development of effector autoaggressive T cells. SJL IL-10(-/-) mice with high numbers of PLP(139-151)-specific precursors in the repertoire did not develop spontaneous EAE, but when they were injected with pertussis toxin, they showed atypical clinical signs of EAE with small numbers of typical mononuclear cell infiltrates predominantly in the meninges. EAE could be inhibited by prior tolerization of the mice with soluble PLP(139-151) peptide. These findings indicate that IL-10 may contribute to the regulation of the endogenous autoimmune repertoire.     

Fine specificity of CD4+ T cell responses to the dominant encephalitogenic PLP 139-151 peptide in SJL/J mice

PLP 139-151(S) is the major encephalitogenic epitope of PLP in the SJL/J mouse. CD4⁺ T cells specific for PLP 139-151(S) induce a relapsing-remitting form of EAE which is similar to the human demyelinating disease MS in both clinical course and histopathology. We are interested in events involved in activation of autoreactive T cells and how to specifically regulate these immune response to both prevent and treat ongoing demyelinating disease. In the current study, we examined the effect of both amino acid substitutions and deletions in the native PLP 139-151(S) peptide to identify which residues are critical for immunogenicity and encephalitogenicity. Conservative and nonconservative substitutions at position 145 diminished or completely destroyed the encephalitogenic potential of the peptide without affecting the ability to recall a proliferative response in lymph node T cells primed with the native PLP 139-151(S) peptide indicating an interesting dichotomy between ability to induce T cell proliferation and ability to induce active clinical disease. In addition, tryptophan at position 144 was identified as a critical TCR contact site as a peptide containing an alanine for tryptophan at this position (A144) primed a unique population of T cells which did not cross react with the native PLP 139-151(S). In addition, A144 was unable to stimulate PLP 139-151(S)-specific T cells in vitro or to induce active relapsing EAE in vivo. The significance of these results to the potential development of new strategies for preventing and treating T cell-mediated autoimmune diseases is discussed.Special issue dedicated to Dr. Majorie B. Lees.     

Parental MHC molecule haplotype expression in (SJL/J x SWR)F1 mice with acute experimental allergic encephalomyelitis induced with two different synthetic peptides of myelin proteolipid protein.

To determine if the Ag that induces an autoimmune disease influences parental MHC haplotype molecule expression in situ in MHC heterozygotes, acute experimental allergic encephalomyelitis (EAE) was induced with different encephalitogenic peptides in (SJL/J x SWR)F1 mice. The mice were sensitized with either a synthetic peptide corresponding to mouse myelin proteolipid protein (PLP) residues 103-116 YKTTICGKGLSATV which induces EAE in SWR (H-2q), but not SJL/J (H-2s) mice or a synthetic peptide corresponding to PLP residues 139-151 HCLGKWLGHPDKF which is encephalitogenic in SJL/J but not SWR mice. Mice were killed when they were moribund or at 30 days after sensitization. Twelve of 18 F1 mice given PLP peptide 103-116 and 12 of 17 mice given PLP peptide 139-151 developed EAE within 2 to 3 wk after sensitization. Cryostat sections of brain samples from F1 and parental mice were immunostained with a panel of mAb identifying H-2s and H-2q class I and II MHC molecules. In brains of controls, class I MHC molecules were expressed on choroid plexus, endothelial cells, and microglia whereas class II MHC molecules were absent. In EAE lesions, class I and II MHC molecules were present on inflammatory and parenchymal cells, but the degree of parental haplotype molecule expression did not vary with the different peptide Ag tested. Thus, in (SJL/J x SWR)F1 mice, myelin PLP peptides 103-116 and 139-151 are co-dominant Ag with respect to clinical and histologic disease and parental haplotype MHC molecule expression. We propose a unifying hypothesis consistent with these results and previous observations of differential Ia expression in (responder x non-responder)F1 guinea pigs. We suggest that MHC molecules may bind locally derived peptide Ag in inflammatory sites and that these interactions influence levels of MHC haplotype molecules on APC.     

IL-4, IL-10, IL-13, and TGF-beta from an altered peptide ligand-specific Th2 cell clone down-regulate adoptive transfer of experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is induced in the SJL/J mouse by adoptive transfer of activated proteolipid protein peptide (PLP) 139-151-specific Th1 cells. T cells responding to altered peptide ligands (APL) of PLP, previously shown to induce Th2 differentiation and regulate disease in PLP-immunized mice, do not transfer EAE. However, the exact mechanism of disease regulation by APL-specific T cells has not been elucidated. In this report, we show that 1F1, a Th2 clone specific for an APL of PLP139-151 can prevent adoptive transfer of EAE when cocultured with PLP-encephalitogenic spleen cells (PLP-spleen). Cytokines from activated 1F1 cells were detected by hybridization of mRNA to oligonucleotide arrays (DNA chip) and by ELISA. The Th2 cytokines found to be present at the highest protein and mRNA levels were evaluated for their role in suppression of adoptive transfer of EAE from PLP-activated spleen cell cultures. Abs to individual cytokines in 1F1 PLP-spleen cocultures suggested that IL-4, IL-13, and TGF-beta played a significant role in suppressing EAE. Abs to the combination of IL-4, IL-10, IL-13, and TGF-beta completely neutralized the protective effect of 1F1. Addition of Th2 cytokines to PLP-spleen cultures showed that IL-13 and TGF-beta were each individually effective and low levels of IL-4 synergized with IL-13 to inhibit disease transfer. IL-5, IL-9, and IL-10 had little or no effect whereas GM-CSF slightly enhanced EAE. Our results demonstrate that Th2 cytokines derived from APL-specific Th2 cells can effectively down-regulate the encephalitogenic potential of PLP-spleen cells if present during their reactivation in culture.     

SJL Mice Infected with Acanthamoeba castellanii Develop Central Nervous System Autoimmunity through the Generation of Cross-Reactive T Cells for Myelin Antigens

We recently reported that Acanthamoeba castellanii (ACA), an opportunistic pathogen of the central nervous system (CNS) possesses mimicry epitopes for proteolipid protein (PLP) 139–151 and myelin basic protein 89–101, and that the epitopes induce experimental autoimmune encephalomyelitis (EAE) in SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of cross-reactive T cells, predominantly for PLP 139–151, as evaluated by T cell proliferation and IA^s/dextramer staining. We verified that PLP 139–151-sensitized lymphocytes generated in infected mice contained a high proportion of T helper 1 cytokine-producing cells, and they can transfer disease to naïve animals. Likewise, the animals first primed with suboptimal dose of PLP 139–151 and later infected with ACA, developed EAE, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in the pathogenesis of CNS diseases such as multiple sclerosis.