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.     

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.     

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.     

Synthetic Peptide dendrimers block the development and expression of experimental allergic encephalomyelitis

Multiple Ag peptides (MAPs) containing eight proteolipid protein (PLP)(139-151) peptides arranged around a dendrimeric branched lysine core were used to influence the expression and development of relapsing experimental allergic encephalomyelitis (EAE) in SJL mice. The PLP(139-151) MAPs were very efficient agents in preventing the development of clinical disease when administered after immunization with the PLP(139-151) monomeric encephalitogenic peptide in CFA. The treatment effect with these MAPs was peptide specific; irrelevant multimeric peptides such as guinea pig myelin basic protein GPBP(72-84) MAP (a dendrimeric octamer composed of the 72-84 peptide) and PLP(178-191) MAP (a dendrimeric octamer composed of the PLP(178-191) peptide) had no treatment effect on PLP(139-151)-induced EAE. PLP(139-151) MAP treatment initiated after clinical signs of paralysis also altered the subsequent course of EAE; it limited developing signs of paralysis and effectively limited the severity and number of disease relapses in MAP-treated mice over a 60-day observation period. PLP(139-151) MAP therapy initiated before disease onset acts to limit the numbers of Th17 and IFN-gamma-producing cells that enter into the CNS. However, Foxp3(+) cells entered the CNS in numbers equivalent for nontreated and PLP(139-151) MAP-treated animals. The net effect of PLP(139-151) MAP treatment dramatically increases the ratio of Foxp3(+) cells to Th17 and IFN-gamma-producing cells in the CNS of PLP(139-151) MAP-treated animals.     

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.     

Functional activation of myelin-specific T cells by virus-induced molecular mimicry

Molecular mimicry is the process by which T cells activated in response to determinants on an infecting microorganism cross-react with self epitopes, leading to an autoimmune disease. Normally, infection of SJL/J mice with the BeAn strain of Theiler's murine encephalomyelitis virus (TMEV) results in a persistent CNS infection, leading to a chronic progressive, CD4(+) T cell-mediated demyelinating disease. Myelin damage is initiated by T cell responses to virus persisting in CNS APCs, and progressive demyelinating disease (50 days postinfection) is perpetuated by myelin epitope-specific CD4(+) T cells activated by epitope spreading. We developed an infectious model of molecular mimicry by inserting a sequence encompassing the immunodominant myelin epitope, proteolipid protein (PLP) 139-151, into the coding region of a nonpathogenic TMEV variant. PLP139-TMEV-infected mice developed a rapid onset paralytic inflammatory, demyelinating disease paralleled by the activation of PLP139-151-specific CD4(+) Th1 responses within 10-14 days postinfection. The current studies demonstrate that the early onset demyelinating disease induced by PLP139-TMEV is the direct result of autoreactive PLP139-151-specific CD4(+) T cell responses. PLP139-151-specific CD4(+) T cells from PLP139-TMEV-infected mice transferred demyelinating disease to naive recipients and PLP139-151-specific tolerance before infection prevented clinical disease. Finally, infection with the mimic virus at sites peripheral to the CNS induced early demyelinating disease, suggesting that the PLP139-151-specific CD4(+) T cells could be activated in the periphery and traffic to the CNS. Collectively, infection with PLP139-151 mimic encoding TMEV serves as an excellent model for molecular mimicry by inducing pathologic myelin-specific CD4(+) T cells via a natural virus infection.     

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.     

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.     

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.     

Pertussis toxin modulates the immune response to neuroantigens injected in incomplete Freund's adjuvant: induction of Th1 cells and experimental autoimmune encephalomyelitis in the presence of high frequencies of Th2 cells

Pertussis toxin (PT) has been widely used to facilitate the induction of experimental autoimmune encephalomyelitis (EAE) in rodents. It has been suggested that this microbial product promotes EAE by opening up the blood-brain barrier and thereby facilitates the migration of pathogenic T cells to the CNS. However, PT has other biological effects that could contribute to its activity in EAE, such as enhancing the cytokine production by T cells and induction of lymphocytosis. In this work, we investigated the effects of PT on the pathogenicity, cytokine differentiation, and clonal sizes of neuroantigen-reactive T cells in EAE in mice. Our results show that PT prevented the protection from EAE conferred by injection of PLPp139-151 in IFA and induced high frequencies of peptide-specific Th1 cells and disease. Interestingly, the mice developed EAE despite the simultaneous vigorous clonal expansion of PLPp139-151-specific Th2 cells. The data indicate that the Th2 cells in this model neither were protective against EAE nor promoted the disease. Furthermore, the results suggested that the effects of the toxin on neuroantigen-reactive T cells were promoted by the PT-induced activation of APCs in lymphoid tissues and the CNS. Together, the results suggest that microbial products, such as PT, could contribute to the initiation of autoimmune disease by modulating the interaction between the innate and adaptive immune system in the response to self Ags.     

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.     

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.     

The immunopathology of acute experimental allergic encephalomyelitis induced with myelin proteolipid protein. T cell receptors in inflammatory lesions.

To determine whether there is predominance of T cells expressing a particular TCR V beta chain in the inflammatory lesions of an autoimmune disease model, TCR expression was analyzed in central nervous system (CNS) tissues of mice with experimental allergic encephalomyelitis (EAE). Acute EAE was induced in SJL/J mice either by sensitization with a synthetic peptide corresponding to myelin proteolipid protein residues 139-151 or by adoptive transfer of myelin proteolipid protein peptide 139-151-specific encephalitogenic T cell clones. Mice were killed when they showed clinical signs of EAE or by 40 days after sensitization or T cell transfer. Cryostat CNS and lymphoid tissue sections were immunostained with a panel of mAb to T cell markers and proportions of stained cells were counted in inflammatory foci. In mice with both actively induced and adoptively transferred EAE, infiltrates consisted of many CD3+, TCR alpha beta+, and CD4+ cells, fewer CD8+ cells, and small numbers of TCR gamma delta+ cells. Approximately 30% of CD45+ leukocytes in the inflammatory foci were T cells. Cells expressing TCR V beta 2, 3, 4, 6, 7 and 14 were detected in the infiltrates, whereas TCR V beta 8 and 11, which that are deleted in SJL mice, were absent. When EAE was induced by transfer of T cell clones that use either V beta 2, 6, 10, or 17, there was also a heterogeneous accumulation of T cells in the lesions. Similar proportions of TCR V beta+ and gamma delta+ cells were detected in EAE lesions and in the spleens of the mice. Thus, at the time that clinical signs are present in acute EAE, peripherally derived, heterogeneous TCR V beta+ cells are found in CNS lesions, even when the immune response is initiated to a short peptide Ag or by a T cell clone using a single TCR V beta.     

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.     

Specific T regulatory cells display broad suppressive functions against experimental allergic encephalomyelitis upon activation with cognate antigen

To date, very few Ag-based regimens have been defined that could expand T regulatory (Treg) cells to reverse autoimmunity. Additional understanding of Treg function with respect to specificity and broad suppression should help overcome these limitations. Ig-proteolipid protein (PLP)1, an Ig carrying a PLP1 peptide corresponding to amino acid residues 139-151 of PLP, displayed potent tolerogenic functions and proved effective against experimental allergic encephalomyelitis (EAE). In this study, we took advantage of the Ig-PLP1 system and the PLP1-specific TCR transgenic 5B6 mouse to define a regimen that could expand Ag-specific Treg cells in vivo and tested for effectiveness against autoimmunity involving diverse T cell specificities. The findings indicate that in vivo exposure to aggregated Ig-PLP1 drives PLP1-specific 5B6 TCR transgenic cells to evolve as Treg cells expressing CD25, CTLA-4, and Foxp3 and producing IL-10. These Treg cells were able to suppress PLP1 peptide-induced EAE in both SJL/J and F(1) (SJL/J x C57BL/6) mice. However, despite being effective against disease induced with a CNS homogenate, the Treg cells were unable to counter EAE induced by a myelin basic protein or a myelin oligodendrocyte glycoprotein peptide. Nevertheless, activation with Ag before transfer into the host mice supports suppression of both myelin oligodendrocyte glycoprotein- and myelin basic protein peptide-induced EAE. Thus, it is suggested that activation of Treg cells by the cognate autoantigen is necessary for operation of broad suppressive functions.     

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.     

Brain-derived heat shock protein 70-peptide complexes induce NK cell-dependent tolerance to experimental autoimmune encephalomyelitis

Heat shock proteins (Hsp) are markedly up-regulated at sites of inflammation during autoimmune diseases like experimental autoimmune encephalomyelitis (EAE). In this study, we show that Hsp70-peptide complexes (pc) isolated from brains of mice with EAE prevented the development of EAE clinically and pathologically when administered before proteolipid protein 139-151 (PLP139-151) immunization. In contrast, pure Hsp70 or Hsp70-pc derived from brains of healthy mice or other inflamed tissue did not modulate the expression of EAE. In animals in which EAE had been suppressed by Hsp70-pc, lymphocytes showed increased cell death in response to PLP139-151 that correlated with elevated IFN-gamma and NO production. Coculture of spleen cells from Hsp70-pc immunized mice with spleen cells from untreated EAE mice, in addition to depletion experiments, showed that NK cells reduced reactivity to PLP139-151. Transfer of NK cells from Hsp70-pc-immunized mice to recipients sensitized for EAE abolished disease development. Thus, we propose that Hsp70 demonstrate the ability to bind to peptides generated during brain inflammation and to induce a regulatory NK cell population that is capable of preventing subsequent autoimmunization for EAE.     

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.     

C57BL/6J小鼠实验性自身免疫性脑脊髓炎模型的建立及其病理特征

目的探讨C57BL/6J小鼠建立实验性自身免疫性脑脊髓炎（EAE）模型的可能性及其发病特点。方法使用PLP139-151抗原及其C57BL/6J小鼠自制脑脊髓匀浆（spinal cord homogenate,SCH）免疫C57BL/6J小鼠,使用完全福（氏）免疫佐剂为免疫佐剂,并在尾静脉注射百日咳杆菌,建立EAE模型,与经典的PLP139-151免疫的SJL/J小鼠EAE模型进行对比。结果PLP139-151免疫C57BL/6J小鼠仅有一只小鼠表现为尾部张力明显降低;自制SCH免疫C57BL/6J小鼠可见明显脱髓鞘改变。与PLP139-151免疫SJL/J小鼠组相比发病率较低（P〈0.05）,神经功能评分比较没有明显差异（P〉0.05）,但发病时间长于PLP139-151免疫SJL/J小鼠组（P〈0.05）。结论SCH免疫C57BL/6J小鼠的EAE动物模型,主要表现为急性单相病程,从临床表现和病理学特点来看符合人类MS的病理特点,值得在以后的研究中进一步研究探讨。