Pre-emptive targeting of the epitope spreading cascade with genetically modified regulatory T cells during autoimmune demyelinating disease.

Epitope spreading or endogenous self-priming has been implicated in mediating the progression of autoimmune disease. In the present study we created an immune-deviated, epitope spreading response in SWXJ mice after the onset of experimental autoimmune encephalomyelitis, a prototypic autoimmune animal model widely used in multiple sclerosis research. We established an immunoregulatory spreading repertoire by transferring T cells genetically modified to produce high levels of IL-10 in response to a dominant epitope spreading determinant. Installation of a Th2/Tr1-like spreading repertoire resulted in a marked and prolonged inhibition of disease progression and demyelination characterized by 1) bystander inhibition of the recall response to the priming immunogen, and 2) a Th1-->Tr1 immune-deviated spreading response involving a shift in the source of IL-10 production from the transferred regulatory population to the host-derived, endogenously primed repertoire. Thus, our data provide a rationale for cell-based therapeutic intervention in multiple sclerosis by showing that pre-emptive targeting of the epitope spreading cascade with regulatory T cells effectively induces an immune-deviated spreading response capable of inhibiting ongoing inflammatory autoreactivity and disease progression.

Differential DM20 mRNA expression distinguishes two distinct patterns of spontaneous recovery from murine autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for multiple sclerosis (MS) mediated by T cells responding to CNS myelin proteins. Immunization of SWXJ mice with the immunodominant p139-151 peptide of myelin proteolipid protein (PLP) results in a relapsing-remitting pattern of EAE characterized by incomplete remyelination during clinical recovery. In the present study we observed two distinct clinical patterns of spontaneous remission during recovery from EAE, viz., sustained remission involving continuous neurologic improvement and aborted remission involving modest transient clinical improvement. We hypothesized that the ability to recover from autoimmune demyelination was directly linked to remyelination events that recapitulated developmental processes. Quantitative immunocytochemistry of CNS tissue showed decreased demyelination in mice undergoing sustained remission compared to mice undergoing aborted remission. Quantitative RT-PCR analysis showed elevated expression of DM20, the developmental isoform of PLP, in CNS tissue from mice undergoing sustained remission compared to mice undergoing aborted recovery. Moreover, DM20 expression was similarly elevated in CNS tissue from mice undergoing sustained recovery from EAE relapse. Our data indicate that expression of the developmental DM20 isoform of PLP is intimately associated with decreased demyelination and sustained clinical recovery from EAE. Thus, DM20 gene expression may provide an appropriate molecular target for promoting CNS remyelination and may serve as a useful marker for predicting clinical outcome and assessing the effectiveness of strategies aimed at promoting CNS tissue repair during autoimmune demyelinating disease.

Modulation of the IL-10/IL-12 cytokine circuit by interferon-beta inhibits the development of epitope spreading and disease progression in murine autoimmune encephalomyelitis.

IFN-beta has been shown to be effective in the treatment of multiple sclerosis (MS). However, the primary mechanism by which IFN-beta mediates its therapeutic effect remains unclear. Recent studies indicate that under defined conditions, IFN-beta may downregulate DC expression of IL-12. We and others have shown that IFN-beta may also downregulate IL-10. In light of the recently proposed paradigm that an IL-10/IL-12 immunoregulatory circuit controls susceptibility to autoimmune disease, we examined the effect of IFN-beta on the development and behavior of the autoreactive T cell repertoire during experimental autoimmune encephalomyelitis (EAE), an animal model sharing many features with MS. SWXJ mice were immunized with the immunodominant p139-151 determinant of myelin proteolipid protein (PLP), and at onset of EAE were treated every other day with IFN-beta. After eight weeks of treatment, we assessed autoreactivity and observed no significant IFN-beta effect on splenocyte proliferation or splenocyte production of IFN-gamma, IL-2, IL-4, or IL-5 in response to the priming determinant used to initiate disease. However, in IFN-beta treated mice, the cytokine profile in response to the priming immunogen was significantly skewed toward an increased production of IL-10 and a concurrent decreased production of IL-12. Moreover, the in vivo modulation of the IL-10/IL-12 immunoregulatory circuit in response to the priming immunogen was accompanied by an aborted development of epitope spreading. Our results indicate that IFN-beta induces a reciprocal modulation of the IL-10/IL-12 cytokine circuit in vivo. This skewed autoreactivity establishes an inflammatory microenvironment that effectively prevents endogenous self-priming thereby inhibiting the progression of disease associated with epitope spreading.

Gene therapy in experimental autoimmune encephalomyelitis.

Gene therapy traditionally has been associated with "gene replacement." where exogenous recombinant DNA is introduced ex vivo into somatic cells that are then introduced back into the patient as a way to correct an inherited genetic defect. However, several novel gene therapy strategies for treating autoimmune diseases recently have emerged. Strategies involving the use of several types of DNA vaccines, the application of various viral vectors, and the use of diverse cellular vectors have shown promise in inhibiting autoimmune-mediated inflammation and repairing tissue damaged as a result of autoimmune attack. In the current review, we examine and discuss the development and proposed use of emerging gene therapy strategies for the treatment of autoimmune disease with specific emphasis on experimental autoimmune encephalomyelitis (EAE), an animal model widely used in multiple sclerosis (MS) research.

Alterations in cardiac function and gene expression during autoimmune myocarditis in mice.

Although myocarditis has been implicated in the pathogenesis of heart failure, a definitive relationship between myocardial inflammation, cardiac dysfunction, and changes in myocyte gene expression has not been established. In this study, we examined the hypothesis that myocardial inflammation and replacement fibrosis following an autoimmune response can progress to cardiac dysfunction and may result in progression to the heart failure phenotype. SWXJ mice were immunized with cardiac myosin on day 0 and day 7, in order to induce an autoimmune response to the myosin protein. Cardiac catheterization via the right carotid artery was performed on days 14, 21, 28, 35, and 42, using a 1.4F Millar transducer-tipped catheter. Hearts were weighed, and cross-sections were cut and stained with either haematoxylin and eosin or Masson's trichrome, in order to identify areas of inflammation and/or fibrosis. Myocardial gene expression was determined by Northern blot analysis. In mice with histological evidence of myocarditis, the heart weight/body weight ratio increased beginning on day 14, and cardiac function decreased beginning on day 21. Myocardial inflammation was accompanied by significant fibrosis beginning on day 21. Quantitation of mRNA showed expression of ventricular atrial naturietic factor, as well as a decrease in myosin heavy chain alpha, beginning on day 21. These data demonstrate that autoimmune inflammation of the heart results in significant cardiac dysfunction, leading to phenotypic alterations similar to those demonstrated in human heart failure and animal models of heart failure.

The type IV phosphodiesterase specific inhibitor mesopram inhibits experimental autoimmune encephalomyelitis in rodents.

Experimental autoimmune encephalomyelitis (EAE) is an autoimmune disease with pathological features reminiscent of those seen in multiple sclerosis and thus serves as an animal model for this disease. Inhibition of type IV phosphodiesterase (PDE IV) in animals with this disease has been shown to result in amelioration of disease symptoms. Here we describe the immunomodulatory activity of the novel potent and selective PDE IV inhibitor mesopram. In vitro, mesopram selectively inhibits the activity of type 1 helper T (Th1) cells without affecting cytokine production or proliferation of type 2 helper T (Th2) cells. Administration of mesopram to rodents inhibits EAE in various models. Clinically, EAE is completely suppressed by mesopram in Lewis rats. This is accompanied by a reduction of inflammatory lesions in spinal cord and brain. RT-PCR analysis revealed a marked reduction in the expression of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in the brains of these animals. Furthermore, the ex vivo production of Th1 cytokines by activated spleen cells derived from mesopram-treated animals is significantly reduced compared to vehicle-treated controls. Amelioration of the clinical symptoms is also observed during chronic EAE in mesopram-treated SJL mice as well as in relapsing-remitting EAE in SWXJ mice using a therapeutic treatment regimen. These data demonstrate the anti-inflammatory activity of mesopram and provide a rationale for its clinical development.

T cell design for therapy in autoimmune demyelinating disease.

It has become increasingly more evident that a considerable refinement of currently used reagents and conditions will be needed before an effective gene therapy strategy can be used in the treatment of human autoimmune diseases. Such refinements will focus on optimizing three basic requirements for effective gene therapy, viz.: (1) targeted delivery of the therapeutic gene and/or its gene product in a reliable, efficient manner; (2) long-term expression of the therapeutic gene; and (3) regulated expression of the therapeutic gene so that it is activated only when needed. Using an experimental autoimmune encephalomyelitis mouse model, we have examined the potential for using the T cell as a gene therapy vector for targeted, long-term, regulated delivery of therapeutic transgene factors to the autoimmune inflammatory milieu. Our data indicate that the autoreactive T cell may serve as a useful endogenous vector for antigen-inducible, site-specific delivery of a variety of therapeutic transgene factors capable of mediating both inhibition of autoimmune inflammation and regeneration and/or protection of damaged tissue.

Epitope spreading: a mechanism for progression of autoimmune disease.

Autoimmune diseases are typically characterized by a persistent inflammatory self-recognition process that ultimately leads to chronic progressive disability. Over the past several years we have addressed the fundamental question of why autoimmune diseases are chronic. Our working hypothesis in these studies has been that autoimmunity involves a continuous acquisition of new self-recognition events, thereby providing an inflammatory steady-state that leads to chronicity. This acquired T cell neoautoreactivity is commonly referred to as epitope spreading. By studying multiple sclerosis (MS) and its related animal model, experimental autoimmune encephalomyelitis (EAE), we have found that chronic progression of autoimmune disease is invariably linked to the development of an epitope-spreading process that manifests as a cascade of inflammatory T cell neoautoreactivities to a sequential series of predictable new target self-antigens. However, our most recent observations indicate that the emergence of epitope spreading is accompanied by a concurrent regression of the established primary autoreactivity associated with disease onset. Thus, our studies indicate that progression of autoimmune disease involves a shifting of T cell autoreactivity from primary initiating self-determinants to defined cascades of secondary determinants that sustain the inflammatory self-recognition process during progression to chronicity. Our data support the view that the natural development of self-recognition during autoimmune disease may best be understood when considered in the temporal context of an "epitope du jour" and "moving target" perspective.

Shaping of the autoreactive T-cell repertoire by a splice variant of self protein expressed in thymic epithelial cells.

Intrathymic expression of tissue-specific self antigens may be involved in immunological tolerance and protection from autoimmune disease. We have analyzed the role of T-cell tolerance to proteolipid protein (PLP), the main protein of the myelin sheath, in susceptibility to experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Intrathymic expression of PLP was largely restricted to the shorter splice variant, DM20. Expression of DM20 by thymic epithelium was sufficient to confer T-cell tolerance to all epitopes of PLP in EAE-resistant C57BL/6 mice. In contrast, the major T-cell epitope in SJL/J mice was only encoded by the central nervous system-specific exon of PLP, but not by thymic DM20. Thus, lack of tolerance to this epitope offers an explanation for the exquisite susceptibility of SJL/J mice to EAE. As PLP expression in the human thymus is also restricted to the DM20 isoform, these findings have implications for selection of the autoimmune T-cell repertoire in multiple sclerosis.

Visinin-like protein (VILIP) is a neuron-specific calcium-dependent double-stranded RNA-binding protein.

Double-stranded RNA-binding proteins function in regulating the stability, translation, and localization of specific mRNAs. In this study, we have demonstrated that the neuron-specific, calcium-binding protein, visinin-like protein (VILIP) contains one double-stranded RNA-binding domain, a protein motif conserved among many double-stranded RNA-binding proteins. We showed that VILIP can specifically bind double-stranded RNA, and this interaction specifically requires the presence of calcium. Mobility shift studies indicated that VILIP binds double-stranded RNA as a single protein-RNA complex with an apparent equilibrium dissociation constant of 9.0 x 10(-6) M. To our knowledge, VILIP is the first double-stranded RNA-binding protein shown to be calcium-dependent. Furthermore, VILIP specifically binds the 3'-untranslated region of the neurotrophin receptor, trkB, an mRNA localized to hippocampal dendrites in an activity-dependent manner. Given that VILIP is also expressed in the hippocampus, these data suggest that VILIP may employ a novel, calcium-dependent mechanism to regulate its binding to important localized mRNAs in the central nervous system.

Regression and spreading of self-recognition during the development of autoimmune demyelinating disease.

The autoimmune T cell repertoire in experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) is characterized by CD4(+)T cells of the Th1 phenotype that recognize peptide determinants of central nervous system (CNS) myelin proteins in an MHC class II-restricted manner. Our recent studies and those performed by others have shown that progression to chronicity in EAE and MS is accompanied by a broadening of the T cell repertoire with time. This acquired neo-autoreactivity is commonly referred to as epitope spreading and is presumably the result of endogenous priming to new self-determinants during the CNS inflammation that accompanies disease onset and relapse. In the present study we extend our earlier observations by showing that disease progression in both EAE and MS is accompanied by two concurrent events, viz. (1) the spontaneous regression of the primary established autoimmune repertoire associated with disease onset, and (2) the emergence of the epitope spreading cascade associated with disease progression. Our data show that disease initiation and disease progression in both EAE and MS are typically associated with distinctly different autoimmune T cell repertoires. Our data support the view that the natural development of self-recognition during autoimmune disease may best be understood when considered in the temporal context of an 'epitope du jour' and 'moving target' perspective.

Th2 T cells expressing transgene PDGF-A serve as vectors for gene therapy in autoimmune demyelinating disease.

We hypothesized that T cells can be genetically modified to express growth factor transgene products capable of inducing oligodendrocyte progenitor proliferation. Autoreactive T cells isolated from SWXJ mice immunized with the p139-151 determinant of myelin proteolipid protein (PLP) were transfected with an antigen-inducible transgene for platelet-derived growth factor-A (PDGF), a growth factor important in regulating the development of oligodendrocytes. Isolated antigen-specific T cell clones expressed the PDGF transgene when stimulated with PLP 139-151 peptide and produced biologically active PDGF capable of inducing proliferation of oligodendrocyte progenitor cells. Furthermore, upon adoptive transfer, the PDGF transfected T cells migrated to the CNS and ameliorated ongoing disease. Our data indicate that autoreactive memory Th2 cells can be genetically modified so that upon engagement with self antigen they produce regenerative growth factors capable of mediating tissue repair during autoimmune disease.

Treatment with BBB022A or rolipram stabilizes the blood-brain barrier in experimental autoimmune encephalomyelitis: an additional mechanism for the therapeutic effect of type IV phosphodiesterase inhibitors.

We examined the treatment effects of two structurally distinct phosphodiesterase type IV (PDE IV) inhibitors, BBB022 and rolipram, in murine and rat models of experimental autoimmune encephalomyelitis (EAE). Based on our data, we propose a mechanism of action which may supplement immunomodulatory effects of PDE IV inhibitors. In particular, PDE inhibitors promote elevation of intracellular cAMP levels, increasing the electrical resistance of endothelial monolayers by stabilizing intercellular junctional complexes. Such an effect on central nervous system (CNS) vascular endothelium has the potential to reduce disease severity in EAE, because both inflammatory cells and humoral factors readily cross a disrupted blood-brain barrier (BBB). In this report, we demonstrate the capacity of BBB022 and rolipram to decrease clinical severity of EAE. further, PDE IV inhibitors significantly reduced BBB permeability in the spinal cords of mice with EAE. These results provide evidence that PDE IV-inhibitors may exert therapeutic effects in EAE by modifying cerebrovascular endothelial permeability, reducing tissue edema as well as entry of inflammatory cells and factors.

Spontaneous regression of primary autoreactivity during chronic progression of experimental autoimmune encephalomyelitis and multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is a widely used animal model for multiple sclerosis (MS). EAE is typically initiated by CD4(+) T helper cell type 1 (Th1) autoreactivity directed against a single priming immunodominant myelin peptide determinant. Recent studies have shown that clinical progression of EAE involves the accumulation of neo-autoreactivity, commonly referred to as epitope spreading, directed against peptide determinants not involved in the priming process. This study directly addresses the relative roles of primary autoreactivity and secondary epitope spreading in the progression of both EAE and MS. To this end we serially evaluated the development of several epitope-spreading cascades in SWXJ mice primed with distinctly different encephalitogenic determinants of myelin proteolipid protein. In a series of analogous experiments, we examined the development of epitope spreading in patients with isolated monosymptomatic demyelinating syndrome as their disease progressed to clinically definite MS. Our results indicate that in both EAE and MS, primary proliferative autoreactivity associated with onset of clinical disease invariably regresses with time and is often undetectable during periods of disease progression. In contrast, the emergence of sustained secondary autoreactivity to spreading determinants is consistently associated with disease progression in both EAE and MS. Our results indicate that chronic progression of EAE and MS involves a shifting of autoreactivity from primary initiating self-determinants to defined cascades of secondary determinants that sustain the self-recognition process during disease progression.

Gene therapy for autoimmune demyelinating disease of the central nervous system.

Gene therapy is currently being explored as a new therapeutic treatment of autoimmune disease. The genetic modification of autoreactive memory T cells (T cell-mediated gene therapy) and autoimmune target tissue (target tissue gene therapy) to produce immunoregulatory cytokines offers a promising way to regulate autoimmunity. Furthermore, regenerative gene therapy offers the possibility of delivering growth factors to damaged autoimmune target tissue as a way of mediating repair. In the current review we discuss the different experimental models that are being used to test the efficacy of gene therapy in that treatment of autoimmune disease. We also discuss the importance of regulating transgene expression to ensure the therapeutic transgene products are delivered specifically to the autoimmune milieu in an antigen-inducible, non-constitutive manner.

Digitized image analysis reveals diffuse abnormalities in normal-appearing white matter during acute experimental autoimmune encephalomyelitis.

Demyelination of the central nervous system is a hallmark of multiple sclerosis and its widely used animal model, experimental autoimmune encephalomyelitis (EAE). Recent studies using magnetic resonance imaging and spectroscopy on multiple sclerosis patients have revealed abnormalities of central nervous system normal-appearing white matter suggesting that micro-demyelination and/or extensive membrane turnover accompanies and perhaps precedes the appearance of manifest inflammatory lesions. In the present study, we induced EAE in SWXJ mice and analyzed digitized images of immunocytochemically stained spinal cord for detection of myelin proteolipid protein (PLP). We found that digitized image analysis is a highly sensitive, objective methodology for measuring the extent of myelin loss during EAE. Our data show that two-thirds of the measured reduction of myelin PLP occurring in EAE spinal cord could be attributed to a loss of myelin in normal-appearing white matter. The marked decrease in detection of PLP was accompanied by a corresponding decrease in PLP mRNA in the central nervous system. Our results indicate that during acute EAE, diffuse myelin abnormalities extend far beyond visibly detectable inflammatory foci and are characterized by a global decrease in the expression of myelin genes and their encoded proteins.

The epitope spreading cascade during progression of experimental autoimmune encephalomyelitis and multiple sclerosis.

We have made the following observations regarding self-recognition during the development and progression of murine experimental autoimmune encephalomyelitis (EAE) and human multiple sclerosis (MS): 1) chronic progression of EAE is accompanied by a sequential, predictable cascade of neo-autoreactivity, commonly referred to as epitope spreading, presumably caused by endogenous self-priming during autoimmune-mediated tissue damage; 2) there is an invariant relationship between the progression of EAE and the emergence of epitope spreading; 3) progression of EAE can be inhibited by the induction of antigen-specific tolerance to spreading determinants after onset of initial neurologic symptoms; 4) CD4+ Th 1 cells responding to spreading determinants are autonomously encephalitogenic; 5) epitope spreading occurs during the development of MS and in some cases involves HLA-DP class II-restricted self-recognition; and 6) progression of both EAE and MS is accompanied by the decline of primary T-cell autoreactivity associated with disease onset and by the concurrent emergence of the epitope spreading cascade. Our studies directly challenge the traditional view that EAE and MS are initiated and maintained by autoreactivity directed against a single predominant myelin protein or determinant. Our results indicate that progression of EAE and MS involves a shifting of T-cell autoreactivity from primary initiating self-determinants to defined cascades of secondary determinants that sustain the inflammatory self-recognition process during disease progression.

Expression of chemokines RANTES, MIP-1alpha and GRO-alpha correlates with inflammation in acute experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an investigator-initiated disorder that serves as an animal model for the common human demyelinating disease multiple sclerosis. Both diseases are typified by disseminated perivascular and submeningeal cuffs in the central nervous system (CNS). It was shown recently that chemokines are integral to the pathogenesis of EAE. In the present study we analyzed the gene expression of three chemokines, RANTES, MIP-1alpha and GRO-alpha, at the onset of acute EAE, and correlated that expression with the intensity of inflammatory changes in the CNS. We showed that all three chemokines are upregulated simultaneously with symptom onset of acute EAE, and that chemokine expression correlates with the intensity of inflammation in the CNS. This consistent relationship supports the hypothesis that chemokines are relevant to leukocyte accumulation in CNS parenchyma.

In vivo effects of interferon beta-1a on immunosuppressive cytokines in multiple sclerosis.

Recombinant interferon beta (IFNbeta) benefits patients with relapsing remitting multiple sclerosis (MS), but the mechanisms of action are unknown. We studied in vivo immunologic effects of IFNbeta treatment and their relationship to clinical efficacy. Cytokines were measured in blood and CSF from MS patients participating in a placebo-controlled phase III clinical trial and an open-label phase IV [corrected] tolerability study of IFNbeta-1a. Additionally, immunologic studies were conducted in animals with proteolipid protein (PLP)-induced chronic relapsing experimental autoimmune encephalomyelitis. Single intramuscular (IM) injections of IFNbeta-1a (6 MIU, 30 microg) were associated with significant in vivo upregulation of interleukin-10 (IL-10) and IL-4 but not IFNgamma mRNA in peripheral blood mononuclear cells. Forty-eight hours after each IFNbeta-1a injection, serum IL-10 levels increased and remained elevated for 1 week. IFNbeta-1a recipients in the placebo-controlled phase III clinical trial showed significantly increased concentrations of CSF IL-10 after 2 years of treatment. This response correlated with a favorable therapeutic response. Exposure of PLP-reactive murine T-cell lines to IFNbeta resulted in increased antigen-driven expression of IL-4 and IL-10 and reduced encephalitogenicity. IFNbeta-1a injections induce systemic and intrathecal immunosuppressive cytokines. Myelin-specific T cells treated with IFNbeta-1a demonstrate increased immunosuppressive cytokine expression and reduced encephalitogenicity. The relationship between increased CSF IL-10 and response to therapy suggests that induction of IL-10 is a mechanism underlying IFNbeta-1a effects in MS patients.

HLA-DP: a class II restriction molecule involved in epitope spreading during the development of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. It is widely believed that complex polygenic inheritance patterns involving HLA-DR and -DQ class II genes contribute to MS susceptibility, and current evidence indicates that disease risk vs disease outcome may be associated with distinctly different HLA class II alleles. We have recently shown that the early development of MS is accompanied by an extensive plasticity of myelin self-recognition with the acquisition of neo-autoreactivity, or epitope spreading, as a prominent feature. Although we did not observe a common determinant recognized by patients sharing identical HLA-DR or -DQ class II alleles, we did observe epitope spreading to the p50-63 determinant of myelin proteolipid protein (PLP) in two study subjects showing complete disparity at HLA-DR and -DQ but identity at the HLA-DP allele DPB1*0301. In the present study we show that self-recognition during the early stages in the development of MS involves HLA-DP class II restricted responses to the PLP 50-63 spreading determinant. Our results suggest that self-presentation by HLA-DP may play an important role in epitope spreading and in the propagation of self-recognition during the clinical progression of MS.