Controlled Delivery of Single or Multiple Antigens in Tolerogenic Nanoparticles Using Peptide-Polymer Bioconjugates.

Polymeric nanoparticles (NPs) have demonstrated their potential to induce antigen (Ag)-specific immunological tolerance in multiple immune models and are at various stages of commercial development. Association of Ag with NPs is typically achieved through surface coupling or encapsulation methods. However, these methods have limitations that include high polydispersity, uncontrollable Ag loading and release, and possible immunogenicity. Here, using antigenic peptides conjugated to poly(lactide-co-glycolide), we developed Ag-polymer conjugate NPs (acNPs) with modular loading of single or multiple Ags, negligible burst release, and minimally exposed surface Ag. Tolerogenic responses of acNPs were studied in vitro to decouple the role of NP size, concentration, and Ag loading on regulatory T cell (Treg) induction. CD4+CD25+Foxp3+ Treg induction was dependent on NP size, but CD25 expression of CD4+ T cells was not. NP concentration and Ag loading could be modulated to achieve maximal levels of Treg induction. In relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE), a murine model of multiple sclerosis, acNPs were effective in inhibiting disease induced by a single peptide or multiple peptides. The acNPs provide a simple, modular, and well-defined platform, and the NP physicochemical properties offer potential to design and answer complex mechanistic questions surrounding NP-induced tolerance.

Peptide-Conjugated Nanoparticles Reduce Positive Co-stimulatory Expression and T Cell Activity to Induce Tolerance.

Targeted approaches to treat autoimmune diseases would improve upon current therapies that broadly suppress the immune system and lead to detrimental side effects. Antigen-specific tolerance was induced using poly(lactide-co-glycolide) nanoparticles conjugated with disease-relevant antigen to treat a model of multiple sclerosis. Increasing the nanoparticle dose and amount of conjugated antigen both resulted in more durable immune tolerance. To identify active tolerance mechanisms, we investigated downstream cellular and molecular events following nanoparticle internalization by antigen-presenting cells. The initial cell response to nanoparticles indicated suppression of inflammatory signaling pathways. Direct and functional measurement of surface MHC-restricted antigen showed positive correlation with both increasing particle dose from 1 to 100 µg/mL and increasing peptide conjugation by 2-fold. Co-stimulatory analysis of cells expressing MHC-restricted antigen revealed most significant decreases in positive co-stimulatory molecules (CD86, CD80, and CD40) following high doses of nanoparticles with higher peptide conjugation, whereas expression of a negative co-stimulatory molecule (PD-L1) remained high. T cells isolated from mice immunized against myelin proteolipid protein (PLP139-151) were co-cultured with antigen-presenting cells administered PLP139-151-conjugated nanoparticles, which resulted in reduced T cell proliferation, increased T cell apoptosis, and a stronger anti-inflammatory response. These findings indicate several potential mechanisms used by peptide-conjugated nanoparticles to induce antigen-specific tolerance.

Down-regulation of neuronal L1 cell adhesion molecule expression alleviates inflammatory neuronal injury.

In multiple sclerosis (MS), the immune cell attack leads to axonal injury as a major cause for neurological disability. Here, we report a novel role of the cell adhesion molecule L1 in the crosstalk between the immune and nervous systems. L1 was found to be expressed by CNS axons of MS patients and human T cells. In MOG35-55-induced murine experimental neuroinflammation, CD4+ T cells were associated with degenerating axons in the spinal cord, both expressing L1. However, neuronal L1 expression in the spinal cord was reduced, while levels of the transcriptional repressor REST (RE1-Silencing Transcription Factor) were up-regulated. In PLP139-151-induced relapsing-remitting neuroinflammation, L1 expression was low at the peak stage of disease, reached almost normal levels in the remission stage, but decreased again during disease relapse indicating adaptive expression regulation of L1. In vitro, activated CD4+ T cells caused contact-dependent down-regulation of L1, up-regulation of its repressor REST and axonal injury in co-cultured neurons. T cell adhesion to neurons and axonal injury were prevented by an antibody blocking L1 suggesting that down-regulation of L1 ameliorates neuroinflammation. In support of this hypothesis, antibody-mediated blocking of L1 in C57BL/6 mice as well as neuron-specific depletion of L1 in synapsinCre × L1fl/fl mice reduces disease severity and axonal pathology despite unchanged immune cell infiltration of the CNS. Our data suggest that down-regulation of neuronal L1 expression is an adaptive process of neuronal self-defense in response to pro-inflammatory T cells, thereby alleviating immune-mediated axonal injury.

Clínica y diagnóstico de la enfermedad de Pelizaeus-Merzbacher: descripción de cinco casos / Clinical features and diagnosis of Pelizaeus-Merzbacher disease: five case reports

Introducción. La enfermedad de Pelizaeus-Merzbacher es un trastorno hipomielinizante raro debido a alteraciones en el gen PLP1, que lleva a un fallo de la mielinización axonal de los oligodendrocitos en el sistema nervioso central. Existen dos formas descritas según la gravedad de su presentación: connatal y clásica. Se caracteriza por hipotonía neonatal, retraso del desarrollo psicomotor, espasticidad progresiva de predominio en los miembros inferiores y nistagmo, con signos y síntomas piramidales y extrapiramidales, y la forma connatal es mucho más grave. La resonancia magnética muestra leucoencefalopatía hipomielinizante difusa, los potenciales evocados usualmente se alteran y la confirmación se realiza mediante estudio molecular del gen PLP1. Casos clínicos. Se presentan cinco pacientes pediátricos afectados, cuatro con la forma clásica y uno con la forma connatal; se describen las características clínicas, los estudios complementarios y se realiza una revisión concisa de la bibliografía. Conclusión. Esta enfermedad tiene una evolución progresiva y casi invariable, lo cual es la clave clínica para diferenciarla de otras entidades como la parálisis cerebral infantil, neuropatías periféricas, esclerosis múltiple, entre otras, además de los hallazgos característicos en las neuroimágenes. Es necesario sospechar este diagnóstico y confirmar alteraciones en el gen PLP1 con el fin de obtener una incidencia real de esta entidad, probablemente subestimada, como otras leucodistrofias (AU)

Introduction. Pelizaeus-Merzbacher disease is an infrequent hypomyelinating disorder caused by alterations in the PLP1 gene, which leads to a fault in the axonal myelination of the oligodendrocytes in the central nervous system. Two forms have been reported, according to the severity of the presentation: connatal and classic. It is characterised by neonatal hypotonia, delayed psychomotor development, progressive spasticity predominantly in the lower limbs and nystagmus, with pyramidal and extrapyramidal signs and symptoms; the connatal form is far more severe. Magnetic resonance imaging shows diffuse hypomyelinating leukoencephalopathy, evoked potentials are usually altered and confirmation is obtained through a molecular study of the PLP1 gene. Case reports. We present the cases of five paediatric patients, four of whom had the classic form and one with the connatal form. The clinical characteristics and complementary studies are described, and a concise review of the literature is carried out. Conclusion. This disease has a progressive and almost unvarying course, which is the clinical key to be able to differentiate it from other entities such as infantile cerebral palsy, peripheral neuropathies or multiple sclerosis, among others, in addition to the characteristic neuroimaging findings. It is necessary to suspect this diagnosis and confirm alterations in the PLP1 gene with the aim of obtaining a real incidence of this entity, which is probably underestimated, like other leukodystrophies (AU)

Oral Administration of Lactococcus lactis Expressing Synthetic Genes of Myelin Antigens in Decreasing Experimental Autoimmune Encephalomyelitis in Rats.

BACKGROUND: Multiple sclerosis is a human autoimmunological disease that causes neurodegeneration. One of the potential ways to stop its development is induction of oral tolerance, whose effect lies in decreasing immune response to the fed antigen. It was shown in animal models that administration of specific epitopes of the three main myelin proteins - myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and proteolipid protein (PLP) - results in induction of oral tolerance and suppression of disease symptoms. Use of bacterial cells to produce and deliver antigens to gut mucosa seems to be an attractive method for oral tolerance induction in treatment of diseases with autoimmune background. MATERIAL AND METHODS: Synthetic genes of MOG35-55, MBP85-97, and PLP139-151 myelin epitopes were generated and cloned in Lactococcus lactis under a CcpA-regulated promoter. The tolerogenic effect of bacterial preparations was tested on experimental autoimmune encephalomyelitis, which is the animal model of MS. EAE was induced in rats by intradermal injection of guinea pig spinal cord homogenate into hind paws. RESULTS: Rats were administered preparations containing whole-cell lysates of L. lactis producing myelin antigens using different feeding schemes. Our study demonstrates that 20-fold, but not 4-fold, intragastric administration of autoantigen-expressing L. lactis cells under specific conditions reduces the clinical symptoms of EAE in rats. CONCLUSIONS: The present study evaluated the use of myelin antigens produced in L. lactis in inhibiting the onset of experimental autoimmune encephalomyelitis in rats. Obtained results indicate that application of such recombinant cells can be an attractive method of oral tolerance induction.

Eluding anaphylaxis allows peptide-specific prevention of the relapsing stage of experimental autoimmune encephalomyelitis.

We have used a peptide derived from Acanthamoeba castellanii (ACA) to treat the relapsing phase of EAE that develops in SJL mice following immunization with the PLP 139-151 peptide. The native sequence of the ACA 81-95 peptide that shares key residues with the PLP 139-151 peptide is weakly encephalitogenic in SJL mice but is not recognized by antiserum from SJL mice immunized with PLP 139-151. A single amino acid change to the ACA 81-95 peptide sequence significantly enhanced its encephalitogenicity. When administered to SJL mice as a nonlinear peptide octamer, the modified ACA peptide prevented relapsing episodes of EAE in SJL mice previously immunized with the PLP 139-151 encephalitogenic peptide.

A comparative study of experimental mouse models of central nervous system demyelination.

Several mouse models of multiple sclerosis (MS) are now available. We have established a mouse model, in which ocular infection with a recombinant HSV-1 that expresses murine interleukin (IL)-2 constitutively (HSV-IL-2) causes central nervous system demyelination in different strains of mice. This model differs from most other models, in which it represents a mixture of viral and immune triggers. In the present study, we directly compared MOG35-55, MBP35-47 and PLP190-209 models of experimental autoimmune encephalitis with our HSV-IL-2-induced MS model. Mice with HSV-IL-2- and myelin oligodendrocyte glycoprotein (MOG)-induced demyelinating diseases demonstrated a similar pattern and distribution of demyelination in their brain, spinal cord (SC) and optic nerves (ONs). In contrast, no demyelination was detected in the ONs of myelin basic protein (MBP)- and proteolipid protein (PLP)-injected mice. Interferon-ß (IFN-ß) injections significantly reduced demyelination in brains of all groups, in the SCs of the MOG and MBP groups, and completely blocked it in the SCs of the PLP and HSV-IL-2 groups as well as in ONs of MOG and HSV-IL-2 groups. In contrast to IFN-ß treatment, IL-12p70 protected the HSV-IL-2 group from demyelination, whereas IL-4 was not effective at all in preventing demyelination. MOG-injected mice showed clinical signs of paralysis and disease-related mortality, whereas mice in the other treatment groups did not. Collectively, the results indicate that the HSV-IL-2 model and the MOG model complement each other and, together, provide unique insights into the heterogeneity of human MS.

A regenerative approach to the treatment of multiple sclerosis.

Progressive phases of multiple sclerosis are associated with inhibited differentiation of the progenitor cell population that generates the mature oligodendrocytes required for remyelination and disease remission. To identify selective inducers of oligodendrocyte differentiation, we performed an image-based screen for myelin basic protein (MBP) expression using primary rat optic-nerve-derived progenitor cells. Here we show that among the most effective compounds identifed was benztropine, which significantly decreases clinical severity in the experimental autoimmune encephalomyelitis (EAE) model of relapsing-remitting multiple sclerosis when administered alone or in combination with approved immunosuppressive treatments for multiple sclerosis. Evidence from a cuprizone-induced model of demyelination, in vitro and in vivo T-cell assays and EAE adoptive transfer experiments indicated that the observed efficacy of this drug results directly from an enhancement of remyelination rather than immune suppression. Pharmacological studies indicate that benztropine functions by a mechanism that involves direct antagonism of M1 and/or M3 muscarinic receptors. These studies should facilitate the development of effective new therapies for the treatment of multiple sclerosis that complement established immunosuppressive approaches.

Suppression of MOG- and PLP-induced experimental autoimmune encephalomyelitis using a novel multivalent bifunctional peptide inhibitor.

Previously, bifunctional peptide inhibitors (BPI) with a single antigenic peptide have been shown to suppress experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner. In this study, a multivalent BPI (MVBMOG/PLP) with two antigenic peptides derived from myelin oligodendrocyte glycoprotein (MOG38-50) and myelin proteolipid protein (PLP139-151) was evaluated in suppressing MOG38-50- and PLP139-151-induced EAE. MVBMOG/PLP significantly suppressed both models of EAE even when there was some evidence of epitope spreading in the MOG38-50-induced EAE model. In addition, MVBMOG/PLP was found to be more effective than PLP-BPI and MOG-BPI in suppressing MOG38-50-induced EAE. Thus, the development of MVB molecules with broader antigenic targets can lead to suppression of epitope spreading in EAE.

Dendritic cells regulate amyloid-ß-specific T-cell entry into the brain: the role of perivascular amyloid-ß.

Amyloid-ß (Aß) accumulation in the brain is one of the hallmarks of Alzheimer's disease (AD). T-cell entry into vascular and parenchymal brain areas loaded with Aß has been observed with both beneficial as well as detrimental effects. Using a new AD mouse model, we studied the molecular mechanisms allowing CD4 T cells to specifically target Aß-loaded brain areas. We observed that following Aß immunization, CD11c+ dendritic cells (DCs) and CD4 T cells occurred primarily in the perivascular and leptomeningial spaces of cerebral vessels deposited with Aß. CD11c+ cells expressed high levels of the DC maturation markers DEC-205, MHC class II and CD86. Notably, the majority of cerebral blood vessels were found adjacent to Aß plaques, expressing high levels of the ICAM-1 and VCAM-1 adhesion molecules. We propose that the drainage of Aß to the leptomeningeal and perivascular spaces and its deposition there provide the antigenic source for DCs to stimulate Aß-specific T cells on their way to target amyloid plaques within the brain tissue.

Alteration of T cell cytokine production in PLPp-139-151-induced EAE in SJL mice by an immunostimulatory CpG oligonucleotide.

Experimental autoimmune encephalomyelitis (EAE) is--in certain aspects--regarded as an animal model of the human CNS autoimmune disease multiple sclerosis (MS). While in EAE CNS-autoantigen-specific immunity is induced in a defined way, the initial processes leading to CNS autoimmunity in humans are so far unknown. Despite essential restrictions, which exist regarding the interpretation of EAE data towards MS, EAE might be a useful model to study certain basic aspects of CNS autoimmunity. Studies in MS have demonstrated that established autoimmune pathology can be critically influenced by environmental factors, in particular viral and bacterial infections. To investigate this interaction, EAE as an instrument to study CNS autoimmunity under defined conditions appears to be a suitable experimental tool. For this reason, we here investigated the influence of the Toll-like-receptor (TLR) ligand CpG oligonucleotide (CpG) on already established CNS autoimmunity in murine proteolipid protein (PLP)-induced EAE in SJL mice. CpG were found to co-stimulate PLPp-specific IFN-γ production in the peripheral immune system and in the CNS. However, CpG induced Interleukin (IL)-17 production in the inflamed CNS both alone and in combination with additional PLPp stimulation. These findings might indicate a mechanism by which systemic infections and the microbial stimuli associated with them may influence already existing CNS autoimmune pathology.

Kinetics of IL-17- and interferon-gamma-producing PLPp-specific CD4 T cells in EAE induced by coinjection of PLPp/IFA with pertussis toxin in SJL mice.

Systemic administration of Pertussis toxin (PTX) abrogates T cell tolerance mediated by injection of neuroantigens in incomplete Freund's adjuvant (IFA) and causes experimental autoimmune encephalomyelitis (EAE). PTX concomitantly induces high frequencies of neuroantigen-specific IFN-gamma- and IL-17-producing T cells. Both IL-17 and IFN-gamma have been implicated as a key effector cytokines in the pathogenesis of EAE, possibly with different functions. We therefore investigated potential differences in the temporal and spatial kinetics of the PTX-induced neuroantigen-specific IFN-gamma- and IL-17-producing T cell effector populations. IFN-gamma- and IL-17-producing PLPp-specific T cells initially arose in comparable frequencies in the local draining lymph nodes (drLN) after immunization as measured by cytokine ELISPOT. High frequencies of both IFN-gamma- and IL-17-producing T cells were present in the immune periphery before onset of EAE. The highest frequencies of PTX-induced IFN-gamma- and IL-17-producing PLPp-specific cells coincided in the inflamed CNS during acute EAE. During recovery, both IFN-gamma- and IL-17-producing PLPp-specific T cells simultaneously disappeared from the CNS, whereas high frequencies of these cells remained present in the immune periphery. The functional affinity of both IFN-gamma- and IL-17-producing T cells did not change during EAE. Therefore, autoimmune pathology in this model did not correlate with specific PTX effects either on Th1 or Th17 cells regarding their kinetics and CNS migration.

Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation.

Rapamycin is an oral immunosuppressant drug previously reported to efficiently induce naturally occurring CD4(+)CD25(+)FoxP3(+) regulatory T ((n)T(reg)) cells re-establishing long-term immune self-tolerance in autoimmune diseases. We investigated the effect of rapamycin administration to SJL/j mice affected by PLP(139-151)-induced relapsing-remitting experimental autoimmune encephalomyelitis (RR-EAE). We found that oral or intraperitoneal treatment at the peak of disease or at the end of the first clinical attack, dramatically ameliorated the clinical course of RR-EAE. Treatment suspension resulted in early reappearance of disease. Clinical response was associated with reduced central nervous system demyelination and axonal loss. Rapamycin induced suppression of IFN-gamma, and IL-17 release from antigen-specific T cells in peripheral lymphoid organs. While CD4(+)FoxP3(+) cells were unaffected, we observed disappearance of CD4(+)CD45RB(high) effector T (T(eff)) cells and selective expansion of T(reg) cells bearing the CD4(+)CD45RB(low)FoxP3(+)CD25(+)CD103(+) extended phenotype. Finally, the dual action of rapamycin on both T(eff) and T(reg) cells resulted in modulation of their ratio that closely paralleled disease course. Our data show that rapamycin inhibits RR-EAE, provide evidence for the immunological mechanisms, and indicate this compound as a potential candidate for the treatment of multiple sclerosis.

Immune response to controlled release of immunomodulating peptides in a murine experimental autoimmune encephalomyelitis (EAE) model.

The effects of controlled release on immune response to an immunomodulating peptide were evaluated in a murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). The peptide, Ac-PLP-BPI-NH(2)-2 (Ac-HSLGKWLGHPDKF-(AcpGAcpGAcp)(2)-ITDGEATDSG-NH(2); Ac = acetyl, Acp = epsilon aminocaproic acid) was designed to suppress T-cell activation in response to PLP(139-151), an antigenic peptide in MS. Poly-lactide-co-glycolide (PLGA) microparticles containing Ac-PLP-BPI-NH(2)-2 (8+/-4 microm, 1.4+/-0.2% (w/w)) were prepared by a powder-in oil-in water emulsion-solvent evaporation method, sterilized and administered subcutaneously (s.c.) to SJL/J (H-2(s)) mice in which EAE had been induced by immunization with PLP(139-151). Treatment groups received Ac-PLP-BPI-NH(2)-2: (i) in solution by repeated i.v. or s.c. injection, (ii) in solution co-administered with blank PLGA microparticles, (iii) in solution co-administered with Ac-PLP-BPI-NH(2)-2 loaded microparticles, and (iv) as Ac-PLP-BPI-NH(2)-2 loaded microparticles. Administration of Ac-PLP-BPI-NH(2)-2 as an s.c. solution produced clinical scores and maintenance of body weight comparable to i.v. solution, but with reduced overall survival, presumably due to anaphylaxis. Administration as s.c. microparticles provided a somewhat less effective reduction in symptoms but with no toxicity during treatment. Thus, the results suggest that s.c. administration of Ac-PLP-BPI-NH(2)-2 microparticles can provide pharmacological efficacy and reduction in dosing frequency without increased toxicity.

Role of gut commensal microflora in the development of experimental autoimmune encephalomyelitis.

Mucosal tolerance has been considered a potentially important pathway for the treatment of autoimmune disease, including human multiple sclerosis and experimental conditions such as experimental autoimmune encephalomyelitis (EAE). There is limited information on the capacity of commensal gut bacteria to induce and maintain peripheral immune tolerance. Inbred SJL and C57BL/6 mice were treated orally with a broad spectrum of antibiotics to reduce gut microflora. Reduction of gut commensal bacteria impaired the development of EAE. Intraperitoneal antibiotic-treated mice showed no significant decline in the gut microflora and developed EAE similar to untreated mice, suggesting that reduction in disease activity was related to alterations in the gut bacterial population. Protection was associated with a reduction of proinflammatory cytokines and increases in IL-10 and IL-13. Adoptive transfer of low numbers of IL-10-producing CD25(+)CD4(+) T cells (>75% FoxP3(+)) purified from cervical lymph nodes of commensal bacteria reduced mice and in vivo neutralization of CD25(+) cells suggested the role of regulatory T cells maintaining peripheral immune homeostasis. Our data demonstrate that antibiotic modification of gut commensal bacteria can modulate peripheral immune tolerance that can protect against EAE. This approach may offer a new therapeutic paradigm in the treatment of multiple sclerosis and perhaps other autoimmune conditions.

Decreased severity of experimental autoimmune encephalomyelitis during resveratrol administration is associated with increased IL-17+IL-10+ T cells, CD4(-) IFN-gamma+ cells, and decreased macrophage IL-6 expression.

Experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis, is induced after injection of PLP(139-151) myelin peptide in complete Freund's adjuvant into SJL/J mice. During EAE, T cells and macrophages infiltrate the brain, produce cytokines IL-17, IFN-gamma, TNF-alpha, or IL-6, and bring about autoimmune neuroinflammation. However, infiltrating T cells which simultaneously produce IL-17 and IL-10 or infiltrating CD4(-) NKT cells that produce IFN-gamma protect against EAE. Resveratrol, a plant polyphenol, exhibits anti-inflammatory properties. To determine if resveratrol can relieve EAE, SJL/J mice were administered diets enriched in resveratrol at EAE injection. EAE symptoms were significantly less compared with controls in mice fed resveratrol. At day 56 of EAE, splenic T cells from mice fed 0%, 0.04% or 0.08% resveratrol that were restimulated with PLP(139-151) produced similar levels while splenic T cells from mice fed 0.02% resveratrol produced significantly higher levels of IL-17, IFN-gamma, and TNF-alpha. At peak EAE (day 14), mice fed resveratrol had higher numbers of IL-17+ T cells, IL-17+/IL-10+ T cells, and CD4(-)IFN-gamma+ cells in the brain and spleen compared with controls. Adoptive transfer of day 14 EAE encephalogenic T cells into mice fed resveratrol reduced the severity of EAE. In addition, resveratrol directly suppressed expression of IL-6 and IL-12/23 p40 but increased expression of IL-12 p35 and IL-23 p19 from macrophages. Therefore resveratrol protection against EAE is not associated with declines in IL-17+ T cells but is associated with rises in IL-17+/IL-10+ T cells and CD4(-)IFN-gamma+ and with repressed macrophage IL-6 and IL-12/23 p40 expression.

Soluble mannosylated myelin peptide inhibits the encephalitogenicity of autoreactive T cells during experimental autoimmune encephalomyelitis.

We have previously shown that immunization with a mannosylated myelin peptide in complete adjuvant induces tolerance instead of disease in experimental autoimmune encephalomyelitis (EAE), a rodent model for multiple sclerosis. In this report we demonstrate that treatment with a soluble mannosylated epitope of proteolipid protein (M-PLP(139-151)) significantly inhibits disease mediated by autoreactive myelin-specific T cells during EAE. Treatment with M-PLP(139-151), applied in different EAE models, significantly reduced the incidence of disease and the severity of clinical symptoms. Delayed-type hypersensitivity responses were abolished after peptide treatment, emphasizing the impact on peripheral T-cell reactivity. Histological analysis of spinal cord tissue from mice treated with M-PLP(139-151) revealed the presence of only few macrophages and T cells. Moreover, little expression of interferon-gamma, interleukin-23, or major histocompatibility complex class II antigen was detected. Immune modulation by M-PLP(139-151) was primarily antigen-specific because an irrelevant mannosylated peptide showed no significant effect on delayed-type hypersensitivity responses or on the course of EAE. Therefore, mannosylated antigens may represent a novel therapeutic approach for antigen-specific modulation of autoreactive T cells in vivo.

CD4+ T cell expressed CD80 regulates central nervous system effector function and survival during experimental autoimmune encephalomyelitis.

CD80 expressed on the surface of APCs provides a positive costimulatory signal to naive CD4+ T cells during activation. Therefore, it was hypothesized that treatment of SJL mice with various forms of anti-CD80 mAb during remission from the acute phase of relapsing experimental autoimmune encephalomyelitis (R-EAE) would ameliorate disease progression. We previously reported that treatment of SJL mice with anti-CD80 Fab during R-EAE remission blocked activation of T cells specific for endogenous myelin epitopes, inhibiting epitope spreading and clinical disease progression; however, treatment with the native form of the same anti-CD80 mAb exacerbated disease progression. The current data show that intact anti-CD80 mAb binds both CNS-infiltrating CD4+ T cells and CD11c+ dendritic cells and that exacerbation of R-EAE directly correlates with increased survival and activity of myelin-specific CD4+ T cells, while the percentage of CD11c+ dendritic cells in the CNS and their APC activity was not altered. In vitro data show that cross-linking CD80 on the surface of CD4+ T cells activated in the presence of Th1-promoting cytokines increases the level of T cell activation, effector function, and survival by directly up-regulating the expression levels of transcripts for T-bet, IFN-gamma, and Bcl-xL. These findings indicate a novel regulatory role for CD80-mediated intracellular signals in CD4+ T cells and have important implications for using anti-costimulatory molecule mAb therapy in established autoimmune disease.

Severe disease, unaltered leukocyte migration, and reduced IFN-gamma production in CXCR3-/- mice with experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a CD4(+) Th1 T cell-mediated disease of the CNS, used to study certain aspects of multiple sclerosis. CXCR3, the receptor for CXCL10, CXCL9, and CXCL11, is preferentially expressed on activated Th1 T cells and has been proposed to govern the migration of lymphocytes into the inflamed CNS during multiple sclerosis and EAE. Unexpectedly, CXCL10-deficient mice were susceptible to EAE, leaving uncertain what the role of CXCR3 and its ligands might play in this disease model. In this study, we report that CXCR3(-/-) mice exhibit exaggerated severity of EAE compared with wild-type (CXCR3(+/+)) littermate mice. Surprisingly, there were neither quantitative nor qualitative differences in CNS-infiltrating leukocytes between CXCR3(+/+) and CXCR3(-/-) mice with EAE. Despite these equivalent inflammatory infiltrates, CNS tissues from CXCR3(-/-) mice with EAE showed worsened blood-brain barrier disruption and more von Willebrand factor-immunoreactive vessels within inflamed spinal cords, as compared with CXCR3(+/+) mice. Spinal cords of CXCR3(-/-) mice with EAE demonstrated decreased levels of IFN-gamma, associated with reduced inducible NO synthase immunoreactivity, and lymph node T cells from CXCR3(-/-) mice primed with MOG(35-55) secreted less IFN-gamma in Ag-driven recall responses than cells from CXCR3(+/+) animals. CXCR3(-/-) lymph node T cells also showed enhanced Ag-driven proliferation, which was reduced by addition of IFN-gamma. Taken with prior findings, our data show that CXCL10 is the most relevant ligand for CXCR3 in EAE. CXCR3 does not govern leukocyte trafficking in EAE but modulates T cell IFN-gamma production and downstream events that affect disease severity.

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.