The Median Eminence, A New Oligodendrogenic Niche in the Adult Mouse Brain.

The subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus are known as neurogenic niches. We show that the median eminence (ME) of the hypothalamus comprises BrdU+ newly proliferating cells co-expressing NG2 (oligodendrocyte progenitors) and RIP (pre-myelinating oligodendrocytes), suggesting their differentiation toward mature oligodendrocytes (OLs). ME cells can generate neurospheres (NS) in vitro, which differentiate mostly to OLs compared with SVZ-NS that typically generate neurons. Interestingly, this population of oligodendrocyte progenitors is increased in the ME from experimental autoimmune encephalomyelitis (EAE)-affected mice. Notably, the thrombospondin 1 (TSP1) expressed by astrocytes, acts as negative regulator of oligodendrogenesis in vitro and is downregulated in the ME of EAE mice. Importantly, transplanted ME-NS preferentially differentiate to MBP+ OLs compared with SVZ-NS in Shiverer mice. Hence, discovering the ME as a new site for myelin-producing cells has a great importance for advising future therapy for demyelinating diseases and spinal cord injury.

Circulating Picomolar Levels of CCL2 Downregulate Ongoing Chronic Experimental Autoimmune Encephalomyelitis by Induction of Regulatory Mechanisms.

Multiple sclerosis is an inflammatory disease of the CNS characterized by neurologic impairment resulting from primary demyelination and axonal damage. The pathogenic mechanisms of disease development include Ag-specific T cell activation and Th1 differentiation, followed by T cell and macrophage migration into the CNS. CCL2 is a chemokine that induces migration of monocytes, memory T cells, and dendritic cells. We previously demonstrated that picomolar levels of CCL2 strongly restrict the development of inflammation in models of inflammatory bowel disease. Moreover, CCR2 deficiency in T cells promotes a program inducing the accumulation of Foxp3+ regulatory T cells while decreasing the levels of Th17 cells in vivo. In the current study, the effect of picomolar levels of CCL2 on the autoimmune inflammatory response associated with a multiple sclerosis-like disease in mice was analyzed. We found that low dosages of CCL2 were effective in suppressing MOG-induced experimental autoimmune encephalomyelitis (EAE), and they downregulated chronic EAE. The modulation of EAE by CCL2 was associated with downregulation of Th1/Th17 cells and upregulation of TGF-ß and induction of regulatory CD4+Foxp3 T cells. Most strikingly, these low levels of CCL2 induced formation of highly functional regulatory T cells. Thus, this study strongly supports the potential use of CCL2 as a regulatory mediator for treating inflammatory autoimmune diseases.

CCR2 Regulates the Immune Response by Modulating the Interconversion and Function of Effector and Regulatory T Cells.

Chemokines and chemokine receptors establish a complex network modulating immune cell migration and localization. These molecules were also suggested to mediate the differentiation of leukocytes; however, their intrinsic, direct regulation of lymphocyte fate remained unclear. CCR2 is the main chemokine receptor inducing macrophage and monocyte recruitment to sites of inflammation, and it is also expressed on T cells. To assess whether CCR2 directly regulates T cell responses, we followed the fates of CCR2-/- T cells in T cell-specific inflammatory models. Our in vitro and in vivo results show that CCR2 intrinsically mediates the expression of inflammatory T cell cytokines, and its absence on T cells results in attenuated colitis progression. Moreover, CCR2 deficiency in T cells promoted a program inducing the accumulation of Foxp3+ regulatory T cells, while decreasing the levels of Th17 cells in vivo, indicating that CCR2 regulates the immune response by modulating the effector/regulatory T ratio.

EBI2 Is Highly Expressed in Multiple Sclerosis Lesions and Promotes Early CNS Migration of Encephalitogenic CD4 T Cells.

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1ß), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs.

Post-CNS-inflammation expression of CXCL12 promotes the endogenous myelin/neuronal repair capacity following spontaneous recovery from multiple sclerosis-like disease.

BACKGROUND: Demyelination and axonal degeneration, hallmarks of multiple sclerosis (MS), are associated with the central nervous system (CNS) inflammation facilitated by C-X-C motif chemokine 12 (CXCL12) chemokine. Both in MS and in experimental autoimmune encephalomyelitis (EAE), the deleterious CNS inflammation has been associated with upregulation of CXCL12 expression in the CNS. We investigated the expression dynamics of CXCL12 in the CNS with progression of clinical EAE and following spontaneous recovery, with a focus on CXCL12 expression in the hippocampal neurogenic dentate gyrus (DG) and in the corpus callosum (CC) of spontaneously recovered mice, and its potential role in promoting the endogenous myelin/neuronal repair capacity. METHODS: CNS tissue sections from mice with different clinical EAE phases or following spontaneous recovery and in vitro differentiated adult neural stem cell cultures were analyzed by immunofluorescent staining and confocal imaging for detecting and enumerating neuronal progenitor cells (NPCs) and oligodendrocyte precursor cells (OPCs) and for expression of CXCL12. RESULTS: Our expression dynamics analysis of CXCL12 in the CNS with EAE progression revealed elevated CXCL12 expression in the DG and CC, which persistently increases following spontaneous recovery even though CNS inflammation has subsided. Correspondingly, the numbers of NPCs and OPCs in the DG and CC, respectively, of EAE-recovered mice increased compared to that of naïve mice (NPCs, p < 0.0001; OPCs, p < 0.00001) or mice with active disease (OPCs, p < 0.0005). Notably, about 30 % of the NPCs and unexpectedly also OPCs (~50 %) express CXCL12, and their numbers in DG and CC, respectively, are higher in EAE-recovered mice compared with naïve mice and also compared with mice with ongoing clinical EAE (CXCL12(+) NPCs, p < 0.005; CXCL12(+) OPCs, p < 0.0005). Moreover, a significant proportion (>20 %) of the CXCL12(+) NPCs and OPCs co-express the CXCL12 receptor, CXCR4, and their numbers significantly increase with recovery from EAE not only relative to naïve mice (p < 0.0002) but also to mice with ongoing EAE (p < 0.004). CONCLUSIONS: These data link CXCL12 expression in the DG and CC of EAE-recovering mice to the promotion of neuro/oligodendrogenesis generating CXCR4(+) CXCL12(+) neuronal and oligodendrocyte progenitor cells endowed with intrinsic neuro/oligondendroglial differentiation potential. These findings highlight the post-CNS-inflammation role of CXCL12 in augmenting the endogenous myelin/neuronal repair capacity in MS-like disease, likely via CXCL12/CXCR4 autocrine signaling.

Role of a Novel Human Leukocyte Antigen-DQA1*01:02;DRB1*15:01 Mixed Isotype Heterodimer in the Pathogenesis of "Humanized" Multiple Sclerosis-like Disease.

Gene-wide association and candidate gene studies indicate that the greatest effect on multiple sclerosis (MS) risk is driven by the HLA-DRB1*15:01 allele within the HLA-DR15 haplotype (HLA-DRB1*15:01-DQA1*01:02-DQB1*0602-DRB5*01:01). Nevertheless, linkage disequilibrium makes it difficult to define, without functional studies, whether the functionally relevant effect derives from DRB1*15:01 only, from its neighboring DQA1*01:02-DQB1*06:02 or DRB5*01:01 genes of HLA-DR15 haplotype, or from their combinations or epistatic interactions. Here, we analyzed the impact of the different HLA-DR15 haplotype alleles on disease susceptibility in a new "humanized" model of MS induced in HLA-transgenic (Tg) mice by human oligodendrocyte-specific protein (OSP)/claudin-11 (hOSP), one of the bona fide potential primary target antigens in MS. We show that the hOSP-associated MS-like disease is dominated by the DRB1*15:01 allele not only as the DRA1*01:01;DRB1*15:01 isotypic heterodimer but also, unexpectedly, as a functional DQA1*01:02;DRB1*15:01 mixed isotype heterodimer. The contribution of HLA-DQA1/DRB1 mixed isotype heterodimer to OSP pathogenesis was revealed in (DRB1*1501xDQB1*0602)F1 double-Tg mice immunized with hOSP(142-161) peptide, where the encephalitogenic potential of prevalent DRB1*1501/hOSP(142-161)-reactive Th1/Th17 cells is hindered due to a single amino acid difference in the OSP(142-161) region between humans and mice; this impedes binding of DRB1*1501 to the mouse OSP(142-161) epitope in the mouse CNS while exposing functional binding of mouse OSP(142-161) to DQA1*01:02;DRB1*15:01 mixed isotype heterodimer. This study, which shows for the first time a functional HLA-DQA1/DRB1 mixed isotype heterodimer and its potential association with disease susceptibility, provides a rationale for a potential effect on MS risk from DQA1*01:02 through functional DQA1*01:02;DRB1*15:01 antigen presentation. Furthermore, it highlights a potential contribution to MS risk also from interisotypic combination between products of neighboring HLA-DR15 haplotype alleles, in this case the DQA1/DRB1 combination.

Cannabidiol, a non-psychoactive cannabinoid, leads to EGR2-dependent anergy in activated encephalitogenic T cells.

BACKGROUND: Cannabidiol (CBD), the main non-psychoactive cannabinoid, has been previously shown by us to ameliorate clinical symptoms and to decrease inflammation in myelin oligodendrocyte glycoprotein (MOG)35-55-induced mouse experimental autoimmune encephalomyelitis model of multiple sclerosis as well as to decrease MOG35-55-induced T cell proliferation and IL-17 secretion. However, the mechanisms of CBD anti-inflammatory activities are unclear. METHODS: Here we analyzed the effects of CBD on splenocytes (source of accessory T cells and antigen presenting cells (APC)) co-cultured with MOG35-55-specific T cells (TMOG) and stimulated with MOG35-55. Using flow cytometry, we evaluated the expression of surface activation markers and inhibitory molecules on T cells and B cells. TMOG cells were purified using CD4 positive microbead selection and submitted for quantitative PCR and microarray of mRNA transcript analyzes. Cell signaling studies in purified TMOG were carried out using immunoblotting. RESULTS: We found that CBD leads to upregulation of CD69 and lymphocyte-activation gene 3 (LAG3) regulatory molecules on CD4(+)CD25(-) accessory T cells. This subtype of CD4(+)CD25(-)CD69(+)LAG3(+) T cells has been recognized as induced regulatory phenotype promoting anergy in activated T cells. Indeed, we observed that CBD treatment results in upregulation of EGR2 (a key T cell anergy inducer) mRNA transcription in stimulated TMOG cells. This was accompanied by elevated levels of anergy promoting genes such as IL-10 (anti-inflammatory cytokine), STAT5 (regulatory factor), and LAG3 mRNAs, as well as of several enhancers of cell cycle arrest (such as Nfatc1, Casp4, Cdkn1a, and Icos). Moreover, CBD exposure leads to a decrease in STAT3 and to an increase in STAT5 phosphorylation in TMOG cells, positive and negative regulators of Th17 activity, respectively. In parallel, we observed decreased levels of major histocompatibility complex class II (MHCII), CD25, and CD69 on CD19(+) B cells following CBD treatment, showing diminished antigen presenting capabilities of B cells and reduction in their pro-inflammatory functions. CONCLUSIONS: Our data suggests that CBD exerts its immunoregulatory effects via induction of CD4(+)CD25(-)CD69(+)LAG3(+) cells in MOG35-55-activated APC/TMOG co-cultures. This is accompanied by EGR2-dependent anergy of stimulated TMOG cells as well as a switch in their intracellular STAT3/STAT5 activation balance leading to the previously observed decrease in Th17 activity.

DQB1*06:02-Associated Pathogenic Anti-Myelin Autoimmunity in Multiple Sclerosis-Like Disease: Potential Function of DQB1*06:02 as a Disease-Predisposing Allele.

Susceptibility to multiple sclerosis (MS) has been linked mainly to the HLA-DRB1 locus, with the HLA-DR15 haplotype (DRB1*1501-DQA1*0102-DQB1*0602-DRB5*0101) dominating MS risk in Caucasians. Although genes in the HLA-II region, particularly DRB1*1501, DQA1*0102-DQB1*0602, are in tight linkage disequilibrium, genome-wide-association, and gene candidate studies identified the DRB1*15:01 allele as the primary risk factor in MS. Many genetic and immune-functional studies have indicated DRB1*15:01 as a primary risk factor in MS, while only some functional studies suggested a disease-modifying role for the DRB5*01 or DQB1*06 alleles. In this respect, the susceptibility of DRB1*15:01-transgenic (Tg) mice to myelin basic protein- or myelin oligodendrocyte glycoprotein-induced MS-like disease is consistent with primary contribution of DRB1*15:01 to HLA-DR15+ MS. The studies summarized here show that susceptibility to MS-like disease, induced in HLA-"humanized" mice by myelin oligodendrocytic basic protein or by the proteolipid protein, one of the most prominent encephalitogenic target antigens implicated in human MS, is determined by DQB1*06:02, rather than by the DRB1*15:01 allele. These findings not only offer a rationale for a potential role for DQB1*06:02 in predisposing susceptibility to MS, but also suggest a more complex and differential functional role for HLA-DR15 alleles, depending on the primary target myelin antigen. However, the conflict between these findings in HLA-Tg mice and the extensive genome-wide-association studies, which could not detect any significant effect from the DQB1*06:02 allele on MS risk, is rather puzzling. Functional analysis of MS PBLs for DQB1*06:02-associated anti-myelin autoimmunity may indicate whether or not DQB1*06:02 is associated with MS pathogenesis.

From classic to spontaneous and humanized models of multiple sclerosis: impact on understanding pathogenesis and drug development.

Multiple sclerosis (MS), a demyelinating disease of the central nervous system (CNS), presents as a complex disease with variable clinical and pathological manifestations, involving different pathogenic pathways. Animal models, particularly experimental autoimmune encephalomyelitis (EAE), have been key to deciphering the pathophysiology of MS, although no single model can recapitulate the complexity and diversity of MS, or can, to date, integrate the diverse pathogenic pathways. Since the first EAE model was introduced decades ago, multiple classic (induced), spontaneous, and humanized EAE models have been developed, each recapitulating particular aspects of MS pathogenesis. The advances in technologies of genetic ablation and transgenesis in mice of C57BL/6J background and the development of myelin-oligodendrocyte glycoprotein (MOG)-induced EAE in C57BL/6J mice yielded several spontaneous and humanized EAE models, and resulted in a plethora of EAE models in which the role of specific genes or cell populations could be precisely interrogated, towards modeling specific pathways of MS pathogenesis/regulation in MS. Collectively, the numerous studies on the different EAE models contributed immensely to our basic understanding of cellular and molecular pathways in MS pathogenesis as well as to the development of therapeutic agents: several drugs available today as disease modifying treatments were developed from direct studies on EAE models, and many others were tested or validated in EAE. In this review, we discuss the contribution of major classic, spontaneous, and humanized EAE models to our understanding of MS pathophysiology and to insights leading to devising current and future therapies for this disease.

Bispecificity for myelin and neuronal self-antigens is a common feature of CD4 T cells in C57BL/6 mice.

The recognition of multiple ligands by a single TCR is an intrinsic feature of T cell biology, with important consequences for physiological and pathological processes. Polyspecific T cells targeting distinct self-antigens have been identified in healthy individuals as well as in the context of autoimmunity. We have previously shown that the 2D2 TCR recognizes the myelin oligodendrocyte glycoprotein epitope (MOG)35-55 as well as an epitope within the axonal protein neurofilament medium (NF-M15-35) in H-2(b) mice. In this study, we assess whether this cross-reactivity is a common feature of the MOG35-55-specific T cell response. To this end, we analyzed the CD4 T cell response of MOG35-55-immunized C57BL/6 mice for cross-reactivity with NF-M15-35. Using Ag recall responses, we established that an important proportion of MOG35-55-specific CD4 T cells also responded to NF-M15-35 in all mice tested. To study the clonality of this response, we analyzed 22 MOG35-55-specific T cell hybridomas expressing distinct TCR. Seven hybridomas were found to cross-react with NF-M15-35. Using an alanine scan of NF-M18-30 and an in silico predictive model, we dissected the molecular basis of cross-reactivity between MOG35-55 and NF-M15-35. We established that NF-M F24, R26, and V27 proved important TCR contacts. Strikingly, the identified TCR contacts are conserved within MOG38-50. Our data indicate that due to linear sequence homology, part of the MOG35-55-specific T cell repertoire of all C57BL/6 mice also recognizes NF-M15-35, with potential implications for CNS autoimmunity.

An immunomodulating motif of the HIV-1 fusion protein is chirality-independent: implications for its mode of action.

An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between D- and L-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a D-enantiomer of ISLAD CM (termed ISLAD D-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35-55)-specific line T-cells to the same extent as the l-motif form. Moreover, the D- and L-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD D-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35-55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the D-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies.

Cannabinoids decrease the th17 inflammatory autoimmune phenotype.

Cannabinoids, the Cannabis constituents, are known to possess anti-inflammatory properties but the mechanisms involved are not understood. Here we show that the main psychoactive cannabinoid, Δ-9-tetrahydrocannabinol (THC), and the main nonpsychoactive cannabinoid, cannabidiol (CBD), markedly reduce the Th17 phenotype which is known to be increased in inflammatory autoimmune pathologies such as Multiple Sclerosis. We found that reactivation by MOG35-55 of MOG35-55-specific encephalitogenic T cells (cells that induce Experimental Autoimmune Encephalitis when injected to mice) in the presence of spleen derived antigen presenting cells led to a large increase in IL-17 production and secretion. In addition, we found that the cannabinoids CBD and THC dose-dependently (at 0.1-5 µM) suppressed the production and secretion of this cytokine. Moreover, the mRNA and protein of IL-6, a key factor in Th17 induction, were also decreased. Pretreatment with CBD also resulted in increased levels of the anti-inflammatory cytokine IL-10. Interestingly, CBD and THC did not affect the levels of TNFα and IFNγ. The downregulation of IL-17 secretion by these cannabinoids does not seem to involve the CB1, CB2, PPARγ, 5-HT1A or TRPV1 receptors. In conclusion, the results show a unique cannabinoid modulation of the autoimmune cytokine milieu combining suppression of the pathogenic IL-17 and IL-6 cytokines along with boosting the expression of the anti-inflammatory cytokine IL-10.

A highly conserved sequence associated with the HIV gp41 loop region is an immunomodulator of antigen-specific T cells in mice.

Modulation of T-cell responses by HIV occurs via distinct mechanisms, 1 of which involves inactivation of T cells already at the stage of virus-cell fusion. Hydrophobic portions of the gp41 protein of the viral envelope that contributes to membrane fusion may modulate T-cell responsiveness. Here we found a highly conserved sequence (termed "ISLAD") that is associated with the membranotropic gp41 loop region. We showed that ISLAD has the ability to bind the T-cell membrane and to interact with the T-cell receptor (TCR) complex. Furthermore, ISLAD inhibited T-cell proliferation and interferon-γ secretion that resulted from TCR engagement through antigen-presenting cells. Moreover, administering ISLAD (10 µg per mouse) to an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis reduced the severity of the disease. This was related to the inhibition of pathogenic T-cell proliferation and to reduced pro-inflammatory cytokine secretion in the lymph nodes of ISLAD-treated EAE mice. The data suggest that T-cell inactivation by HIV during membrane fusion may lie in part in this conserved sequence associated with the gp41 loop region.

DQB1*0602 rather than DRB1*1501 confers susceptibility to multiple sclerosis-like disease induced by proteolipid protein (PLP).

BACKGROUND: Multiple sclerosis (MS) is associated with pathogenic autoimmunity primarily focused on major CNS-myelin target antigens including myelin basic protein (MBP), proteolipidprotein (PLP), myelin oligodendrocyte protein (MOG). MS is a complex trait whereby the HLA genes, particularly class-II genes of HLA-DR15 haplotype, dominate the genetic contribution to disease-risk. Due to strong linkage disequilibrium in HLA-II region, it has been hard to establish precisely whether the functionally relevant effect derives from the DRB1*1501, DQA1*0102-DQB1*0602, or DRB5*0101 loci of HLA-DR15 haplotype, their combinations, or their epistatic interactions. Nevertheless, most genetic studies have indicated DRB1*1501 as a primary risk factor in MS. Here, we used 'HLA-humanized' mice to discern the potential relative contribution of DRB1*1501 and DQB1*0602 alleles to susceptibility to "humanized" MS-like disease induced by PLP, one of the most prominent and encephalitogenic target-antigens implicated in human MS. METHODS: The HLA-DRB1*1501- and HLA-DQB1*0602-Tg mice (MHC-II(-/-)), and control non-HLA-DR15-relevant-Tg mice were immunized with a set of overlapping PLP peptides or with recombinant soluble PLP for induction of "humanized" MS-like disease, as well as for ex-vivo analysis of immunogenic/immunodominant HLA-restricted T-cell epitopes and associated cytokine secretion profile. RESULTS: PLP autoimmunity in both HLA-DR15-Tg mice was focused on 139-151 and 175-194 epitopes. Strikingly, however, the HLA-DRB1*1501-transgenics were refractory to disease induction by any of the overlapping PLP peptides, while HLA-DQB1*0602 transgenics were susceptible to disease induction by PLP139-151 and PLP175-194 peptides. Although both transgenics responded to both peptides, the PLP139-151- and PLP175-194-reactive T-cells were directed to Th1/Th17 phenotype in DQB1*0602-Tg mice and towards Th2 in DRB1*1501-Tg mice. CONCLUSIONS: While genome studies map a strong MS susceptibility effect to the region of DRB1*1501, our findings offer a rationale for potential involvement of pathogenic DQ6-associated autoimmunity in MS. Moreover, that DQB1*0602, but not DRB1*1501, determines disease-susceptibility to PLP in HLA-transgenics, suggests a potential differential, functional role for DQB1*0602 as a predisposing allele in MS. This, together with previously demonstrated disease-susceptibility to MBP and MOG in DRB1*1501-transgenics, also suggests a differential role for DRB1*1501 and DQB1*0602 depending on target antigen and imply a potential complex 'genotype/target antigen/phenotype' relationship in MS heterogeneity.

'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS) yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and "epitope spread", have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such "multi-epitope-targeting" approach in murine experimental autoimmune encephalomyelitis (EAE) associated with a single ("classical") or multiple ("complex") anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc) encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as "multi-epitope-targeting" agents. Y-MSPc was superior to peptide(s) in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells). Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of "classical" or "complex EAE" or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a "multi-epitope-targeting" strategy is required for effective immune-specific therapy of organ-specific autoimmune diseases associated with complex and dynamic pathogenic autoimmunity, such as MS; our data further demonstrate that the "multi-epitope-targeting" approach to therapy is optimized through specifically designed multi-epitope-proteins, rather than myelin peptide cocktails, as "multi-epitope-targeting" agents. Such artificial multi-epitope proteins can be tailored to other organ-specific autoimmune diseases.

Cannabidiol inhibits pathogenic T cells, decreases spinal microglial activation and ameliorates multiple sclerosis-like disease in C57BL/6 mice.

BACKGROUND AND PURPOSE: Cannabis extracts and several cannabinoids have been shown to exert broad anti-inflammatory activities in experimental models of inflammatory CNS degenerative diseases. Clinical use of many cannabinoids is limited by their psychotropic effects. However, phytocannabinoids like cannabidiol (CBD), devoid of psychoactive activity, are, potentially, safe and effective alternatives for alleviating neuroinflammation and neurodegeneration. EXPERIMENTAL APPROACH: We used experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) in C57BL/6 mice, as a model of multiple sclerosis. Using immunocytochemistry and cell proliferation assays we evaluated the effects of CBD on microglial activation in MOG-immunized animals and on MOG-specific T-cell proliferation. KEY RESULTS: Treatment with CBD during disease onset ameliorated the severity of the clinical signs of EAE. This effect of CBD was accompanied by diminished axonal damage and inflammation as well as microglial activation and T-cell recruitment in the spinal cord of MOG-injected mice. Moreover, CBD inhibited MOG-induced T-cell proliferation in vitro at both low and high concentrations of the myelin antigen. This effect was not mediated via the known cannabinoid CB(1) and CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: CBD, a non-psychoactive cannabinoid, ameliorates clinical signs of EAE in mice, immunized against MOG. Suppression of microglial activity and T-cell proliferation by CBD appeared to contribute to these beneficial effects.

Involvement of phosphodiesterases in autoimmune diseases.

We have previously shown that several phosphodiesterase (PDE) subtypes are up-regulated in muscles and lymph node cells (LNC) of rats with experimental autoimmune myasthenia gravis (EAMG). In the present study we investigated PDE expression during the course of EAMG and experimental allergic encephalomyelitis (EAE) and found that the up-regulated expression of selected PDE subtypes in both experimental models is correlated with disease severity. In EAMG, PDE expression is correlated also with muscle damage. A similar up-regulation of PDE was also observed in the respective human diseases, MG and multiple sclerosis (MS). Our findings suggest that change in PDE expression levels is a general phenomenon in autoimmune diseases and may also be used as a marker for disease severity.

The myelin-associated oligodendrocytic basic protein (MOBP) as a relevant primary target autoantigen in multiple sclerosis.

Multiple sclerosis (MS) is a disease of the human CNS, characterized by perivascular inflammation, demyelination and axonal damage. Although the etiology of MS is unknown, it is believed that the disease results from destructive autoimmune mechanisms, presumably initiated by abnormal activation of potentially pathogenic autoimmune T-cells recognizing CNS components. The myelin-associated oligodendrocyte basic protein (MOBP), a relatively abundant CNS-specific myelin protein, which plays a role in stabilizing the myelin sheath in the CNS, has recently been implicated in the pathogenesis of MS. Here we review studies showing that MOBP is as an important candidate target antigen in MS as the other widely studied target antigens, myelin basic protein (MBP), proteolipid protein (PLP), and myelin oligodendrocyte glycoprotein (MOG). The studies summarized below indicate that T-cell autoimmunity against MOBP can be detected in MS patients; T-cells reactive against MOBP can be pathogenic in several mouse strains as well as in the "humanized" HLA-DR15-Tg mice; and, that the HLA-DQ6-restricted, but not HLA-DR15-restricted, MOBP-reactive T-cells cause in HLA-DR15-Tg mice MS-like clinical disease associated with perivascular and parenchymal infiltration, demyelination, axonal loss, and optic neuritis. Accordingly, the MOBP should be considered a bona fide primary target antigen in MS, in addition to MBP, PLP, and MOG.

HLA-DQB1*0602 determines disease susceptibility in a new "humanized" multiple sclerosis model in HLA-DR15 (DRB1*1501;DQB1*0602) transgenic mice.

The susceptibility to multiple sclerosis (MS), a chronic neurological autoimmune disease that primarily targets CNS myelin, has long been associated with HLA class-II genes. Although several other HLA and non-HLA disease predisposing alleles have been identified, alleles of the HLA-DR15 haplotype (DRB1*1501, DRB5*0101, and DQB1*0602) remain the strongest susceptibility factor. Many studies have suggested that the HLA-DRB1*1501 allele determines MS-associated susceptibility. However, due to strong linkage disequilibrium within the HLA class II region, it has been difficult to unequivocally determine the relative roles of the DRB1*1501 and DQB1*0602 products. In this study we use HLA class-II transgenic mice to illuminate the relative contributions of the DRB1*1501 and DQB1*0602 alleles or their combination to susceptibility toward a new "humanized" MS-like disease induced by myelin-associated oligodendrocytic basic protein (MOBP). Although many immunological studies have focused overwhelmingly on the role of the HLA-DRB1*1501 product in MS, we show that HLA-DRB1*1501 transgenics are refractory to MOBP disease induction, whereas the HLA-DQB1*0602 transgenics are susceptible via T cells reactive against MOBP15-36 and MOBP55-77 encephalitogenic epitopes. Although both transgenics react against these epitopes, the MOBP15-36- and MOBP55-77-reactive T cells are of Th2-type in HLA-DRB1*1501 transgenics and are pathogenic Th1/Th17 cells in the HLA-DQB1*0602 transgenic mice. This new humanized model of MS further implicates autoimmunity against MOBP in MS pathogenesis, provides the first evidence of pathogenic HLA-DQ-associated anti-myelin autoimmunity, and is the first to offer a rationale for HLA-DQB1*0602 association with MS. These findings have important bearing on the candidacy of the DQB1*0602 allele as a genetic risk factor for MS.