Abbreviations used: CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; MBP, myelin basic protein; MS, multiple sclerosis; NS, nonsense. The extent to which myelin-specific Th1 and Th17 cells contribute to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) is controversial. Combinations of interleukin (IL)-1, IL-6, and IL-23 with transforming growth factor  were used to differentiate myelin-specific T cell receptor transgenic T cells into Th17 cells, none of which could induce EAE, whereas Th1 cells consistently transferred disease. However, IL-6 was found to promote the differentiation of encephalitogenic Th17 cells. Further analysis of myelinspecific T cells that were encephalitogenic in spontaneous EAE and actively induced EAE demonstrated that T-bet expression was critical for pathogenicity, regardless of cytokine expression by the encephalitogenic T cells. These data suggest that encephalitogenicity of myelin-specific T cells appears to be mediated by a pathway dependent on T-bet and not necessarily pathway-specific end products, such as interferon  and IL-17. T-bet is

Pro-inflammatory T cells mediate autoimmune demyelination in multiple sclerosis. However, the factors driving their development and multiple sclerosis susceptibility are incompletely understood. We investigated how micro-RNAs, newly described as post-transcriptional regulators of gene expression, contribute to pathogenic T-cell differentiation in multiple sclerosis. miR-128 and miR-27b were increased in naïve and miR-340 in memory CD4(+) T cells from patients with multiple sclerosis, inhibiting Th2 cell development and favouring pro-inflammatory Th1 responses. These effects were mediated by direct suppression of B lymphoma Mo-MLV insertion region 1 homolog (BMI1) and interleukin-4 (IL4) expression, resulting in decreased GATA3 levels, and a Th2 to Th1 cytokine shift. Gain-of-function experiments with these micro-RNAs enhanced the encephalitogenic potential of myelin-specific T cells in experimental autoimmune encephalomyelitis. In addition, treatment of multiple sclerosis patient T cells with oligonucleotide micro-RNA inhibitors led to the restoration of Th2 responses. These data illustrate the biological significance and therapeutic potential of these micro-RNAs in regulating T-cell phenotypes in multiple sclerosis.

CD99 is a critical regulator of leukocyte transendothelial migration (TEM). Watson et al. describe the CD99 signaling pathway responsible. This involves a complex of CD99 with the A-kinase anchoring protein ezrin and soluble adenylyl cyclase that activates protein kinase A during leukocyte TEM.

Leukocyte transendothelial migration (TEM; diapedesis) is a critical event in immune surveillance and inflammation. Most TEM occurs at endothelial cell borders (paracellular). However, there is indirect evidence to suggest that at the tight junctions of the blood-brain barrier (BBB), leukocytes migrate directly through the endothelial cell body (transcellular). Why leukocytes migrate through the endothelial cell body rather than the cell borders is unknown. To test the hypothesis that the tightness of endothelial cell junctions influences the pathway of diapedesis, we developed an in vitro model of the BBB that possessed ten-fold higher electrical resistance than standard culture conditions and strongly expressed the BBB tight junction proteins claudin-5 and claudin-3. We found that paracellular TEM was still the predominant pathway (≥98%) and TEM was dependent on PECAM-1 and CD99. We show that endothelial tight junctions expressing claudin-5 are dynamic and undergo rapid remodeling during TEM. Membrane from the endothelial lateral border recycling compartment (LBRC) is mobilized to the exact site of tight junction remodeling. This preserves the endothelial barrier by sealing the intercellular gaps with membrane and engaging the migrating leukocyte with unligated adhesion molecules (PECAM-1 and CD99) as it crosses the cell border. These findings provide new insights into leukocyte-endothelial interactions at the BBB and suggest that tight junctions are more dynamic than previously appreciated.

Objective To determine if suppressing Nogo-A, an axonal inhibitory protein, will promote functional recovery in a murine model of multiple sclerosis (MS). Methods A small interfering RNA was developed to specifically suppress Nogo-A (siRNA-NogoA). The siRNA-NogoA silencing effect was evaluated in vitro and in vivo via immunohistochemistry. The siRNA was administered intravenously in two models of experimental autoimmune encephalomyelitis (EAE). Axonal repair was measured by upregulation of GAP43. ELISA, flow cytometry and 3H-thymidine incorporation was used to determine immunological changes in myelin-specific T cells in mice with EAE. Results The siRNA-NogoA suppressed Nogo-A expression in vitro and in vivo. Systemic administration of siRNA-NogoA ameliorated EAE and promoted axonal repair as demonstrated by enhanced GAP43+ axons in the lesions. Myelin-specific T cell proliferation and cytokine production were unchanged in the siRNA-NogoA treated mice. Interpretation Silencing Nogo-A in EAE promotes functional recovery. The therapeutic benefit appears to be mediated by axonal growth and repair, and is not attributable to changes in the encephalitogenic capacity of the myelin-specific T cells. Silencing Nogo-A may be a therapeutic option for MS patients to prevent permanent functional deficits caused by immune-mediated axonal damage.

Aquaporin-4 (AQP4)-specific T cells are expanded in neuromyelitis optica (NMO) patients and exhibit Th17 polarization. However, their pathogenic role in CNS autoimmune inflammatory disease is unclear. Although multiple AQP4 T-cell epitopes have been identified in WT C57BL/6 mice, we observed that neither immunization with those determinants nor transfer of donor T cells targeting them caused CNS autoimmune disease in recipient mice. In contrast, robust proliferation was observed following immunization of AQP4-deficient (AQP4 , but not WT, mice induced paralysis in recipient WT and B-cell-deficient mice. AQP4-specific Th17-polarized cells induced more severe disease than Th1-polarized cells. Clinical signs were associated with opticospinal infiltrates of T cells and monocytes. Fluorescent-labeled donor T cells were detected in CNS lesions. Visual system involvement was evident by changes in optical coherence tomography. Fine mapping of AQP4 p201-220 and p135-153 epitopes identified peptides within p201-220 but not p135-153, which induced clinical disease in 40% of WT mice by direct immunization. Our results provide a foundation to evaluate how AQP4-specific T cells contribute to AQP4-targeted CNS autoimmunity (ATCA) and suggest that pathogenic AQP4-specific T-cell responses are normally restrained by central tolerance, which may be relevant to understanding development of AQP4-reactive T cells in NMO.is a rare, disabling, and sometimes fatal CNS autoimmune inflammatory demyelinating disease that causes attacks of paralysis and visual loss (1). Immunologic, epidemiologic, and pathologic evidence suggests T cells have an important role in the etiology of NMO (1-3). Pathogenic aquaporin-4 (AQP4)-specific antibodies in NMO serum are predominantly IgG1, a T-cell-dependent IgG subclass (4), and T-cell-mediated CNS inflammation permits CNS entry of those antibodies (5, 6). NMO susceptibility is associated with allelic MHC II genes, in particular HLA-DR17 (DRB1*0301) in certain populations (7). AQP4-specific T cells have been identified in patients (8, 9), and T cells specific for dominant AQP4 epitopes exhibit Th17 polarization (8). It is therefore important to understand factors that control development and regulate the expression of AQP4-specific T cells in NMO.One cannot feasibly test whether AQP4-reactive T cells participate directly in CNS inflammation in NMO patients. Animal models can permit in vivo evaluation of the role of AQP4-specific T cells in CNS autoimmunity. Although multiple AQP4 T-cell epitopes have been identified in WT mice and rats (10-13), attempts to create AQP4-targeted experimental NMO ("ENMO") with clinical manifestations of CNS autoimmune disease by either direct immunization of those determinants or adoptive transfer of T cells targeting them have been unsuccessful (11-13).Recently, it was observed that immunization of AQP4-deficient (AQP4 −/− ) mice with AQP4 peptide (p) 135-153 elicited T-cell proliferation and that those T cells induced mild clinical disease in 70% of recipient WT mice (14...

Myelin-specific effector Th1 cells are able to perpetuate CNS inflammation in experimental autoimmune encephalomyelitis, an animal model representative of multiple sclerosis. Although the effects of cytokines in the CNS microenvironment on naive CD4+ T cells have been well described, much less is known about their ability to influence Ag-experienced effector cells. TGF-β is a multifunctioning cytokine present in the healthy and inflamed CNS with well-characterized suppressive effects on naive T cell functions. However, the effects of TGF-β on effector Th1 cells are not well defined. Using myelin-specific TCR transgenic mice, we demonstrate that TGF-β elicits differential effects on naive versus effector Th1 cells. TGF-β enhances cellular activation, proliferation, and cytokine production of effector Th1 cells; however, adoptive transfer of these cells into naive mice showed a reduction in encephalitogenicity. We subsequently demonstrate that the reduced encephalitogenic capacity is due to the ability of TGF-β to promote the self-regulation of Th1 effector cells via IL-10 production. These data demonstrate a mechanism by which TGF-β is able to suppress the encephalitogenicity of myelin-specific Th1 effector cells that is unique from its suppression of naive T cells.

SUMMARY Effector Th1 cells perpetuate inflammatory damage in a number of autoimmune diseases, including MS and its animal model EAE. Recently, a self-regulatory mechanism was described where effector Th1 cells produce the immunomodulatory cytokine IL-10 to dampen the inflammatory response in both normal and autoimmune inflammation. While the presence of TGF-β has been suggested to enhance and stabilize an IFN-γ+IL-10+ phenotype, the molecular mechanism is poorly understood. Additionally, in the context of adoptive transfer EAE, it is unclear if IL-10 acts on the transferred Th1 cells or on cells within the host. In the present study, using myelinspecific TCR-Tg mice, repetitive Ag stimulation of effector Th1 cells in the presence of TGF-β increased the population of IFN-γ+IL-10+ cells, which correlated with a decrease in EAE severity. Additionally, TGF-β signaling caused binding of smad4 to the IL-10 promoter, providing molecular evidence for TGF-β-mediated IL-10 production from Th1 effector cells. Lastly, this study demonstrates that IL-10 reduced encephalitogenic markers such as IFN-γ and T-bet on Th1 effector cells expressing the IL-10R, but also prevented recruitment of both transferred and host-derived inflammatory T cells. These data establish a regulatory mechanism by which highly activated Th1 effector cells modulate their pathogenicity through induction of IL-10.