Laquinimod is an orally administered compound that is under investigation in relapsing-remitting multiple sclerosis. To understand the mechanism by which laquinimod exerts its clinical effects, we have performed human and murine studies assessing its immunomodulatory properties. In experimental autoimmune encephalomyelitis, the therapeutic administration of laquinimod beginning during the recovery of SJL mice, prevented further relapses as expected and strongly reduced infiltration of CD4+ and CD8+ T cells in the central nervous system. We hypothesized that this beneficial effect was mediated by dendritic cells, since we and others found a modulation of different dendritic cell subsets under treatment. According to the findings on antigen-presenting cells in the murine system, we found a reduced capacity of human monocyte-derived dendritic cells treated with therapeutic concentrations of laquinimod, upon maturation with lipopolysaccharide, to induce CD4+ T cell proliferation and secretion of pro-inflammatory cytokines. Furthermore, laquinimod treatment of mature dendritic cells resulted in a decreased chemokine production by both murine and human dendritic cells, associated with a decreased monocyte chemo-attraction. In laquinimod-treated patients with multiple sclerosis we consistently found reduced chemokine and cytokine secretion by conventional CD1c+ dendritic cells upon lipopolysaccharide stimulation. Similarly to the animal model of relapsing-remitting multiple sclerosis, dendritic cell subsets were altered in patients upon laquinimod treatment, as the number of conventional CD1c+ and plasmacytoid CD303+ dendritic cells were decreased within peripheral blood mononuclear cells. Moreover, laquinimod treatment in patients with multiple sclerosis and mice modified the maturation of dendritic cells demonstrated by an upregulation of CD86 expression in vivo. Our data suggest that inhibition of the NF-κB pathway is responsible for the changes observed in dendritic cell maturation and functions. These findings indicate that laquinimod exhibits its disease-modulating activity in multiple sclerosis by downregulating immunogenicity of dendritic cell responses. We suggest that monitoring dendritic cell properties in multiple sclerosis should be implemented in future therapeutic trials.

Deubiquitination of NF-B members by IntroductionCYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. 1 CYLD removes lysine-63 polyubiquitin chains from distinct members of the NF-B pathway 2-4 and mutations in CYLD dysregulate NF-B activity.Complete deletion of CYLD in mice (CYLD ko ) renders them susceptible to skin tumors, 5 but the CYLD ko mice do not display alterations of the immune system, as we could show for B-6 and T-cell development (A.W., N.H., S. Reissig, manuscript in preparation). Other knockout mouse models of CYLD point to a role for flCYLD in immunity, including hyperinduction of IFN␣ in virus-infected dendritic cells (DCs) 7 as well as protection from infections in its absence. 8 However, mice exclusively expressing the naturally occurring short splice variant of CYLD, sCYLD, are characterized by lymphomegaly, splenomegaly, and dramatic increases in B-cell numbers 6 caused by aberrant NF-B signaling. Because this pathway is important for the function of DCs, 9 which orchestrate innate and adaptive immune responses, we investigated the effect of sCYLD overexpression on DC function. We found CYLD ex7/8 DCs to be hyperresponsive to LPS, capable of inducing superior T-cell expansion on DC immunization in vivo, and able to suppress tolerance induced by ␣-DEC-205:OVA administration. As a molecular basis for this phenotype, we identified increased nuclear Bcl-3, p50, and p65 in resting and stimulated CYLD ex7/8 bone marrowderived DCs (BMDCs). Methods Mice and BMDC generationC57BL/6 wild-type (WT), CYLD ko , 5 and CYLD ex7/8 6 mice 6 to 8 weeks old were used as recipients and for BMDC generation as previously described. 10 St42 TCR Tg mice have been previously described. 11 OT-I mice 12 were obtained from Christian Kurts (Institute for Molecular Medicine & Experimental Immunology, Bonn, Germany). Approval for these studies was obtained from the review board of the Federal State of Rhineland-Palatinate, Germany. All animal experiments were in accordance with the guidelines of the Central Animal Facility Institution of the University of Mainz. Flow cytometryFACSCanto cytometer and FlowJo software (Tree Star, Ashland, OR) using ␣-CD8 and ␣-CD90.1/CD45.1 (eBioscience, San Diego, CA) was used. ␣-IL-6, IL-10, and TNF-␣ antibodies from Cytometric Bead Array Flex system (Becton Dickinson, Franklin Lakes, NJ) were used and analyzed with FCAP Array Software (BD Biosciences, Mountain View, CA). Immune toleranceRecipient mice were given 10 6 adoptively transferred OT-I cells, immunized with 20 g DEC-205:OVA, and boosted with 50 g OVA protein (Sigma-Aldrich, St Louis, MO) as previously described. 13Western blot analysis and electrophoretic mobility shift assay Western blot analysis was performed as previously described. 8 Nuclear and cytoplasmic fractions were prepared according to standard procedures. 14,15 NF-B Consensus Oligonucleotide was purchased from For personal use only. on May 7, 2018. by guest www.bloodjournal.org ...

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αβ T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, thereby regulating NF-κB-mediated gene expression. This study thus reveals intrinsic functions of Bcl-3 in Treg cells, identifies Bcl-3 as a potential prognostic marker for colitis and illustrates the mechanism by which Bcl-3 regulates NF-κB activity in Tregs to prevent colitis.