Long-term real-world effectiveness and safety of fingolimod over 5 years in Germany.

OBJECTIVE: To evaluate the 5-year real-world benefit-risk profile of fingolimod in patients with relapsing-remitting MS (RRMS) in Germany. METHODS: Post-Authorization Non-interventional German sAfety study of GilEnyA (PANGAEA) is a non-interventional real-world study to prospectively assess the effectiveness and safety of fingolimod in routine clinical practice in Germany. The follow-up period comprised 5 years. Patients were included if they had been diagnosed with RRMS and had been prescribed fingolimod as part of clinical routine. There were no exclusion criteria except the contraindications for fingolimod as defined in the European label. The effectiveness and safety analysis set comprised 4032 and 4067 RRMS patients, respectively. RESULTS: At the time of the 5-year follow-up of PANGAEA, 66.57% of patients still continued fingolimod therapy. Annualized relapse rates decreased from baseline 1.5 ± 1.15 to 0.42 ± 0.734 at year 1 and 0.21 ± 0.483 at year 5, and the disability status remained stable, as demonstrated by the Expanded Disability Status Scale mean change from baseline (0.1 ± 2.51), the decrease of the Multiple Sclerosis Severity Score from 5.1 ± 2.59 at baseline to 3.9 ± 2.31 at the 60-months follow-up, and the percentage of patients with 'no change' in the Clinical Global Impression scale at the 60-months follow-up (78.11%). Adverse events (AE) occurring in 75.04% of patients were in line with the known safety profile of fingolimod and were mostly non-serious AE (33.62%) and non-serious adverse drug reactions (50.59%; serious AE 4.98%; serious ADR 10.82%). CONCLUSIONS: PANGAEA demonstrated the sustained beneficial effectiveness and safety of fingolimod in the long-term real-world treatment of patients with RRMS.

The Change of Fingolimod Patient Profiles over Time: A Descriptive Analysis of Two Non-Interventional Studies PANGAEA and PANGAEA 2.0.

(1) Background: Fingolimod (Gilenya®) was the first oral treatment for patients with relapsing-remitting multiple sclerosis (RRMS). Since its approval, the treatment landscape has changed enormously. (2) Methods: Data of PANGAEA and PANGAEA 2.0, two German real-world studies, were descriptively analysed for possible evolution of patient profiles and treatment behavior. Both are prospective, multi-center, non-interventional, long-term studies on fingolimod use in RRMS in real life. Data of 4229 PANGAEA patients (recruited 2011-2013) and 2441 PANGAEA 2.0 patients (recruited 2015-2018) were available. Baseline data included demographics, RRMS characteristics and disease severity. (3) Results: The mean age of PANGAEA and PANGAEA 2.0 patients was similar (38.8 vs. 39.2 years). Patients in PANGAEA 2.0 had shorter disease duration (7.1 vs. 8.2 years) and fewer relapses in the year before baseline (1.2 vs. 1.6). Disease severity at baseline estimated by EDSS and SDMT was lower in PANGAEA 2.0 patients compared to PANGAEA (EDSS difference 1.0 points; SDMT difference 3.3 points). (4) Conclusions: The results hint at an influence of changes in the treatment guidelines and the label on fingolimod patients profiles over time. Patients tended to have lower disease activity at fingolimod initiation, suggesting an earlier intervention. This indicates increased experience in using fingolimod for sub-optimally treated RRMS patients and a change in mindset towards an early treatment optimization.

Descriptive Analysis of Real-World Data on Fingolimod Long-Term Treatment of Young Adult RRMS Patients.

Background: Fingolimod (Gilenya®) is approved for adult and pediatric patients with highly active relapsing-remitting multiple sclerosis (RRMS). Objectives: The objective was to describe the effectiveness of fingolimod in young adults compared to older patients in clinical practice. Methods: PANGAEA is the largest prospective, multi-center, non-interventional, long-term study evaluating fingolimod in RRMS. We descriptively analyzed demographics, MS characteristics, and severity in two subgroups of young adults (≤20 and >20 to ≤30 years) and older patients (>30 years). Results: Young adults had lower Expanded Disability Status Scale (EDSS) scores compared to older patients (1.8 and 2.3 vs. 3.2) at baseline. The mean EDSS scores remained stable over 5 years in all subgroups. Young adults had higher annual relapse rates (2.0 and 1.7 vs. 1.4) at study entry, which were reduced by approximately 80% in all subgroups over 5 years. The proportion of patients with no clinical disease activity in year 4 was 52.6 and 73.4 vs. 66.9% in patients ≤20, >20 to ≤30 years and >30 years, respectively. The symbol digit modalities test score increased by 15.25 ± 8.3 and 8.3 ± 11.3 (mean ± SD) from baseline in patients >20 to ≤30 and >30 years. Conclusions: Real-world evidence suggests a long-term treatment benefit of fingolimod in young RRMS patients.

Treatment of spontaneous EAE by laquinimod reduces Tfh, B cell aggregates, and disease progression.

OBJECTIVE: To evaluate the influence of oral laquinimod, a candidate multiple sclerosis (MS) treatment, on induction of T follicular helper cells, development of meningeal B cell aggregates, and clinical disease in a spontaneous B cell-dependent MS model. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice by immunization with recombinant myelin oligodendrocyte glycoprotein (rMOG) protein. Spontaneous EAE was evaluated in C57BL/6 MOG p35-55-specific T cell receptor transgenic (2D2) × MOG-specific immunoglobulin (Ig)H-chain knock-in (IgHMOG-ki [Th]) mice. Laquinimod was administered orally. T cell and B cell populations were examined by flow cytometry and immunohistochemistry. RESULTS: Oral laquinimod treatment (1) reduced CD11c+CD4+ dendritic cells, (2) inhibited expansion of PD-1+CXCR5+BCL6+ T follicular helper and interleukin (IL)-21-producing activated CD4+CD44+ T cells, (3) suppressed B cell CD40 expression, (4) diminished formation of Fas+GL7+ germinal center B cells, and (5) inhibited development of MOG-specific IgG. Laquinimod treatment not only prevented rMOG-induced EAE, but also inhibited development of spontaneous EAE and the formation of meningeal B cell aggregates. Disability progression was prevented when laquinimod treatment was initiated after mice developed paralysis. Treatment of spontaneous EAE with laquinimod was also associated with increases in CD4+CD25hiFoxp3+ and CD4+CD25+IL-10+ regulatory T cells. CONCLUSIONS: Our observations that laquinimod modulates myelin antigen-specific B cell immune responses and suppresses both development of meningeal B cell aggregates and disability progression in spontaneous EAE should provide insight regarding the potential application of laquinimod to MS treatment. Results of this investigation demonstrate how the 2D2 × Th spontaneous EAE model can be used successfully for preclinical evaluation of a candidate MS treatment.

Dimethyl fumarate treatment induces adaptive and innate immune modulation independent of Nrf2.

Dimethyl fumarate (DMF) (BG-12, Tecfidera) is a fumaric acid ester (FAE) that was advanced as a multiple sclerosis (MS) therapy largely for potential neuroprotection as it was recognized that FAEs are capable of activating the antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, DMF treatment in randomized controlled MS trials was associated with marked reductions in relapse rate and development of active brain MRI lesions, measures considered to reflect CNS inflammation. Here, we investigated the antiinflammatory contribution of Nrf2 in DMF treatment of the MS model, experimental autoimmune encephalomyelitis (EAE). C57BL/6 wild-type (WT) and Nrf2-deficient (Nrf2(-/-)) mice were immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (p35-55) for EAE induction and treated with oral DMF or vehicle daily. DMF protected WT and Nrf2(-/-) mice equally well from development of clinical and histologic EAE. The beneficial effect of DMF treatment in Nrf2(-/-) and WT mice was accompanied by reduced frequencies of IFN-γ and IL-17-producing CD4(+) cells and induction of antiinflammatory M2 (type II) monocytes. DMF also modulated B-cell MHC II expression and reduced the incidence of clinical disease in a B-cell-dependent model of spontaneous CNS autoimmunity. Our observations that oral DMF treatment promoted immune modulation and provided equal clinical benefit in acute EAE in Nrf2(-/-) and WT mice, suggest that the antiinflammatory activity of DMF in treatment of MS patients may occur through alternative pathways, independent of Nrf2.

Glatiramer acetate treatment negatively regulates type I interferon signaling.

OBJECTIVE: Glatiramer acetate (GA; Copaxone), a disease-modifying therapy for multiple sclerosis (MS), promotes development of anti-inflammatory (M2, type II) monocytes that can direct differentiation of regulatory T cells. We investigated the innate immune signaling pathways that participate in GA-mediated M2 monocyte polarization. METHODS: Monocytes were isolated from myeloid differentiation primary response gene 88 (MyD88)-deficient, Toll-IL-1 receptor domain-containing adaptor inducing interferon (IFN)-ß (TRIF)-deficient, IFN-α/ß receptor subunit 1 (IFNAR1)-deficient, and wild-type (WT) mice and human peripheral blood. GA-treated monocytes were stimulated with Toll-like receptor ligands, then evaluated for activation of kinases and transcription factors involved in innate immunity, and secretion of proinflammatory cytokines. GA-treated mice were evaluated for cytokine secretion and susceptibility to experimental autoimmune encephalomyelitis. RESULTS: GA-mediated inhibition of proinflammatory cytokine production by monocytes occurred independently of MyD88 and nuclear factor-κB, but was blocked by TRIF deficiency. Furthermore, GA did not provide clinical benefit in TRIF-deficient mice. GA inhibited activation of p38 mitogen-activated protein kinase, an upstream regulator of activating transcription factor (ATF)-2, and c-Jun N-terminal kinase 1, which regulates IFN regulatory factor 3 (IRF3). Consequently, nuclear translocation of ATF-2 and IRF3, components of the IFN-ß enhanceosome, was impaired. Consistent with these observations, GA inhibited production of IFN-ß in vivo in WT mice, but did not modulate proinflammatory cytokine production by monocytes from IFNAR1-deficient mice. CONCLUSION: Our results demonstrate that GA inhibits the type I IFN pathway in M2 polarization of monocytes independently of MyD88, providing an important mechanism connecting innate and adaptive immune modulation in GA therapy and valuable insight regarding its potential use with other MS treatments.

The ubiquitin-modifying enzyme A20 restricts ubiquitination of the kinase RIPK3 and protects cells from necroptosis.

A20 is an anti-inflammatory protein linked to multiple human diseases; however, the mechanisms by which A20 prevents inflammatory disease are incompletely defined. We found that A20-deficient T cells and fibroblasts were susceptible to caspase-independent and kinase RIPK3-dependent necroptosis. Global deficiency in RIPK3 significantly restored the survival of A20-deficient mice. A20-deficient cells exhibited exaggerated formation of RIPK1-RIPK3 complexes. RIPK3 underwent physiological ubiquitination at Lys5 (K5), and this ubiquitination event supported the formation of RIPK1-RIPK3 complexes. Both the ubiquitination of RIPK3 and formation of the RIPK1-RIPK3 complex required the catalytic cysteine of A20's deubiquitinating motif. Our studies link A20 and the ubiquitination of RIPK3 to necroptotic cell death and suggest additional mechanisms by which A20 might prevent inflammatory disease.

Liver-specific Fas silencing prevents galactosamine/lipopolysaccharide-induced liver injury.

Acute liver failure (ALF) is a life threatening disease for which only few treatment options exist. The molecular pathways of disease progression are not well defined, but the death receptor Fas (CD95/Apo-1) appears to play a pivotal role in hepatocyte cell death and the development of ALF. Here, we explored posttranscriptional gene silencing of Fas by RNAi to inhibit pathophysiological gene expression. For targeting Fas expression in mice, Fas siRNA was formulated with the liver-specific siRNA delivery system DBTC. Treatment of mice with DBTC/siRNA(Fas) reduced Fas expression in the liver, but not in the spleen, lung, kidney or heart. Furthermore, silencing of Fas receptor was effective in blocking or reducing several aspects of ALF when it was tested in mice exposed to galactosamine/lipopolysaccharide (G/L), a well-known model of ALF. The application of DBTC/siRNA(Fas) 48 h prior G/L exposure resulted in amelioration of hepatic perfusion, reduction of hepatocellular death and increase of survival rate. The administration of DBTC/siRNA(Fas) formulation further diminished the inflammatory response upon G/L challenge, as indicated by a marked decrease of TNFα mRNA expression. However, IL-6 plasma concentration remained unaffectedly by DBTC/siRNA(Fas) formulation. Since the specific silencing of hepatic Fas expression only partially protected from inflammation, but completely attenuated apoptotic and necrotic cell death as well as microcirculatory dysfunction, the development of therapeutic strategies with DBTC lipoplex formulations to treat ALF should be combined with anti-inflammatory strategies to reach maximal therapeutic efficacy.

MOG transmembrane and cytoplasmic domains contain highly stimulatory T-cell epitopes in MS.

OBJECTIVE: Recently, we reported that the 218 amino acid murine full-length myelin oligodendrocyte glycoprotein (MOG) contains novel T-cell epitopes p119-132, p181-195, and p186-200, located within its transmembrane and cytoplasmic domains, and that p119-132 is its immunodominant encephalitogenic T-cell epitope in mice. Here, we investigated whether the corresponding human MOG sequences contain T-cell epitopes in patients with multiple sclerosis (MS) and healthy controls (HC). METHODS: Peripheral blood T cells from patients with MS and HC were examined for proliferation to MOG p119-130, p181-195, p186-200, and p35-55 by fluorescence-activated cell sorting analysis using carboxylfluorescein diacetate succinimidyl ester dilution assay. Intracellular production of proinflammatory cytokines was analyzed by flow cytometry. RESULTS: MOG p119-130, p181-195, and p186-200 elicited significantly greater T-cell responses than p35-55 in patients with MS. T cells from patients with MS proliferated significantly more strongly to MOG p119-130 and p186-200 than did T cells from HC. Further, MOG p119-130-specific T cells exhibited Th17 polarization, suggesting this T-cell epitope may be relevant to MS pathogenesis. CONCLUSIONS: Transmembrane and cytoplasmic MOG domains contain potent T-cell epitopes in MS. Recognition of these determinants is important when evaluating T-cell responses to MOG in MS and may have implications for development of myelin antigen-based therapeutics.

Immunodominant T-cell epitopes of MOG reside in its transmembrane and cytoplasmic domains in EAE.

OBJECTIVE: Studies evaluating T-cell recognition of myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis (MS) and its model, experimental autoimmune encephalomyelitis (EAE), have focused mostly on its 117 amino acid (aa) extracellular domain, especially peptide (p) 35-55. We characterized T-cell responses to the entire 218 aa MOG sequence, including its transmembrane and cytoplasmic domains. METHODS: T-cell recognition in mice was examined using overlapping peptides and intact full-length mouse MOG. EAE was evaluated by peptide immunization and by adoptive transfer of MOG epitope-specific T cells. Frequency of epitope-specific T cells was examined by ELISPOT. RESULTS: Three T-cell determinants of MOG were discovered in its transmembrane and cytoplasmic domains, p119-132, p181-195, and p186-200. Transmembrane MOG p119-132 induced clinical EAE, CNS inflammation, and demyelination as potently as p35-55 in C57BL/6 mice and other H-2(b) strains. p119-128 contained its minimal encephalitogenic epitope. p119-132 did not cause disease in EAE-susceptible non-H-2(b) strains, including Biozzi, NOD, and PL/J. MOG p119-132-specific T cells produced Th1 and Th17 cytokines and transferred EAE to wild-type recipient mice. After immunization with full-length MOG, a significantly higher frequency of MOG-reactive T cells responded to p119-132 than to p35-55, demonstrating that p119-132 is an immunodominant encephalitogenic epitope. MOG p181-195 did not cause EAE, and MOG p181-195-specific T cells could not transfer EAE into wild-type or highly susceptible T- and B-cell-deficient mice. CONCLUSIONS: Transmembrane and cytoplasmic domains of MOG contain immunodominant T-cell epitopes in EAE. A CNS autoantigen can also contain nonpathogenic stimulatory T-cell epitopes. Recognition that a myelin antigen contains multiple encephalitogenic and nonencephalitogenic determinants may have implications for therapeutic development in MS.

Laquinimod, an up-and-coming immunomodulatory agent for treatment of multiple sclerosis.

Laquinimod is a novel oral drug that is currently being evaluated for the treatment of relapsing-remitting multiple sclerosis (RRMS). Although the mode of action of laquinimod remains to be fully elucidated, current knowledge indicates that laquinimod exerts beneficial activities both on the peripheral immune system and within the central nervous system (CNS). The immunomodulatory properties have been deciphered primarily from studies of laquinimod in the animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Data indicate that laquinimod has a primary effect on innate immunity. Laquinimod modulates the function of various myeloid antigen presenting cell populations, which then downregulate proinflammatory T cell responses. Further, data also indicate that laquinimod acts directly on resident cells within the CNS to reduce demyelination and axonal damage. Results from clinical trials that tested laquinimod in RRMS demonstrated that it reduced relapse rate and the mean cumulative number of active lesions, and had a more marked reduction in disability progression than relapse rate. Laquinimod treatment was associated with an excellent safety and tolerability profile. These data indicate that laquinimod will offer a valuable new treatment option for RRMS patients.

MHC class II-dependent B cell APC function is required for induction of CNS autoimmunity independent of myelin-specific antibodies.

Whether B cells serve as antigen-presenting cells (APCs) for activation of pathogenic T cells in the multiple sclerosis model experimental autoimmune encephalomyelitis (EAE) is unclear. To evaluate their role as APCs, we engineered mice selectively deficient in MHC II on B cells (B-MHC II(-/-)), and to distinguish this function from antibody production, we created transgenic (Tg) mice that express the myelin oligodendrocyte glycoprotein (MOG)-specific B cell receptor (BCR; IgH(MOG-mem)) but cannot secrete antibodies. B-MHC II(-/-) mice were resistant to EAE induced by recombinant human MOG (rhMOG), a T cell- and B cell-dependent autoantigen, and exhibited diminished Th1 and Th17 responses, suggesting a role for B cell APC function. In comparison, selective B cell IL-6 deficiency reduced EAE susceptibility and Th17 responses alone. Administration of MOG-specific antibodies only partially restored EAE susceptibility in B-MHC II(-/-) mice. In the absence of antibodies, IgH(MOG-mem) mice, but not mice expressing a BCR of irrelevant specificity, were fully susceptible to acute rhMOG-induced EAE, also demonstrating the importance of BCR specificity. Spontaneous opticospinal EAE and meningeal follicle-like structures were observed in IgH(MOG-mem) mice crossed with MOG-specific TCR Tg mice. Thus, B cells provide a critical cellular function in pathogenesis of central nervous system autoimmunity independent of their humoral involvement, findings which may be relevant to B cell-targeted therapies.

Tob1 plays a critical role in the activation of encephalitogenic T cells in CNS autoimmunity.

Reliable biomarkers corresponding to disease progression or therapeutic responsiveness in multiple sclerosis (MS) have not been yet identified. We previously reported that low expression of the antiproliferative gene TOB1 in CD4⁺ T cells of individuals presenting with an initial central nervous system (CNS) demyelinating event (a clinically isolated syndrome), correlated with high risk for progression to MS. We report that experimental autoimmune encephalomyelitis (EAE) in Tob1⁻/ ⁻ mice was associated with augmented CNS inflammation, increased infiltrating CD4⁺ and CD8⁺ T cell counts, and increased myelin-reactive Th1 and Th17 cells, with reduced numbers of regulatory T cells. Reconstitution of Rag1⁻/ ⁻mice with Tob1⁻/⁻ CD4⁺ T cells recapitulated the aggressive EAE phenotype observed in Tob1⁻/⁻ mice. Furthermore, severe spontaneous EAE was observed when Tob1⁻/⁻ mice were crossed to myelin oligodendrocyte glycoprotein­specific T cell receptor transgenic (2D2) mice. Collectively, our results reveal a critical role for Tob1 in adaptive T cell immune responses that drive development of EAE, thus providing support for the development of Tob1 as a biomarker for demyelinating disease activity.

Aquaporin 4-specific T cells in neuromyelitis optica exhibit a Th17 bias and recognize Clostridium ABC transporter.

OBJECTIVE: Aquaporin 4 (AQP4)-specific autoantibodies in neuromyelitis optica (NMO) are immunoglobulin (Ig)G1, a T cell-dependent Ig subclass, indicating that AQP4-specific T cells participate in NMO pathogenesis. Our goal was to identify and characterize AQP4-specific T cells in NMO patients and healthy controls (HC). METHODS: Peripheral blood T cells from NMO patients and HC were examined for recognition of AQP4 and production of proinflammatory cytokines. Monocytes were evaluated for production of T cell-polarizing cytokines and expression of costimulatory molecules. RESULTS: T cells from NMO patients and HC proliferated to intact AQP4 or AQP4 peptides (p11-30, p21-40, p61-80, p131-150, p156-170, p211-230, and p261-280). T cells from NMO patients demonstrated greater proliferation to AQP4 than those from HC, and responded most vigorously to p61-80, a naturally processed immunodominant determinant of intact AQP4. T cells were CD4(+), and corresponding to association of NMO with human leukocyte antigen (HLA)-DRB1*0301 and DRB3, AQP4 p61-80-specific T cells were HLA-DR restricted. The T-cell epitope within AQP4 p61-80 was mapped to 63-76, which contains 10 residues with 90% homology to a sequence within Clostridium perfringens adenosine triphosphate-binding cassette (ABC) transporter permease. T cells from NMO patients proliferated to this homologous bacterial sequence, and cross-reactivity between it and self-AQP4 was observed, supporting molecular mimicry. In NMO, AQP4 p61-80-specific T cells exhibited Th17 polarization, and furthermore, monocytes produced more interleukin 6, a Th17-polarizing cytokine, and expressed elevated CD40 and CD80 costimulatory molecules, suggesting innate immunologic dysfunction. INTERPRETATION: AQP4-specific T-cell responses are amplified in NMO, exhibit a Th17 bias, and display cross-reactivity to a protein of an indigenous intestinal bacterium, providing new perspectives for investigating NMO pathogenesis.

Laquinimod, a quinoline-3-carboxamide, induces type II myeloid cells that modulate central nervous system autoimmunity.

Laquinimod is a novel oral drug that is currently being evaluated for the treatment of relapsing-remitting (RR) multiple sclerosis (MS). Using the animal model for multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), we examined how laquinimod promotes immune modulation. Oral laquinimod treatment reversed established RR-EAE and was associated with reduced central nervous system (CNS) inflammation, decreased Th1 and Th17 responses, and an increase in regulatory T cells (Treg). In vivo laquinimod treatment inhibited donor myelin-specific T cells from transferring EAE to naive recipient mice. In vivo laquinimod treatment altered subpopulations of myeloid antigen presenting cells (APC) that included a decrease in CD11c(+)CD11b(+)CD4(+) dendritic cells (DC) and an elevation of CD11b(hi)Gr1(hi) monocytes. CD11b(+) cells from these mice exhibited an anti-inflammatory type II phenotype characterized by reduced STAT1 phosphorylation, decreased production of IL-6, IL-12/23 and TNF, and increased IL-10. In adoptive transfer, donor type II monocytes from laquinimod-treated mice suppressed clinical and histologic disease in recipients with established EAE. As effects were observed in both APC and T cell compartments, we examined whether T cell immune modulation occurred as a direct effect of laquinimod on T cells, or as a consequence of altered APC function. Inhibition of Th1 and Th17 differentiation was observed only when type II monocytes or DC from laquinimod-treated mice were used as APC, regardless of whether myelin-specific T cells were obtained from laquinimod-treated or untreated mice. Thus, laquinimod modulates adaptive T cell immune responses via its effects on cells of the innate immune system, and may not influence T cells directly.

Electrostatically Stabilized Magnetic Nanoparticles - An Optimized Protocol to Label Murine T Cells for in vivo MRI.

We present a novel highly efficient protocol to magnetically label T cells applying electrostatically stabilized very small superparamagnetic iron oxide particles (VSOP). Our long-term aim is to use magnetic resonance imaging (MRI) to investigate T cell dynamics in vivo during the course of neuroinflammatory disorders such as experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Encephalitogenic T cells were co-incubated with VSOP, or with protamine-complexed VSOP (VProt), respectively, at different conditions, optimizing concentrations and incubation times. Labeling efficacy was determined by atomic absorption spectrometry as well as histologically, and evaluated on a 7 T MR system. Furthermore, we investigated possible alterations of T cell physiology caused by the labeling procedure. T cell co-incubation with VSOP resulted in an efficient cellular iron uptake. T2 times of labeled cells dropped significantly, resulting in prominent hypointensity on T2*-weighted scans. Optimal labeling efficacy was achieved by VProt (1 mM Fe/ml, 8 h incubation; T2 time shortening of ∼80% compared to untreated cells). Although VSOP promoted T cell proliferation and altered the ratio of T cell subpopulations toward a CD4(+) phenotype, no effects on CD4 T cell proliferation or phenotypic stability were observed by labeling in vitro differentiated Th17 cells with VProt. Yet, high concentrations of intracellular iron oxide might induce alterations in T cell function, which should be considered in cell tagging studies. Moreover, we demonstrated that labeling of encephalitogenic T cells did not affect pathogenicity; labeled T cells were still capable of inducing EAE in susceptible recipient mice.

MAPK3 deficiency drives autoimmunity via DC arming.

DC are professional APC that instruct T cells during the inflammatory course of EAE. We have previously shown that MAPK3 (Erk1) is important for the induction of T-cell anergy. Our goal was to determine the influence of MAPK3 on the capacity of DC to arm T-cell responses in autoimmunity. We report that DC from Mapk3(-/-) mice have a significantly higher membrane expression of CD86 and MHC-II and--when loaded with the myelin oligodendrocyte glycoprotein--show a superior capacity to prime naïve T cells towards an inflammatory phenotype than Mapk3(+/+) DC. Nonetheless and as previously described, Mapk3(-/-) mice were only slightly but not significantly more susceptible to myelin oligodendrocyte glycoprotein-induced EAE than WT littermate mice. However, Mapk3(+/+) mice engrafted with Mapk3(-/-) BM (KO-->WT) developed a severe form of EAE, in direct contrast to WT-->KO mice, which were even less sick than control WT-->WT mice. An infiltration of DC and accumulation of Th17 cells was also observed in the CNS of KO-->WT mice. Therefore, triggering of MAPK3 in the periphery might be a therapeutic option for the treatment of neuroinflammation since absence of this kinase in the immune system leads to severe EAE.

Activation of kinin receptor B1 limits encephalitogenic T lymphocyte recruitment to the central nervous system.

Previous proteomic and transcriptional analyses of multiple sclerosis lesions revealed modulation of the renin-angiotensin and the opposing kallikrein-kinin pathways. Here we identify kinin receptor B1 (Bdkrb1) as a specific modulator of immune cell entry into the central nervous system (CNS). We demonstrate that the Bdkrb1 agonist R838 (Sar-[D-Phe]des-Arg(9)-bradykinin) markedly decreases the clinical symptoms of experimental autoimmune encephalomyelitis (EAE) in SJL mice, whereas the Bdkrb1 antagonist R715 (Ac-Lys-[D-betaNal(7), Ile(8)]des-Arg(9)-bradykinin) resulted in earlier onset and greater severity of the disease. Bdkrb1-deficient (Bdkrb1(-/-)) C57BL/6 mice immunized with a myelin oligodendrocyte glycoprotein fragment, MOG(35-55), showed more severe disease with enhanced CNS-immune cell infiltration. The same held true for mixed bone marrow-chimeric mice reconstituted with Bdkrb1(-/-) T lymphocytes, which showed enhanced T helper type 17 (T(H)17) cell invasion into the CNS. Pharmacological modulation of Bdkrb1 revealed that in vitro migration of human T(H)17 lymphocytes across blood-brain barrier endothelium is regulated by this receptor. Taken together, these results suggest that the kallikrein-kinin system is involved in the regulation of CNS inflammation, limiting encephalitogenic T lymphocyte infiltration into the CNS, and provide evidence that Bdkrb1 could be a new target for the treatment of chronic inflammatory diseases such as multiple sclerosis.

Differential immune cell dynamics in the CNS cause CD4+ T cell compartmentalization.

In the course of autoimmune CNS inflammation, inflammatory infiltrates form characteristic perivascular lymphocyte cuffs by mechanisms that are not yet well understood. Here, intravital two-photon imaging of the brain in anesthetized mice, with experimental autoimmune encephalomyelitis, revealed the highly dynamic nature of perivascular immune cells, refuting suggestions that vessel cuffs are the result of limited lymphocyte motility in the CNS. On the contrary, vessel-associated lymphocyte motility is an actively promoted mechanism which can be blocked by CXCR4 antagonism. In vivo interference with CXCR4 in experimental autoimmune encephalomyelitis disrupted dynamic vessel cuffs and resulted in tissue-invasive migration. CXCR4-mediated perivascular lymphocyte movement along CNS vessels was a key feature of CD4(+) T cell subsets in contrast to random motility of CD8(+) T cells, indicating a dominant role of the perivascular area primarily for CD4(+) T cells. Our results visualize dynamic T cell motility in the CNS and demonstrate differential CXCR4-mediated compartmentalization of CD4(+) T-cell motility within the healthy and diseased CNS.