Ulf Schulze-Topphoff

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

Antigen presentation, but not antibody secretion, by B cells drives CNS autoimmunity induced by immunization with human MOG.

Aquaporin 4-Specific T Cells in Neuromyelitis Optica Exhibit a Th17 Bias and Recognize Clostridium ABC Transporter

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).

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 A20s 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.

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 CD11bhiGr1hi 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.

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

Significance Dimethyl fumarate (DMF) (BG-12, Tecfidera), a fumaric acid ester (FAE), is a commonly prescribed oral therapy for multiple sclerosis (MS), a CNS autoimmune inflammatory demyelinating disease that may result in sustained neurologic damage. It is thought that the benefit of DMF in MS therapy is mediated through activation of the antioxidative transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, the role of Nrf2 in the antiinflammatory effects of DMF has not been fully elucidated. Here, we investigated the role of Nrf2 in DMF treatment of the MS model, experimental autoimmune encephalomyelitis (EAE), and demonstrated DMF can modulate T cells, B cells, and antigen-presenting cells, and reduce clinical and histologic EAE, 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.

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

Loss of antiproliferative gene TOB1 results in more severe EAE driven by augmented pathogenic T cell responses.

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.

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-2b strains. p119-128 contained its minimal encephalitogenic epitope. p119-132 did not cause disease in EAE-susceptible non-H-2b 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.

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.

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.