Anne Waschbisch

Effects of Natalizumab Treatment on Foxp3+ T Regulatory Cells

Background Natalizumab, a monoclonal humanized antibody targeting the alpha-4 chain of very late activation antigen 4 (VLA-4) exerts impressive therapeutic effects in patients with relapsing-remitting multiple sclerosis. Our objective was to study impacts of Natalizumab therapy on Foxp3+ T regulatory cells (Tregs) in multiple sclerosis (MS) patients. Methodology A combined approach of in vitro and ex vivo experiments using T cells isolated from the peripheral blood of healthy donors and Natalizumab treated MS patients was chosen. We determined binding of Natalizumab and its effects on the frequency, transmigratory behaviour and suppressive function of Tregs. Principal Findings Binding of Natalizumab and expression of CD49d (alpha-4 chain of VLA-4) differed between non-regulatory and regulatory cells. Albeit Foxp3+ Tregs had lower levels of CD49d, Natalizumab blocked the transmigration of Foxp3+ Tregs similar to non-regulatory T cells. The frequency of peripheral blood Tregs was unaffected by Natalizumab treatment. Natalizumab does not alter the suppressive capacity of CD4+CD25highCD127lowFoxp3+ Tregs under in vitro conditions. Furthermore, the impaired function of Tregs in MS patients is not restored by Natalizumab treatment. Conclusions We provide a first detailed analysis of Natalizumab effects on the regulatory T cell population. Our prospective study shows that Foxp3+ Tregs express lower levels of VLA-4 and bind less Natalizumab. We further the understanding of the mechanisms of action of Natalizumab by demonstrating that unlike other immunomodulatory drugs the beneficial therapeutic effects of the monoclonal antibody are largely independent of alterations in Treg frequency or function.

FOXP3+ T regulatory cells in idiopathic inflammatory myopathies.

FOXP3+ T regulatory cells (Tregs) are considered key players in the maintenance of immune homeostasis. Here we studied the presence and potential role of FOXP3+ Tregs in myositis. CD3 and FOXP3 expression in dermatomyositis, polymyositis and inclusion body myositis was assessed by immunohistochemistry and real-time PCR. FOXP3+ Tregs were found in close proximity to effector cells and their numbers correlated with the degree of inflammation. Despite divergent pathogenetic concepts, we observed no differences in the frequency of FOXP3 immunoreactive cells or FOXP3 mRNA expression between different myositis entities. Functional assays using human myoblasts as targets of CD8+ cells demonstrate that Tregs are capable to inhibit the lytic activity of cytotoxic cells. Our data suggest that FOXP3 Tregs serve to counterbalance muscle destruction by cytotoxic T cells in myositis.

Patient preferences for disease-modifying drugs in multiple sclerosis therapy: a choice-based conjoint analysis

Objectives: With an increasing number of disease-modifying treatments (DMTs) for multiple sclerosis (MS), patient preferences will gain importance in the decision-making process. We assessed patients’ implicit preferences for oral versus parenteral DMTs and identified factors influencing patients’ treatment preference. Methods: Patients with relapsing–remitting MS (n = 156) completed a questionnaire assessing treatment preferences, whereby they had to decide between pairs of hypothetical treatment scenarios. Based on this questionnaire a choice-based conjoint analysis was conducted. Results: Treatment frequency and route of administration showed a stronger influence on patient preference compared with frequency of mild side effects. The latter attribute was more important for treatment-naïve patients compared with DMT-experienced patients. The higher the Extended Disability Status Scale score, the more likely pills, and the less likely fewer side effects were preferred. Pills were preferred over injections by 93% of patients, when treatment frequency and frequency of side effects were held constant. However, preference switched to injections when pills had to be taken three times daily and injections only once per week. Injections were also preferred when pills were associated with frequent side effects. Conclusions: Our results suggest that route of administration and treatment frequency play an important role in the patients’ preference for a given DMT.

Frequency of regulatory T cells is not affected by transient B cell depletion using anti-CD20 antibodies in rheumatoid arthritis.

Objectives Transient B cell depletion with the monoclonal anti-CD20 antibody rituximab has shown favourable clinical responses in patients with rheumatoid arthritis (RA). Recently a characteristic regeneration pattern of B cell subpopulations has been reported. However, little is known about the impact of B-cell depletion on peripheral T cells in particular regulatory T cells. Materials and Methodology 17 patients with RA having failed anti-TNF were treated with rituximab. Four colour staining was performed using CD19, CD3, CD4, CD8, CD16, CD56, CD25, HLA-DR, HLA-G and intracellular Foxp3 at five time points spanning up to 12 months after rituximab. In addition, quantification of the soluble form of the HLA class I molecule HLA-G by ELISA has been performed. Results Peripheral B cell depletion lasted 6 to 9 months. The absolute number of CD3+, CD4+ and CD8+ lymphocytes showed no significant changes up to 1 year after B-cell depletion compared to before therapy. Only the relative frequency for CD3 and CD4 showed a significant increase (p < 0.05). In particular, CD4+CD25++ and Foxp3 positive regulatory T cells remained constant. The percentage of HLA-G positive cells in the CD4+ or CD8+ population did not change significantly either. The amount of sHLA-G remained without significant changes. Conclusion Absolute T cell counts showed no significant changes after rituximab compared to the time point before therapy.In particular, the frequency of regulatory T cells with a CD4+CD25++ phenotype as well as positive Foxp3 expression were numerically stable. Additionally, HLA-G positive regulatory T cells and soluble levels of HLA-G showed no significant changes.

Human muscle cells express the costimulatory molecule B7‐H3, which modulates muscle–immune interactions

OBJECTIVE Interactions between the family of B7 ligands and their receptors are increasingly recognized as crucial for stimulation and/or inhibition of immune responses. The present study was undertaken to examine the expression and functional relevance of B7 homolog 3 (B7-H3), a novel B7 homolog attributed significant immunoregulatory functions, in human muscle cells in vivo and in vitro. METHODS Thirty-five muscle biopsy specimens obtained from patients with polymyositis, dermatomyositis, inclusion body myositis, or noninflammatory myopathies and normal controls were analyzed by immunohistochemistry for B7-H3 expression. The expression of B7-H3 protein on primary human myoblasts and TE671 muscle rhabdomyosarcoma cells was studied by flow cytometry and Western blot analysis. B7-H3 small interfering RNA (siRNA) was used to study the impact of knockdown of B7-H3 on CD8+ cell-mediated lysis in skeletal muscle cells. RESULTS B7-H3 was not detectable on normal muscle fibers. In contrast, its expression was markedly increased on muscle fibers from patients with inflammatory myopathies. Cell-surface staining was most prominent in the contact areas between muscle fibers and inflammatory cells. B7-H3 protein was detected on myoblasts cultured from control and myositis patient muscle tissue as well as in TE671 muscle rhabdomyosarcoma cells. Knockdown of B7-H3 by siRNA in TE671 cells enhanced CD8+ T cell-specific lysis, indicating a functional role of B7-H3 in the protection of skeletal muscle from immune-mediated lysis. CONCLUSION Our results demonstrate that human muscle cells express B7-H3, a functional coinhibitory molecule of the B7 family. B7-H3 may play an important role in muscle-immune interactions, providing further evidence of the active role of muscle cells in local immunoregulatory processes.

Intercellular exchanges of membrane fragments (trogocytosis) between human muscle cells and immune cells: A potential mechanism for the modulation of muscular immune responses

Trogocytosis is a cell-contact dependent intercellular transfer of membrane fragments and associated molecules. We studied trogocytosis in the interaction of T cells with human skeletal muscle cells modeling muscle-immune cell interactions under pathophysiological conditions i.e. myositis. Human myoblasts donate membrane fragments to T cells. Acquisition of muscle-derived membrane molecules depended on T-cell activation, was independent of T-cell receptor engagement, sensitive to inhibition of actin polymerization and amplified by protein kinase C activation. Single-cell patch clamping was used to demonstrate the change in membrane capacitance upon incorporation of membrane fragments in T cells. Membrane uptake was fast and temporarily, but had clear functional consequences: T cells after intimate contact with myoblasts stimulated the proliferation of autologous T cells. Our observations raise the hypothesis that trogocytosis may modulate the outcome of T-T interactions within the micromilieu of skeletal muscle.

Human myoblasts modulate the function of antigen-presenting cells

Muscle biopsy specimens of myositis patients were analyzed for the presence of dendritic cells (DC) and macrophages (MPh) by immunohistochemistry. The interaction of DC and myoblasts (MB) was studied by coculture and effects on DC phenotype and function were assessed by flow cytometry and T-cell proliferation assays. Effects of MB-lysates on the phagocytic capacity of MPh were analyzed in bead-incorporation assays. Myositis specimens revealed a tendency towards more immature DC. MB modulated the maturation state of DC and DC recovered from MB-coculture had an inhibitory effect on T-cell proliferation. MB-lysates strongly stimulated MPh phagocytosis. Hypothetically, MB might modulate APC, counterbalancing immune-mediated damage.

Vascular pathology in multiple sclerosis: mind boosting or myth busting?

The investigation of central nervous system vascular changes in the pathophysiology of multiple sclerosis (MS) is a time-honored concept. Yet, recent reports on changes in venous cerebrospinal outflow, the advent of new magnetic resonance imaging techniques and the investigation of immunomodulatory properties of several vascular mediators on the molecular level have added new excitement to hypotheses centering around vascular pathology as determining factor in the pathophysiology of MS. Here we critically review the concept of chronic cerebrospinal venous insufficiency in MS patients and describe new imaging techniques including perfusion weighted imaging, susceptibility weighted imaging and diffusion weighted imaging which reveal central nervous system hypoperfusion, perivascular iron deposition and diffuse structural changes in the MS brain. On a molecular basis, vascular mediators represent interesting targets connecting vascular pathology with immunomodulation. In summary, the relation of venous changes to the pathophysiology of MS may not be as simple as initially described and it certainly seems awkward to think of the complex disease MS solely as result of a simple venous outflow obstruction. Yet, the investigation of new vascular concepts as one variable in the pathophysiology of the autoimmune attack seems very worthwhile and may add to a better understanding of this devastating disorder.

Fingolimod effects in neuroinflammation: Regulation of astroglial glutamate transporters?

Fingolimod is an oral sphingosine-1-phosphate-receptor modulator which reduces the recirculation of immune cells and may also directly target glial cells. Here we investigate effects of fingolimod on expression of astroglial glutamate transporters under pro-inflammatory conditions. In astrocyte cell culture, the addition of pro-inflammatory cytokines led to a significant downregulation of glutamate transporters glutamate transporter-1 (slc1a2/SLC1A2) and glutamate aspartate transporter (slc1a3/SLC1A3) expression on the mRNA or protein level. In this setting, the direct application of fingolimod-1 phosphate (F1P) on astrocytes did not change expression levels of slc1a2 and slc1a3 mRNA. The analysis of both transporters on the protein level by Western Blot and immunocytochemistry did also not reveal any effect of F1P. On a functional level, the addition of conditioned supernatants from F1P treated astrocytes to neuronal cell culture did not result in increased neurite growth. In experimental autoimmune encephalomyelitis as a model of multiple sclerosis, fingolimod treatment reduced T cell and macrophages/microglia mediated inflammation and also diminished astrocyte activation. At the same time, fingolimod restored the reduced expression of slc1a2 and slc1a3 in the inflamed spinal cord on the mRNA level and of SLC1A2 and SLC1A3 on the protein level, presumably via indirect, anti-inflammatory mechanisms. These findings provide further evidence for a predominantly peripheral effect of the compound in neuroinflammation.

Visual Search as a Tool for a Quick and Reliable Assessment of Cognitive Functions in Patients with Multiple Sclerosis

Background Despite the high frequency of cognitive impairment in multiple sclerosis, its assessment has not gained entrance into clinical routine yet, due to lack of time-saving and suitable tests for patients with multiple sclerosis. Objective The aim of the study was to compare the paradigm of visual search with neuropsychological standard tests, in order to identify the test that discriminates best between patients with multiple sclerosis and healthy individuals concerning cognitive functions, without being susceptible to practice effects. Methods Patients with relapsing remitting multiple sclerosis (n = 38) and age-and gender-matched healthy individuals (n = 40) were tested with common neuropsychological tests and a computer-based visual search task, whereby a target stimulus has to be detected amongst distracting stimuli on a touch screen. Twenty-eight of the healthy individuals were re-tested in order to determine potential practice effects. Results Mean reaction time reflecting visual attention and movement time indicating motor execution in the visual search task discriminated best between healthy individuals and patients with multiple sclerosis, without practice effects. Conclusions Visual search is a promising instrument for the assessment of cognitive functions and potentially cognitive changes in patients with multiple sclerosis thanks to its good discriminatory power and insusceptibility to practice effects.