Johanna Breuer

L-Selectin is a possible biomarker for individual PML risk in natalizumab-treated MS patients

Objective: To find biomarkers identifying patients at risk for the development of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. Methods: Patients were recruited from 10 European and US cohorts. Of 289 patients with multiple sclerosis (MS), 224 had been treated with natalizumab (18–80 months), 21 received other immune-modulatory treatments, and 28 were untreated. We had access to samples from 16 natalizumab PML patients. Eight of these patients had given blood before the diagnosis of PML. We also analyzed non-natalizumab-treated patients who developed PML (n = 10) and age- and sex-matched healthy donors (n = 31). All flow cytometric assessments were done on previously cryopreserved, viable peripheral blood mononuclear cells. Results: The percentage of l-selectin-expressing CD4+ T cells was significantly lower in patients treated long-term with natalizumab (40.2%) when compared with patients not receiving natalizumab treatment (47.2%; p = 0.016) or healthy controls (61.0%; p < 0.0001). An unusually low percentage (9-fold lower; 4.6%) was highly correlated with the risk of developing PML in the patient group with available pre-PML samples when compared with non-PML natalizumab-treated patients (p ≤ 0.0001). Samples were gathered between 4 and 26 months before PML diagnosis. Conclusions: The cell-based assessment of the percentage of l-selectin-expressing CD4 T cells could provide an urgently needed biomarker for individual PML risk assessment.

Ultraviolet B light attenuates the systemic immune response in central nervous system autoimmunity

Environmental conditions (eg, latitude) play a critical role in the susceptibility and severity of many autoimmune disorders, including multiple sclerosis (MS). Here, we investigated the mechanisms underlying the beneficial effects of immune regulatory processes induced in the skin by moderate ultraviolet B (UVB) radiation on central nervous system (CNS) autoimmunity.

PML risk stratification using anti-JCV antibody index and L-selectin.

Background: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters. Objective: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification. Methods: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients). Results: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor “CD62L low” increasing a patient’s relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group. Conclusions: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence.

Blood coagulation factor XII drives adaptive immunity during neuroinflammation via CD87-mediated modulation of dendritic cells

Aberrant immune responses represent the underlying cause of central nervous system (CNS) autoimmunity, including multiple sclerosis (MS). Recent evidence implicated the crosstalk between coagulation and immunity in CNS autoimmunity. Here we identify coagulation factor XII (FXII), the initiator of the intrinsic coagulation cascade and the kallikrein–kinin system, as a specific immune cell modulator. High levels of FXII activity are present in the plasma of MS patients during relapse. Deficiency or pharmacologic blockade of FXII renders mice less susceptible to experimental autoimmune encephalomyelitis (a model of MS) and is accompanied by reduced numbers of interleukin-17A-producing T cells. Immune activation by FXII is mediated by dendritic cells in a CD87-dependent manner and involves alterations in intracellular cyclic AMP formation. Our study demonstrates that a member of the plasmatic coagulation cascade is a key mediator of autoimmunity. FXII inhibition may provide a strategy to combat MS and other immune-related disorders.

The TASK1 channel inhibitor A293 shows efficacy in a mouse model of multiple sclerosis

The two-pore domain potassium channel TASK1 (KCNK3) has recently emerged as an important modulator in autoimmune CNS inflammation. Previously, it was shown that T lymphocytes obtained from TASK1(-/-) mice display impaired T cell effector functions and that TASK1(-/-) mice show a significantly reduced disease severity in myelin oligodendrocyte glycoprotein (MOG(35-55)) peptide induced experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We here evaluate a potent and specific TASK1 channel inhibitor, A293, which caused a dose-dependent reduction of T cell effector functions (cytokine production and proliferation). This effect was abolished in CD4(+) T cells from TASK1(-/-) mice but not in cells from TASK3(-/-) mice. In electrophysiological measurements, A293 application induced a significant reduction of the outward current of wildtype T lymphocytes, while there was no effect in TASK1(-/-) cells. Preventive and therapeutic application of A293 significantly ameliorated the EAE disease course in wildtype mice while it had no significant effect in TASK1(-/-) mice and was still partly effective in TASK3(-/-) mice. In summary, our findings support the concept of TASK1 as an attractive drug target for autoimmune disorders.

CD8 + T-cell pathogenicity in Rasmussen encephalitis elucidated by large-scale T-cell receptor sequencing

Rasmussen encephalitis (RE) is a rare paediatric epilepsy with uni-hemispheric inflammation and progressive neurological deficits. To elucidate RE immunopathology, we applied T-cell receptor (TCR) sequencing to blood (n=23), cerebrospinal fluid (n=2) and brain biopsies (n=5) of RE patients, and paediatric controls. RE patients present with peripheral CD8+ T-cell expansion and its strength correlates with disease severity. In addition, RE is the only paediatric epilepsy with prominent T-cell expansions in the CNS. Consistently, common clones are shared between RE patients, who also share MHC-I alleles. Public RE clones share Vβ genes and length of the CDR3. Rituximab/natalizumab/basiliximab treatment does not change TCR diversity, stem cell transplantation replaces the TCR repertoire with minimal overlap between donor and recipient, as observed in individual cases. Our study supports the hypothesis of an antigen-specific attack of peripherally expanded CD8+ lymphocytes against CNS structures in RE, which might be ameliorated by restricting access to the CNS.

CD4+NKG2D+ T cells exhibit enhanced migratory and encephalitogenic properties in neuroinflammation.

Migration of encephalitogenic CD4+ T lymphocytes across the blood-brain barrier is an essential step in the pathogenesis of multiple sclerosis (MS). We here demonstrate that expression of the co-stimulatory receptor NKG2D defines a subpopulation of CD4+ T cells with elevated levels of markers for migration, activation, and cytolytic capacity especially when derived from MS patients. Furthermore, CD4+NKG2D+ cells produce high levels of proinflammatory IFN-γ and IL-17 upon stimulation. NKG2D promotes the capacity of CD4+NKG2D+ cells to migrate across endothelial cells in an in vitro model of the blood-brain barrier. CD4+NKG2D+ T cells are enriched in the cerebrospinal fluid of MS patients, and a significant number of CD4+ T cells in MS lesions coexpress NKG2D. We further elucidated the role of CD4+NKG2D+ T cells in the mouse system. NKG2D blockade restricted central nervous system migration of T lymphocytes in vivo, leading to a significant decrease in the clinical and pathologic severity of experimental autoimmune encephalomyelitis, an animal model of MS. Blockade of NKG2D reduced killing of cultivated mouse oligodendrocytes by activated CD4+ T cells. Taken together, we identify CD4+NKG2D+ cells as a subpopulation of T helper cells with enhanced migratory, encephalitogenic and cytotoxic properties involved in inflammatory CNS lesion development.

Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis.

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune‐mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1‐deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)35–55‐induced EAE, a model of human MS. The lack of PLD1 reduced chemokine‐mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1‐deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1‐dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.

The two‐pore domain K2P channel TASK2 drives human NK‐cell proliferation and cytolytic function

Natural killer (NK) cells are a subset of cytotoxic lymphocytes that recognize and kill tumor‐ and virus‐infected cells without prior stimulation. Killing of target cells is a multistep process including adhesion to target cells, formation of an immunological synapse, and polarization and release of cytolytic granules. The role of distinct potassium channels in this orchestrated process is still poorly understood. The current study reveals that in addition to the voltage‐gated KV1.3 and the calcium‐activated KCa3.1 channels, human NK cells also express the two‐pore domain K2P channel TASK2 (TWIK‐related acid‐sensitive potassium channel). Expression of Task2 varies among NK‐cell subsets and depends on their differentiation and activation state. Despite its different expression in TASK2highCD56brightCD16− and TASK2lowCD56dimCD16+ NK cells, TASK2 is involved in cytokine‐induced proliferation and cytolytic function of both subsets. TASK2 is crucial for leukocyte functional antigen (LFA‐1) mediated adhesion of both resting and cytokine‐activated NK cells to target cells, an early step in killing of target cells. With regard to the following mechanism, TASK2 plays a role in release of cytotoxic granules by resting, but not IL‐15‐induced NK cells. Taken together, our data exhibit two‐pore potassium channels as important players in NK‐cell activation and effector function.

Liver X receptor activation promotes differentiation of regulatory T cells

The nuclear receptor Liver X Receptor (LXR) is a ligand-activated transcription factor that has been implicated in control of chronic inflammation by downregulating pro-inflammatory T cell responses. An impaired function of regulatory T cells, a subset of CD4+ T cells with a crucial role in maintaining lymphocytes homeostasis and immune regulation, is frequently observed in chronic inflammatory diseases. We observed that pharmacological activation of LXR in T cells not only resulted in a thorough suppression of Th1 and Th17 polarization in vitro, but also significantly induced regulatory T cells (Treg) cell differentiation in a receptor-specific fashion. In line with this, systemic LXR activation by oral treatment of mice with the LXR agonist GW3965 induced gut-associated regulatory T cells in vivo. Importantly, such LXR-activated Tregs had a higher suppressive capacity in functional in vitro coculture assays with effector T cells. Our data thus point towards a dual role of LXR-mediated control of inflammation by suppression of pro-inflammatory T cells and reciprocal induction of regulatory T cells.