Background: Distinct lesion topography in relapsing-remitting multiple sclerosis (RRMS) might be due to different antigen presentation and/or trafficking routes of immune cells into the central nervous system (CNS). Objective: To investigate whether distinct lesion patterns in multiple sclerosis (MS) might be associated with a predominance of distinct circulating T-helper cell subset as well as their innate counterparts. Methods: Flow cytometric analysis of lymphocytes derived from the peripheral blood of patients with exclusively cerebral (n = 20) or predominantly spinal (n = 12) disease manifestation. Results: Patients with exclusively cerebral or preferential spinal lesion manifestation were associated with increased proportions of circulating granulocyte-macrophage colony-stimulating factor (GM-CSF) producing TH1 cells or interleukin (IL)-17-producing TH17 cells, respectively. In contrast, proportions of peripheral IL-17/IL-22-producing lymphoid tissue inducer (LTi), the innate counterpart of TH17 cells, were enhanced in RRMS patients with exclusively cerebral lesion topography. Conclusions: Distinct T-helper and T-helper-like innate lymphoid cell (ILC) subsets are associated with different lesion topography in RRMS.

Objective: To explore the immunomodulatory effects of teriflunomide on lymphocyte subsets, repertoire, and function in patients with relapsing-remitting MS (RRMS). Background: Teriflunomide, an immunomodulator approved for patients with relapsing forms of MS, specifically inhibits proliferation of activated lymphocytes. The TERI-DYNAMIC study (NCT01863888) characterized the immunomodulatory effects of teriflunomide treatment on lymphocyte subsets in patients with RRMS. Design/Methods: Patients with RRMS received once-daily oral teriflunomide 14 mg for 24 weeks. Peripheral blood mononuclear cells were isolated at baseline and Weeks 12 and 24. Lymphocyte subsets, including B cells, T cells, and T-cell subpopulations, were analyzed by flow cytometry. Changes in absolute numbers and the diversity of the CD4 + T-cell receptor repertoire were evaluated by deep sequencing. T-cell function was assessed by ex vivo proliferation and cytokine secretion assays. Results: As expected, teriflunomide treatment for 24 weeks significantly reduced absolute numbers and frequencies of lymphocytes and CD19 + B cells in patients with RRMS (n=38). However, within the CD4 + T-cell population, teriflunomide treatment resulted in a selective decrease in Th1 cells compared with Th17 cell proportions, whereas the percentage of inducible Tregs was increased. With regard to the CD4 + T-cell receptor repertoire, teriflunomide treatment for 24 weeks resulted in a significant reduction of overall clonal diversity reflected by a decrease in the total number of unique clones and a reduction in entropy. Functionally, T cells from patients treated with teriflunomide mounted appropriate proliferative and cytokine responses ex vivo. Conclusions: In patients with RRMS, teriflunomide exerted selective effects on different CD4 + T-cell subsets, while ex vivo T-cell function was maintained, supporting the specific immunomodulatory mechanism of action of teriflunomide. This indicates that teriflunomide treatment results in a correction of an enhanced clonal diversity of the CD4 + T-cell repertoire found in patients with RRMS. Study supported by: Genzyme, a Sanofi company. Disclosure: Dr. Wiendl has received research support from Bayer HealthCare, Biogen Idec, the German Ministry for Education and Research, Deutsche Forschungsgesellschaft, and the Else Kroner Fresenius Foundation. Dr. Gros has received personal compensation for activities with Bayer Healthcare and Genzyme Corporation. Dr. Lindner has nothing to disclose. Dr. Eschborn has nothing to disclose. Dr. Weisser has nothing to disclose. Dr. Posevitz-Fejfar has received personal compensation for activities with Genzyme. Dr. Schulte-Mecklenbeck has nothing to disclose. Dr. Van Wijmeersch has received personal compensation for activities with Bayer Pharmaceuticals Corporation, Biogen Idec, Genzyme Corporation, Sanofi-Aventis Pharmaceuticals, Merck Serono, Novartis, and Teva Neuroscience as a speaker and advisory board me Dr. Hupperts has received personal compensation for activities with Biogen, Merck Serono, Novartis, Sanofi-Aventis as a speaker, advisory board member and/or consultant. Dr. Brette has received personal compensation for activities with Lincoln Pharmaceuticals Ltd. as an employee. Dr. Turner has received personal compensation for activities with Genzyme Corporation as an employee. Dr. Bar-Or has received personal compensation for activities with Bayer, Bayhill Therapeutics, Berlex, Biogen Idec, BioMS, Diogenix, Eli Lilly as a consultant, speaker and advisory board member. Dr. Klotz has received personal compensation from Genzyme, Novartis, Merck Serono, and CSL Behring as a speaker and/or advisor.

SignificanceThe importance of natural killer (NK) cells in the control of autoimmunity has recently attracted considerable attention. The current study revealed NK cells as additional players in controlling T-cell activity in CNS autoimmunity. NK-mediated control of T-cell activity in multiple sclerosis (MS) is dysregulated and caused by impaired DNAX accessory molecule-1/CD155 interaction between NK cells and CD4+T cells. Therapeutic immune modulation of the IL-2 receptor with daclizumab, which has just successfully passed a phase III study in relapsing-remitting MS, not only enhances the cytolytic activity of NK cells but also restores defective NK-mediated immune regulation by increasing the proportion of CD155-expressing CD4+T cells, thus rendering CD4+T cells most likely more sensitive to NK-mediated lysis.

OBJECTIVE:To evaluate the effect of dimethyl fumarate (DMF; Tecfidera, Biogen, Weston, MA) on CD4(+) and CD8(+) T cell subsets in patients with multiple sclerosis (MS).METHODS:Peripheral lymphocyte subsets, including CD4(+) and CD8(+) memory cells and T helper (TH) cells TH1, TH2, TH17, and peripheral regulatory T cell (pTreg) subpopulations were analyzed before and 6 months after onset of DMF treatment.RESULTS:CD4(+) and CD8(+) memory T cells were preferentially decreased compared to naive CD4(+) and CD8(+) T cell populations. Within the CD4(+) memory T cell population, frequencies of TH1 cells were decreased, whereas those of TH2 cells were increased and those of TH17 cells remained unaltered. Accordingly, we observed decreased production of interferon γ, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and interleukin (IL)-22 by CD4(+) T cells under DMF treatment, whereas the frequency of IL-4- and IL-17A-producing CD4(+) T cells remained unchanged. With regard to regulatory T cells, proportions of pTreg increased following DMF treatment.CONCLUSION:Our data demonstrate that DMF treatment of patients with MS affects predominantly memory T cells accompanied by a shift in TH cell populations, resulting in a shift toward anti-inflammatory responses. These findings indicate that monitoring of memory subsets might enhance vigilance of impaired antiviral immunity and that patients with TH1-driven disease might preferentially benefit from DMF treatment.CLASSIFICATION OF EVIDENCE:This study provides Class IV evidence that DMF might preferentially reduce CD4(+) and CD8(+) memory T cells in MS.

BACKGROUND:In light of the increased risk of progressive multifocal encephalopathy (PML) development under long-term treatment with the monoclonal antibody natalizumab which is approved for treatment of active relapsing remitting multiple sclerosis (RRMS), there is a clear need for alternative treatment options with comparable efficacy and reduced PML risk. One such option is fingolimod, a functional sphingosin-1-receptor antagonist that has been approved as first oral drug for treatment of active RRMS. However, the optimal switching design in terms of prevention of disease reoccurrence is still unknown. Moreover, potential additive effects of both drugs on immune functions, especially with regard to migration, have not yet been evaluated.METHODS/DESIGN:This is an exploratory, open-label, monocentric, investigator-initiated clinical trial. Fifteen RRMS patients under stable treatment with natalizumab will receive one last natalizumab infusion followed by a wash-out period of 8 weeks before fingolimod treatment initiation for a period of 24 weeks. Disease activity under natalizumab and during switching will be closely monitored by assessment of relapse rate and disease severity as well as high-frequent high-resolution magnetic resonance imaging including quantitative diffusion tensor imaging. Immunological assays include longitudinal assessment of adhesion molecule expression, functional evaluation of the migratory capacity of immune cells in an in-vitro model of the blood-brain-barrier, and the quality of cellular antiviral immune responses.DISCUSSION:Our trial represents the first detailed and longitudinal functional analysis of key immunological parameters in the process of switching from natalizumab and fingolimod, especially with regard to potential additive effects of both drugs on trafficking and immune surveillance. Moreover, our study will generate valuable information about even subtle disease exacerbations as consequence of natalizumab cessation, which will help to understand whether a switching protocol containing a wash-out period of 8 weeks before fingolimod treatment is appropriate in terms of disease stability.

To evaluate the influence of Fingolimod treatment on B-cell subset composition and function in multiple sclerosis patients and its potential clinical relevance. Subset composition and cytokine production of B cells derived from peripheral blood mononuclear cells from multiple sclerosis patients under Fingolimod treatment, untreated multiple sclerosis patients and healthy controls were analyzed by flow cytometry and ELISA. Migration of lymphocyte subsets across primary human brain microvascular endothelial cells was assessed in an in vitro transmigration assay. Cell numbers and composition of B-cell subsets in cerebrospinal fluid and peripheral blood were determined by flow cytometry. Regulatory B-cell frequencies were correlated with parameters of disease stability. Within the peripheral B-cell compartment of Fingolimod-treated patients, the proportion of regulatory B cells (CD38 + CD27 − CD24 + CD5 + ) was significantly increased as compared to treatment-naïve multiple sclerosis patients and to healthy controls, and significantly more regulatory B cells produced Interleukin-10. Fingolimod treatment enhanced the capacity of regulatory B cells to transmigrate across brain endothelial cells in an in vitro model of the blood-brain-barrier. In line with these findings, the cerebrospinal fluid/blood ratio of total B cells and regulatory B cells was strongly increased by Fingolimod treatment, and patients exhibited increased regulatory B-cell frequencies in the cerebrospinal fluid. Finally, elevated regulatory B-cell percentages in the periphery significantly correlated with clinical and paraclinical disease stability. These data suggest a novel and as yet unrecognized role of Fingolimod in correction of the imbalance between regulatory and effector B-cell functions in multiple sclerosis both by direct effects and indirect partitioning effects on B-cell subpopulations.

Human biospecimen collection, processing and preservation are rapidly emerging subjects providing essential support to clinical as well as basic researchers. Unlike collection of other biospecimens (e.g. DNA and serum), biobanking of viable immune cells, such as peripheral blood mononuclear cells (PBMC) and/or isolated immune cell subsets is still in its infancy. While certain aspects of processing and freezing conditions have been studied in the past years, little is known about the effect of blood transportation on immune cell survival, phenotype and specific functions. However, especially for multicentric and cooperative projects it is vital to precisely know those effects. In this study we investigated the effect of blood shipping and pre-processing delay on immune cell phenotype and function both on cellular and subcellular levels. Peripheral blood was collected from healthy volunteers (n = 9): at a distal location (shipped overnight) and in the central laboratory (processed immediately). PBMC were processed in the central laboratory and analyzed post-cryopreservation. We analyzed yield, major immune subset distribution, proliferative capacity of T cells, cytokine pattern and T-cell receptor signal transduction. Results show that overnight transportation of blood samples does not globally compromise T- cell subsets as they largely retain their phenotype and proliferative capacity. However, NK and B cell frequencies, the production of certain PBMC-derived cytokines and IL-6 mediated cytokine signaling pathway are altered due to transportation. Various control experiments have been carried out to compare issues related to shipping versus pre-processing delay on site. Our results suggest the implementation of appropriate controls when using multicenter logistics for blood transportation aiming at subsequent isolation of viable immune cells, e.g. in multicenter clinical trials or studies analyzing immune cells/subsets. One important conclusion might be that despite changes due to overnight shipment, highly standardized central processing (and analysis) could be superior to multicentric de-central processing with more difficult standardization.

Biopreservation and BiobankingVol. 12, No. 1 Biobank ProfileThe Neuroinflammation Biobank in the Department of Neurology, University Hospital Muenster, GermanyAnita Posevitz-Fejfár and Heinz WiendlAnita Posevitz-FejfárSearch for more papers by this author and Heinz WiendlSearch for more papers by this authorPublished Online:19 Feb 2014https://doi.org/10.1089/bio.2014.1212AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View article"The Neuroinflammation Biobank in the Department of Neurology, University Hospital Muenster, Germany." Biopreservation and Biobanking, 12(1), pp. 74–75FiguresReferencesRelatedDetailsCited byCerebrospinal fluid proteomics indicates immune dysregulation and neuronal dysfunction in antibody associated autoimmune encephalitisJournal of Autoimmunity, Vol. 135EMR-integrated minimal core dataset for routine health care and multiple research settings: A case study for neuroinflammatory demyelinating diseases15 October 2019 | PLOS ONE, Vol. 14, No. 10Imaging matrix metalloproteinase activity in multiple sclerosis as a specific marker of leukocyte penetration of the blood-brain barrierScience Translational Medicine, Vol. 8, No. 364 Volume 12Issue 1Feb 2014 InformationCopyright 2014, Mary Ann Liebert, Inc.To cite this article:Anita Posevitz-Fejfár and Heinz Wiendl.The Neuroinflammation Biobank in the Department of Neurology, University Hospital Muenster, Germany.Biopreservation and Biobanking.Feb 2014.74-75.http://doi.org/10.1089/bio.2014.1212Published in Volume: 12 Issue 1: February 19, 2014Online Ahead of Print:February 12, 2014PDF download

Objective: To evaluate the influence of Fingolimod treatment on B-cell subset composition and function in multiple sclerosis patients and its potential clinical relevance. Methods: Subset composition and cytokine production of B cells derived from peripheral blood mononuclear cells from multiple sclerosis patients under Fingolimod treatment, untreated multiple sclerosis patients and healthy controls were analyzed by flow cytometry and ELISA. Migration of lymphocyte subsets across primary human brain microvascular endothelial cells was assessed in an in vitro transmigration assay. Cell numbers and composition of B-cell subsets in cerebrospinal fluid and peripheral blood were determined by flow cytometry. Regulatory B-cell frequencies were correlated with parameters of disease stability. Results: Within the peripheral B-cell compartment of Fingolimod-treated patients, the proportion of regulatory B cells (CD38(+)CD27(-) CD24(+)CD5(+)) was significantly increased as compared to treatment-naive multiple sclerosis patients and to healthy controls, and significantly more regulatory B cells produced Interleukin-10. Fingolimod treatment enhanced the capacity of regulatory B cells to transmigrate across brain endothelial cells in an in vitro model of the blood-brain-barrier. In line with these findings, the cerebrospinal fluid/blood ratio of total B cells and regulatory B cells was strongly increased by Fingolimod treatment, and patients exhibited increased regulatory B-cell frequencies in the cerebrospinal fluid. Finally, elevated regulatory B-cell percentages in the periphery significantly correlated with clinical and paraclinical disease stability. Interpretation: These data suggest a novel and as yet unrecognized role of Fingolimod in correction of the imbalance between regulatory and effector B-cell functions in multiple sclerosis both by direct effects and indirect partitioning effects on B-cell subpopulations.

Background: The StratifyJCV® test is a qualitative assay to classify MS patients as anti-JC virus (JCV) antibody positive or negative. Quantification of anti-JCV antibody levels in serum and cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients might add to the progressive multifocal leukoencephalopathy (PML) risk assessment. Objective: The objective of this study is to test sera of patients in a quantitative anti-JCV antibody assay, and to compare the results with preexisting data from the StratifyJCV® test. Methods: Sera of a total of 175 MS patients and matched non-MS-controls were tested for anti-JCV antibodies using glutathione S-transferase-tagged-VP1 as antigen. Antibody reactivity was quantified in arbitrary units using human immunoglobulin as standard. Results: The comparison of our assay with StratifyJCV® showed good inter-assay agreement (kappa 0.6), and strong correlation for antibody reactivity ( r 2 = 0.94). Discordant samples had low-reactive positivity, and a higher proportion (13% vs. 4%) tested positive in the StratifyJCV® test only. Conclusions: The method presented is a tool for the reliable quantification of anti-JCV antibodies, which demonstrates good agreement with results from StratifyJCV®. In contrast to StratifyJCV®, we pre-adsorbed all of the sera with BK virus (BKV) VP1 protein to reduce cross-reactivity. This step may account for a higher species-specificity of our assay. As such, our assay might be a promising additional tool for PML risk assessment.

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.

Src family kinases (SFK) control multiple processes during brain development and function. We show here that the phosphoprotein associated with glycosphigolipid-enriched microdomains (PAG)/Csk binding protein (Cbp) modulates SFK activity in the brain. The timing and localization of PAG expression overlap with Fyn and Src, both of which we find associated to PAG. We demonstrate in newborn (P1) mice that PAG negatively regulates Src family kinases (SFK). P1 Pag1(-/-) mouse brains show decreased recruitment of Csk into lipid rafts, reduced phosphorylation of the inhibitory tyrosines within SFKs, and an increase in SFK activity of >/ = 50%. While in brain of P1 mice, PAG and Csk are highly and ubiquitously expressed, little Csk is found in adult brain suggesting altered modes of SFK regulation. In adult brain Pag1-deficiency has no effect upon Csk-distribution or inhibitory tyrosine phosphorylation, but kinase activity is now reduced (-20-30%), pointing to the development of a compensatory mechanism that may involve PSD93. The distribution of the Csk-homologous kinase CHK is not altered. Importantly, since the activities of Fyn and Src are decreased in adult Pag1(-/-) mice, thus presenting the reversed phenotype of P1, this provides the first in vivo evidence for a Csk-independent positive regulatory function for PAG in the brain.

Protective adaptive immune responses rely on TCR-mediated recognition of Ag-derived peptides presented by self-MHC molecules. However, self-Ag (tumor)-specific TCRs are often of too low affinity to achieve best functionality. To precisely assess the relationship between TCR–peptide–MHC binding parameters and T cell function, we tested a panel of sequence-optimized HLA-A*0201/NY–ESO-1157–165–specific TCR variants with affinities lying within physiological boundaries to preserve antigenic specificity and avoid cross-reactivity, as well as two outliers (i.e., a very high- and a low-affinity TCR). Primary human CD8 T cells transduced with these TCRs demonstrated robust correlations between binding measurements of TCR affinity and avidity and the biological response of the T cells, such as TCR cell-surface clustering, intracellular signaling, proliferation, and target cell lysis. Strikingly, above a defined TCR–peptide–MHC affinity threshold (KD < ∼5 μM), T cell function could not be further enhanced, revealing a plateau of maximal T cell function, compatible with the notion that multiple TCRs with slightly different affinities participate equally (codominantly) in immune responses. We propose that rational design of improved self-specific TCRs may not need to be optimized beyond a given affinity threshold to achieve both optimal T cell function and avoidance of the unpredictable risk of cross-reactivity.

The paracaspase MALT1 is pivotal in antigen receptor–mediated lymphocyte activation and lymphomagenesis. MALT1 contains a caspase-like domain, but it is unknown whether this domain is proteolytically active. Here we report that MALT1 had arginine-directed proteolytic activity that was activated after T cell stimulation, and we identify the signaling protein Bcl-10 as a MALT1 substrate. Processing of Bcl-10 after Arg228 was required for T cell receptor–induced cell adhesion to fibronectin. In contrast, MALT1 activity but not Bcl-10 cleavage was essential for optimal activation of transcription factor NF-κB and production of interleukin 2. Thus, the proteolytic activity of MALT1 is central to T cell activation, which suggests a possible target for the development of immunomodulatory or anticancer drugs.

In primary human T cells, anergy induction results in enhanced p59Fyn activity. Because Fyn is the kinase primarily responsible for the phosphorylation of PAG (the phosphoprotein associated with glycosphingolipid-enriched microdomains), which negatively regulates Src-kinase activity by recruiting Csk (the C-terminal Src kinase) to the membrane, we investigated whether anergy induction also affects PAG. Analysis of anergic T cells revealed that PAG is hyperphosphorylated at the Csk binding site, leading to enhanced Csk recruitment and inhibitory tyrosine phosphorylation within Fyn. This together with enhanced phosphorylation of a tyrosine within the SH2 domain of Fyn leads to the formation of a hyperactive conformation, thus explaining the enhanced Fyn kinase activity. In addition, we have also identified the formation of a multiprotein complex containing PAG, Fyn, Sam68, and RasGAP in stimulated T cells. We demonstrate that PAG-Fyn overexpression is sufficient to suppress Ras activation in Jurkat T cells and show that this activity is independent of Csk binding. Thus, in addition to negatively regulating Src family kinases by recruiting Csk, PAG also negatively regulates Ras by recruiting RasGAP to the membrane. Finally, by knocking down PAG, we demonstrate both enhanced Src kinase activity and Ras activation, thereby establishing PAG as an important negative regulator of T-cell activation.