Matthias Mehling

FTY720 therapy exerts differential effects on T cell subsets in multiple sclerosis

Background: The oral immunomodulator FTY720 has shown efficacy in patients with relapsing multiple sclerosis (MS). FTY720 functionally antagonizes sphingosine 1-phosphate receptor-1 (S1P1) on T cells and consequently inhibits S1P/S1P1-dependent lymphocyte egress from secondary lymphoid organs. Little is known about the phenotype and function of T cells remaining in peripheral blood during long-term FTY720 treatment. Methods: T cells from FTY720-treated, interferon-beta (IFNβ)-treated and untreated patients with MS, and healthy donors (HD) were analyzed with respect to T cell subpopulation composition, proliferation, and cytokine production. Results: In FTY720-treated patients (n = 16), peripheral blood CD4+ and CD8+ T cell counts were reduced by approximately 80% and 60% when compared to the other groups (IFNβ: n = 7; untreated: n = 5; HD: n = 10). This related to selective reduction of naïve (CCR7+CD45RA+) and central memory (CCR7+CD45RA−) T cells (TCM), and resulted in a relative increase of peripheral effector memory (CCR7−CD45RA− [TEM] and CCR7−CD45RA+ [TEMRA]) T cells. The remaining blood T cell populations displayed a reduced potential to secrete IL-2 and to proliferate in vitro, but rapidly produced interferon-gamma upon reactivation, confirming a functional TEM/TEMRA phenotype. Neither FTY720 nor FTY720-P directly suppressed proliferation or cytokine production by T cells. Conclusion: Therapeutic dosing of FTY720 reduces naïve T cells and TCM, but not TEM, in blood, without affecting T cell function. This is presumably because naïve T cells and TCM express the homing receptor CCR7, allowing recirculation to secondary lymphoid tissues on a regular basis and, thus, trapping of the cells by FTY720 in lymph nodes.

A PD‐1 polymorphism is associated with disease progression in multiple sclerosis

T cells are considered to play a pivotal role in orchestrating the self‐reactive immune responses in multiple sclerosis (MS). Programmed death 1 (PD‐1) is a member of the B7/CD28 superfamily of costimulatory molecules exerting inhibitory functions on T cells. Recently, an intronic 7146G/A polymorphism within the PD‐1 gene was described and suggested to be associated with autoimmunity. We investigated whether this genetic polymorphism is a genetic modifier for risk and progression of MS. Blood samples from 939 German MS patients (mean age, 39 years; range, 13–71; 566 patients [60%] with relapsing‐remitting MS, 279 (30%) with secondary, and 94 (10%) with primary progressive MS) and 272 healthy white controls were tested. Genotyping was performed by polymerase chain reaction and restriction enzyme digestion; results were confirmed by automatic sequencing. A significant association of the mutated allele with a progressive disease course was detected (44% 7146G vs 56% 7146A, χ2 p = 0.002). Consequences of the PD‐1 mutation for T‐cell function were assessed ex vivo in some patients using microsphere‐stimulated peripheral blood lymphocytes and purified CD4 cells. Importantly, PD‐1–mediated inhibition of T‐cell cytokine secretion (interferon‐γ) is impaired in patients carrying the PD‐1 polymorphism. In conclusion, our data suggest that PD‐1 polymorphism is a genetic modifier of the progression of MS, possibly through inducing a partial defect in PD‐1–mediated inhibition of T‐cell activation. Ann Neurol 2005

Th17 central memory T cells are reduced by FTY720 in patients with multiple sclerosis

Objective: FTY720 is a sphingosine 1-phosphate (S1P) receptor modulator that showed efficacy in phase II and III clinical trials in patients with multiple sclerosis (MS). FTY720 inhibits lymphocyte egress from secondary lymphoid organs into the peripheral circulation, thereby reducing the number of circulating naïve and central memory T cells, but not effector memory T cells in blood. Little is known to which of these memory T-cell subsets interleukin 17 (IL-17)-producing T cells (Th17 cells) belong, which are considered to be key mediators of inflammation in MS, and how they are affected by treatment with FTY720. In this study, we determined the phenotype and frequency of Th17 cells in blood of untreated, FTY720-treated, and interferon-β (IFNβ)-treated patients with MS and healthy donors. Methods: In a prospective observational study, circulating T cells were phenotypically characterized and Th17 cells enumerated in T-cell subsets ex vivo. Production of IL-17 upon activation and expression of the Th17-specific transcription factor RORC2 was assessed in vitro. Results: Th17 cells were found primarily within central memory T cells in all study populations. FTY720 treatment reduced blood central memory T cells, including RORC2+ and IL-17-producing T cells, by >90%. FTY720 did not per se affect IL-17 production when added to activated T cells in vitro. Conclusion: Phenotypic Th17 cells are defined by a central memory T-cell phenotype. FTY720 reduces these Th17 cells in blood. This is presumably because central memory T cells are retained by FTY720 in secondary lymphoid organs.

Altered expression of miR-17-5p in CD4+ lymphocytes of relapsing-remitting multiple sclerosis patients

MicroRNA (miRNA) are a class of post‐transcriptional regulators of gene expression targeting mRNA for translational repression and/or degradation. We analyzed the expression of 365 miRNA in lymphocytes in relapsing–remitting MS patients, and show the first evidence for distinct miRNA expression profiles in CD4+, CD8+ and B cells in MS when compared with those in healthy volunteers. MiR‐17‐5p, which is involved in autoimmunity, was up‐regulated in CD4+ cells from MS patients. This was correlated with alterations in the expression of potential target genes of miR‐17‐5p, i.e. phosphatase and tensin homology and phosphatidyl‐inositol‐3‐kinase regulatory subunit 1, which were down‐regulated upon stimulation of CD4+ cells with anti‐CD3/CD28 in vitro. Functional experiments with a synthetic inhibitor of miR‐17 supported the link between miRNA expression and the altered target gene expression. Moreover, we found distinct responses of deregulated miRNA to stimulation, i.e. miR‐17‐5p and miR‐193a were strongly up‐regulated, in contrast to the down‐regulation of miR‐497, miR‐1 and miR‐126. Other deregulated miRNA did not respond to the stimulation probably due to other, non‐T‐cell activation related, mechanisms in their mode of action. Our findings support the role of miRNA‐dependent regulatory mechanisms in the immunopathogenesis of MS.

Clinical immunology of the sphingosine 1-phosphate receptor modulator fingolimod (FTY720) in multiple sclerosis.

The oral sphingosine 1-phosphate (S1P) receptor (S1PR) modulator fingolimod has been shown to be effective in the treatment of patients with relapsing multiple sclerosis (MS). The drug binds with high affinity to 4 of the 5 G-protein–coupled S1P receptors (S1P1–5). After binding, the receptors are internalized, degraded, and thus functionally antagonized by fingolimod. Under physiologic conditions, S1P1 mediates the egress of lymphocytes from secondary lymphoid organs to the peripheral circulation. Functional antagonism of S1P1 by fingolimod results in a reduction in peripheral lymphocyte counts by inhibiting egress of lymphocytes, including potentially encephalitogenic T cells and their naïve progenitors that would otherwise be present within the circulation. Despite the fingolimod-mediated reduction of lymphocyte counts, fingolimod-treated patients with MS have been shown to have few infections and related complications and were able to mount antigen-specific immune responses in vaccination studies.

Neurofilament heavy chain in CSF correlates with relapses and disability in multiple sclerosis

Objective: Neurodegeneration is now accepted as a pathologic hallmark of multiple sclerosis (MS). We sought to discover whether CSF levels of neurofilament heavy chain protein (NfHSMI35) correlate with disability, disease activity, or specific stages of MS. Methods: An electrochemiluminescence immunoassay was used to retrospectively measure NfHSMI35 in CSF of patients with clinically isolated syndrome (CIS) (n = 63), relapsing-remitting multiple sclerosis (RRMS) (n = 39), secondary progressive multiple sclerosis (SPMS) (n = 25), primary progressive multiple sclerosis (PPMS) (n = 23), or controls (n = 73). Cell count and CSF levels of immunoglobulin and albumin were also measured. Results: CSF levels of NfHSMI35 increased with age in controls (rs = 0.50, p < 0.0001) and CIS (rs = 0.50, p < 0.0001); this effect was less pronounced in RRMS (rs = 0.35, p = 0.027) and absent in SPMS/PPMS. After age correction, NfHSMI35 levels were found to be higher in all disease stages compared to control. Relapses were associated with higher CSF NfHSMI35 values compared with stable disease. NfHSMI35 levels correlated with EDSS scores in patients with CIS and RRMS (rs = 0.33, p = 0.001), and during relapse (rs = 0.35, p = 0.01); the correlation was most prominent in RRMS during relapse (rs = 0.54, p = 0.01). This was not the case for any of the other CSF markers examined. Conclusions: Neuronal loss is a feature of aging, and the age-dependent increase of CSF NfHSMI35 suggests that this loss accelerates over time. For MS, increased NfHSMI35 levels reflect the superimposed presence of further neurodegenerative processes. Evaluation of NfHSMI35 levels is likely to provide a useful surrogate for measuring the rate of neurodegeneration in MS. Furthermore, the dissociation of NfHSMI35 levels with biomarkers of inflammation suggests that the mechanisms responsible for their production are at least partly independent.

A highly sensitive electrochemiluminescence immunoassay for the neurofilament heavy chain protein

BACKGROUND The loss of neurological function is closely related to axonal damage. Neurofilament subunits are concentrated in neurons and axons and have emerged as promising biomarkers for neurodegeneration. Electrochemiluminescence (ECL) based assays are known to be of superior sensitivity and require less sample volume than conventional ELISAs. METHODS We developed an ECL based solid-phase sandwich immunoassay to measure the neurofilament heavy chain protein (NfH(SMI35)) in CSF. We employed commercially available antibodies as previously used in a conventional ELISA (Petzold et al., 2003; Petzold and Shaw, 2007). The optimised and validated assay was applied in a reference cohort and defined patient groups. RESULTS Analytical sensitivity (background plus three SD) of our assay was 2.4 pg/ml. The mean intra-assay coefficient of variation (CV) was 4.8% and the inter-assay CV 8.4%. All measured control and patient samples produced signals well above background. Patients with multiple sclerosis (MS) (median 46.2 pg/ml, n=95), amyotrophic lateral sclerosis (ALS) (160.1 pg/ml, n=50), mild cognitive impairment/Alzheimers disease (MCI/AD) (65.6 pg/ml, n=20), Guillain-Barre syndrome (GBS) (91.0 pg/ml, n=20) or subarachnoid hemorrhage (SAH) (345.0 pg/ml, n=20) had higher CSF NfH(SMI35) values than the reference cohort (27.1 pg/ml, n=73, p<0.0001 for each comparison). CONCLUSION The new ECL based assay for NfH(SMI35) in CSF is superior in terms of sensitivity, precision and accuracy to previously published methods (Petzold et al., 2003; Shaw et al., 2005; Teunissen et al., 2009). The improved performance and small sample volume requirement qualify this method in experimental settings and clinical trials designed to perform a number of tests on limited amounts of material.

Antigen-specific adaptive immune responses in fingolimod-treated multiple sclerosis patients

T cells exit secondary lymphoid organs along a sphingosine1‐phosphate (S1P) gradient and, accordingly, are reduced in blood upon fingolimod‐mediated S1P‐receptor (S1PR)‐blockade. Serving as a model of adaptive immunity, we characterized cellular and humoral immune responses to influenza vaccine in fingolimod‐treated patients with multiple sclerosis (MS) and in untreated healthy controls. Although the mode of action of fingolimod might predict reduced immunity, vaccine‐triggered T cells accumulated normally in blood despite efficient S1PR‐blockade. Concentrations of anti–influenza A/B immunoglobulin (Ig)M and IgG also increased similarly in both groups. These results indicate that fingolimod‐treated individuals can mount vaccine‐specific adaptive immune responses comparable to healthy controls. Ann Neurol 2011

Antimyelin antibodies in clinically isolated syndromes correlate with inflammation in MRI and CSF

ObjectiveWe investigated the correlation of anti-myelin oligodendrocyte glycoprotein- (anti-MOG) and anti-myelin basic protein antibodies (anti-MBP) in serum of CIS patients with inflammatory signs in MRI and in CSF and, as previously suggested, the incidence of more frequent and rapid progression to clinically definite MS (CDMS).Methods133 CIS patients were analysed for anti-MOG and anti-MBP (Western blot). Routine CSF and cranial MRI (quantitatively and qualitatively) measures were analyzed. 55 patients had a follow-up of at least 12 months or until conversion to CDMS.ResultsPatients with anti-MOG and anti-MBP had an increased intrathecal IgG production and CSF white blood cell count (p = 0.048 and p = 0.036). When anti-MBP alone, or both antibodies were present the cranial MRI showed significantly more T2 lesions (p = 0.007 and p = 0.01, respectively). There was a trend for more lesion dissemination in anti-MBP positive patients (p = 0.076). Conversely, anti-MOG- and/or anti-MBP failed to predict conversion to CDMS in our follow-up group (n = 55). Only in female patients with at least one MRI lesion (n = 34) did the presence of anti-MOG correlate with more frequent (p = 0.028) and more rapid (p = 0.0209) transition to CDMS.ConclusionsPresence of anti-MOG or anti-MBP or both was not significantly associated with conversion to CDMS in our CIS cohort. However, patients with anti-MOG and anti-MBP had higher lesion load and more disseminated lesions in cranial MRI as well as higher values for CSF leucocytes and intrathecal IgG production. Our data support a correlation of anti-MOG and anti-MBP to inflammatory signs in MRI and CSF. The prognostic value of these antibodies for CDMS, however, seems to be less pronounced than previously reported.

Fingolimod for Multiple Sclerosis: Mechanism of Action, Clinical Outcomes, and Future Directions

The oral sphingosine 1-phosphate receptor (S1PR) modulator fingolimod functionally antagonizes S1PR hereby blocking lymphocyte egress from secondary lymphoid organs to the peripheral blood circulation. This results in a reduction in peripheral lymphocyte counts, including potentially encephalitogenic T cells. In patients with relapsing multiple sclerosis fingolimod has been shown to be an effective treatment. In phase 2 and phase 3 studies fingolimod-treated patients had reduced disease activity clinically and in MRI. Although severe infectious complications occurred in single cases treated with fingolimod, the frequency of overall infections was comparable in fingolimod-treated patients and controls. Overall, in clinical studies fingolimod was well tolerated and had a favorable safety profile. In follow-up studies with continuous fingolimod, treatment showed sustained efficacy while being well tolerated.