Systems Analysis of Immune Changes after B-cell Depletion in Autoimmune Multiple Sclerosis.

Multiple sclerosis (MS) is a complex genetically mediated autoimmune disease of the central nervous system where anti-CD20-mediated B cell depletion is remarkably effective in the treatment of early disease. While previous studies investigated the effect of B cell depletion on select immune cell subsets using flow cytometry-based methods, the therapeutic impact on patient immune landscape is unknown. In this study, we explored how a therapy-driven " in vivo perturbation " modulates the diverse immune landscape by measuring transcriptomic granularity with single-cell RNA sequencing (scRNAseq). We demonstrate that B cell depletion leads to cell type-specific changes in the abundance and function of CSF macrophages and peripheral blood monocytes. Specifically, a CSF-specific macrophage population with an anti-inflammatory transcriptomic signature and peripheral CD16 + monocytes increased in frequency post-B cell depletion. In addition, we observed increases in TNFα messenger RNA and protein in monocytes post-B cell depletion, consistent with the finding that anti-TNFα treatment exacerbates autoimmune activity in MS. In parallel, B cell depletion also induced changes in peripheral CD4 + T cell populations, including increases in the frequency of TIGIT + regulatory T cells and marked decreases in the frequency of myelin peptide loaded-tetramer binding CD4 + T cells. Collectively, this study provides an exhaustive transcriptomic map of immunological changes, revealing different mechanisms of action contributing to the high efficacy in B cell depletion treatment of MS.

Fatigue, depression, and product tolerability during long-term treatment with intravenous immunoglobulin (Gamunex® 10%) in patients with chronic inflammatory demyelinating polyneuropathy.

INTRODUCTION/AIMS: Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is characterized by progressive weakness and sensory loss, often affecting patient's ability to walk and perform activities of daily living independently. Furthermore, patients often report fatigue and depression which can affect their quality of life. These symptoms were assessed in CIDP patients receiving long-term intravenous immunoglobulin (IVIG) treatment. METHODS: GAMEDIS was a multi-center, prospective, non-interventional study in adult CIDP patients treated with IVIG (10%) and followed for two years. Inflammatory Neuropathy Cause and Treatment (INCAT) disability score, Hughes Disability Scale (HDS), Fatigue Severity Scale (FSS), Beck Depression Inventory II (BDI), Short Form-36 health survey (SF-36) and Work Productivity and Activity Impairment Score Attributable to General Health (WPAI-GH) were assessed at baseline and quarterly. Dosing and treatment intervals, changes in outcome parameters, and adverse events (AEs) were analyzed. RESULTS: 148 evaluable patients were followed for a mean of 83.3 weeks. The mean maintenance IVIG dose was 0.9 g/kg/cycle (mean cycle interval 38 days). Disability and fatigue remained stable throughout the study. Mean INCAT score: 2.4 ± 1.8 at baseline and 2.5 ± 1.9 at study end. HDS: 74.3% healthy/minor symptoms at baseline and 71.6% at study end. Mean FSS: 4.2 ± 1.6 at baseline and 4.1 ± 1.7 at study end. All patients reported minimal/no depression at baseline and throughout. SF-36 and WPAI-GH scores remained stable. Fifteen patients (9.5%) experienced potentially treatment-related AEs. There were no AEs in 99.3% of infusions. DISCUSSION: Long-term treatment of CIDP patients with IVIG 10% in real-world conditions maintained clinical stability on fatigue and depression over 96 weeks. This treatment was well-tolerated and safe.

A genome-wide association study in autoimmune neurological syndromes with anti-GAD65 autoantibodies.

Autoimmune neurological syndromes (AINS) with autoantibodies against the 65 kDa isoform of the glutamic acid decarboxylase (GAD65) present with limbic encephalitis, including temporal lobe seizures or epilepsy, cerebellitis with ataxia, and stiff-person-syndrome or overlap forms. Anti-GAD65 autoantibodies are also detected in autoimmune diabetes mellitus, which has a strong genetic susceptibility conferred by human leukocyte antigen (HLA) and non-HLA genomic regions. We investigated the genetic predisposition in patients with anti-GAD65 AINS. We performed a genome-wide association study (GWAS) and an association analysis of the HLA region in a large German cohort of 1214 individuals. These included 167 patients with anti-GAD65 AINS, recruited by the German Network for Research on Autoimmune Encephalitis (GENERATE), and 1047 individuals without neurological or endocrine disease as population-based controls. Predictions of protein expression changes based on GWAS findings were further explored and validated in the CSF proteome of a virtually independent cohort of 10 patients with GAD65-AINS and 10 controls. Our GWAS identified 16 genome-wide significant (P < 5 × 10-8) loci for the susceptibility to anti-GAD65 AINS. The top variant, rs2535288 [P = 4.42 × 10-16, odds ratio (OR) = 0.26, 95% confidence interval (CI) = 0.187-0.358], localized to an intergenic segment in the middle of the HLA class I region. The great majority of variants in these loci (>90%) mapped to non-coding regions of the genome. Over 40% of the variants have known regulatory functions on the expression of 48 genes in disease relevant cells and tissues, mainly CD4+ T cells and the cerebral cortex. The annotation of epigenomic marks suggested specificity for neural and immune cells. A network analysis of the implicated protein-coding genes highlighted the role of protein kinase C beta (PRKCB) and identified an enrichment of numerous biological pathways participating in immunity and neural function. Analysis of the classical HLA alleles and haplotypes showed no genome-wide significant associations. The strongest associations were found for the DQA1*03:01-DQB1*03:02-DRB1*04:01HLA haplotype (P = 4.39 × 10-4, OR = 2.5, 95%CI = 1.499-4.157) and DRB1*04:01 allele (P = 8.3 × 10-5, OR = 2.4, 95%CI = 1.548-3.682) identified in our cohort. As predicted, the CSF proteome showed differential levels of five proteins (HLA-A/B, C4A, ATG4D and NEO1) of expression quantitative trait loci genes from our GWAS in the CSF proteome of anti-GAD65 AINS. These findings suggest a strong genetic predisposition with direct functional implications for immunity and neural function in anti-GAD65 AINS, mainly conferred by genomic regions outside the classical HLA alleles.

Broader Epstein-Barr virus-specific T cell receptor repertoire in patients with multiple sclerosis.

Epstein-Barr virus (EBV) infection precedes multiple sclerosis (MS) pathology and cross-reactive antibodies might link EBV infection to CNS autoimmunity. As an altered anti-EBV T cell reaction was suggested in MS, we queried peripheral blood T cell receptor ß chain (TCRß) repertoires of 1,395 MS patients, 887 controls, and 35 monozygotic, MS-discordant twin pairs for multimer-confirmed, viral antigen-specific TCRß sequences. We detected more MHC-I-restricted EBV-specific TCRß sequences in MS patients. Differences in genetics or upbringing could be excluded by validation in monozygotic twin pairs discordant for MS. Anti-VLA-4 treatment amplified this observation, while interferon ß- or anti-CD20 treatment did not modulate EBV-specific T cell occurrence. In healthy individuals, EBV-specific CD8+ T cells were of an effector-memory phenotype in peripheral blood and cerebrospinal fluid. In MS patients, cerebrospinal fluid also contained EBV-specific central-memory CD8+ T cells, suggesting recent priming. Therefore, MS is not only preceded by EBV infection, but also associated with broader EBV-specific TCR repertoires, consistent with an ongoing anti-EBV immune reaction in MS.

K2P18.1 translates T cell receptor signals into thymic regulatory T cell development.

It remains largely unclear how thymocytes translate relative differences in T cell receptor (TCR) signal strength into distinct developmental programs that drive the cell fate decisions towards conventional (Tconv) or regulatory T cells (Treg). Following TCR activation, intracellular calcium (Ca2+) is the most important second messenger, for which the potassium channel K2P18.1 is a relevant regulator. Here, we identify K2P18.1 as a central translator of the TCR signal into the thymus-derived Treg (tTreg) selection process. TCR signal was coupled to NF-κB-mediated K2P18.1 upregulation in tTreg progenitors. K2P18.1 provided the driving force for sustained Ca2+ influx that facilitated NF-κB- and NFAT-dependent expression of FoxP3, the master transcription factor for Treg development and function. Loss of K2P18.1 ion-current function induced a mild lymphoproliferative phenotype in mice, with reduced Treg numbers that led to aggravated experimental autoimmune encephalomyelitis, while a gain-of-function mutation in K2P18.1 resulted in increased Treg numbers in mice. Our findings in human thymus, recent thymic emigrants and multiple sclerosis patients with a dominant-negative missense K2P18.1 variant that is associated with poor clinical outcomes indicate that K2P18.1 also plays a role in human Treg development. Pharmacological modulation of K2P18.1 specifically modulated Treg numbers in vitro and in vivo. Finally, we identified nitroxoline as a K2P18.1 activator that led to rapid and reversible Treg increase in patients with urinary tract infections. Conclusively, our findings reveal how K2P18.1 translates TCR signals into thymic T cell fate decisions and Treg development, and provide a basis for the therapeutic utilization of Treg in several human disorders.

Subcortical Volumes as Early Predictors of Fatigue in Multiple Sclerosis.

OBJECTIVE: Fatigue is a frequent and severe symptom in multiple sclerosis (MS), but its pathophysiological origin remains incompletely understood. We aimed to examine the predictive value of subcortical gray matter volumes for fatigue severity at disease onset and after 4 years by applying structural equation modeling (SEM). METHODS: This multicenter cohort study included 601 treatment-naive patients with MS after the first demyelinating event. All patients underwent a standardized 3T magnetic resonance imaging (MRI) protocol. A subgroup of 230 patients with available clinical follow-up data after 4 years was also analyzed. Associations of subcortical volumes (included into SEM) with MS-related fatigue were studied regarding their predictive value. In addition, subcortical regions that have a central role in the brain network (hubs) were determined through structural covariance network (SCN) analysis. RESULTS: Predictive causal modeling identified volumes of the caudate (s [standardized path coefficient] = 0.763, p = 0.003 [left]; s = 0.755, p = 0.006 [right]), putamen (s = 0.614, p = 0.002 [left]; s = 0.606, p = 0.003 [right]) and pallidum (s = 0.606, p = 0.012 [left]; s = 0.606, p = 0.012 [right]) as prognostic factors for fatigue severity in the cross-sectional cohort. Moreover, the volume of the pons was additionally predictive for fatigue severity in the longitudinal cohort (s = 0.605, p = 0.013). In the SCN analysis, network hubs in patients with fatigue worsening were detected in the putamen (p = 0.008 [left]; p = 0.007 [right]) and pons (p = 0.0001). INTERPRETATION: We unveiled predictive associations of specific subcortical gray matter volumes with fatigue in an early and initially untreated MS cohort. The colocalization of these subcortical structures with network hubs suggests an early role of these brain regions in terms of fatigue evolution. ANN NEUROL 2022;91:192-202.

Sunlight exposure exerts immunomodulatory effects to reduce multiple sclerosis severity.

Multiple sclerosis (MS) disease risk is associated with reduced sun-exposure. This study assessed the relationship between measures of sun exposure (vitamin D [vitD], latitude) and MS severity in the setting of two multicenter cohort studies (nNationMS = 946, nBIONAT = 990). Additionally, effect-modification by medication and photosensitivity-associated MC1R variants was assessed. High serum vitD was associated with a reduced MS severity score (MSSS), reduced risk for relapses, and lower disability accumulation over time. Low latitude was associated with higher vitD, lower MSSS, fewer gadolinium-enhancing lesions, and lower disability accumulation. The association of latitude with disability was lacking in IFN-ß-treated patients. In carriers of MC1R:rs1805008(T), who reported increased sensitivity toward sunlight, lower latitude was associated with higher MRI activity, whereas for noncarriers there was less MRI activity at lower latitudes. In a further exploratory approach, the effect of ultraviolet (UV)-phototherapy on the transcriptome of immune cells of MS patients was assessed using samples from an earlier study. Phototherapy induced a vitD and type I IFN signature that was most apparent in monocytes but that could also be detected in B and T cells. In summary, our study suggests beneficial effects of sun exposure on established MS, as demonstrated by a correlative network between the three factors: Latitude, vitD, and disease severity. However, sun exposure might be detrimental for photosensitive patients. Furthermore, a direct induction of type I IFNs through sun exposure could be another mechanism of UV-mediated immune-modulation in MS.

Ocrelizumab Treatment in Patients with Primary Progressive Multiple Sclerosis: Short-term Safety Results from a Compassionate Use Programme in Germany.

OBJECTIVES: In January 2018, the European Union (EU) approved ocrelizumab in relapsing multiple sclerosis (RMS) and as the first disease-modifying therapy (DMT) for patients with primary progressive multiple sclerosis (PPMS) with efficacy proven in a phase 3 randomised controlled trial. Eleven months prior to the European regulatory approval, a compassionate use programme (CUP) made ocrelizumab available to 489 patients with PPMS in Germany, thereby for the first time providing a therapeutic option to patients with PPMS who could not participate in ocrelizumab studies. Here, we report real-world patient characteristics and short-term safety data of patients with PPMS treated with ocrelizumab in this CUP. PATIENTS AND METHODS: This CUP was initiated in February 2017 - shortly before US Food and Drug administration approval in March 2017 - and ended in January 2018, following ocrelizumab approval in the EU. Adult patients (age ≥18 years) with PPMS who had a positive benefit/risk ratio according to the treating physician were eligible for inclusion at German treatment centres. The main exclusion criteria were current/recent treatment with other immune therapies and unresolved/chronic/active infections. Patients received methylprednisolone and an antihistamine before treatment with intravenous ocrelizumab in 6-month cycles. The first ocrelizumab dose was a 300 mg infusion followed by a second 300 mg infusion 2 weeks later; subsequent doses were delivered as a single 600 mg infusion. Adverse events were reported immediately. RESULTS: Of 580 requests received from 104 centres, 525 patients met the eligibility criteria. Thirty-five patients did not participate due to withdrawal by the treating physician, and one due to death prior to treatment. A total of 489 patients received at least one 600 mg dose of ocrelizumab (administered as two 300 mg infusions) and 51 received a second dose. Due to termination of the CUP upon marketing authorisation, the maximum follow-up period was 12 months. Median patient age was 52 years (range: 24-73), and 49% were female. Previous immunomodulatory or immunosuppressive therapies had been received by 41% of patients, with the most commonly used being glucocorticoids, mitoxantrone, interferon-ß and glatiramer acetate. Patients with a previous malignancy, serious disease or infection (42 patients, 9%) had recovered from this prior to the CUP. Nine serious adverse events and 70 non-serious adverse events were reported in 40 patients. Adverse event categories were generally consistent with the known safety profile of ocrelizumab; one patient had carry-over progressive multifocal leukoencephalopathy (PML) due to previous natalizumab treatment. CONCLUSION: This CUP provides first real-world observations of ocrelizumab for the treatment of PPMS in a large patient cohort in Germany, supporting that ocrelizumab is generally well-tolerated in clinical practice. Physicians should be vigilant for early symptoms of PML, as to date, 9 PML cases that were all confounded have been reported in patients treated with ocrelizumab worldwide, with 8 carry-over cases from a prior DMT.

Harnessing Real-World Data to Inform Decision-Making: Multiple Sclerosis Partners Advancing Technology and Health Solutions (MS PATHS).

Background: Multiple Sclerosis Partners Advancing Technology and Health Solutions (MS PATHS) is the first example of a learning health system in multiple sclerosis (MS). This paper describes the initial implementation of MS PATHS and initial patient characteristics. Methods: MS PATHS is an ongoing initiative conducted in 10 healthcare institutions in three countries, each contributing standardized information acquired during routine care. Institutional participation required the following: active MS patient census of ≥500, at least one Siemens 3T magnetic resonance imaging scanner, and willingness to standardize patient assessments, share standardized data for research, and offer universal enrolment to capture a representative sample. The eligible participants have diagnosis of MS, including clinically isolated syndrome, and consent for sharing pseudonymized data for research. MS PATHS incorporates a self-administered patient assessment tool, the Multiple Sclerosis Performance Test, to collect a structured history, patient-reported outcomes, and quantitative testing of cognition, vision, dexterity, and walking speed. Brain magnetic resonance imaging is acquired using standardized acquisition sequences on Siemens 3T scanners. Quantitative measures of brain volume and lesion load are obtained. Using a separate consent, the patients contribute DNA, RNA, and serum for future research. The clinicians retain complete autonomy in using MS PATHS data in patient care. A shared governance model ensures transparent data and sample access for research. Results: As of August 5, 2019, MS PATHS enrolment included participants (n = 16,568) with broad ranges of disease subtypes, duration, and severity. Overall, 14,643 (88.4%) participants contributed data at one or more time points. The average patient contributed 15.6 person-months of follow-up (95% CI: 15.5-15.8); overall, 166,158 person-months of follow-up have been accumulated. Those with relapsing-remitting MS demonstrated more demographic heterogeneity than the participants in six randomized phase 3 MS treatment trials. Across sites, a significant variation was observed in the follow-up frequency and the patterns of disease-modifying therapy use. Conclusions: Through digital health technology, it is feasible to collect standardized, quantitative, and interpretable data from each patient in busy MS practices, facilitating the merger of research and patient care. This approach holds promise for data-driven clinical decisions and accelerated systematic learning.

Alternative splicing of the TNFSF13B (BAFF) pre-mRNA and expression of the BAFFX1 isoform in human immune cells.

Human B cell activating factor (TNFSF13B, BAFF) is a tumor necrosis factor superfamily member. Binding its unique receptor (TNFRSF13C, BAFF-R) mediates gene expression and cell survival in B cells via activation of NFκB pathway. Furthermore, there is data indicating a role in T cell function. A functionally inhibitory isoform (ΔBAFF) resulting from the deletion of exon 3 in the TNFSF13B pre-RNA has already been reported. However, data on the complexity of post-transcriptional regulation is scarce. Here, we report molecular cloning of nine TNFSF13B transcript variants resulting from alternative splicing of the TNFSF13B pre-mRNA including BAFFX1. This variant is characterized by a partial retention of intron 3 of the TNFSF13B gene causing the appearance of a premature stop codon. We demonstrate the expression of the corresponding BAFFX1 protein in Jurkat T cells, in ex vivo human immune cells and in human tonsillar tissue. Thereby we contribute to the understanding of TNFSF13B gene regulation and reveal that BAFF is regulated through a post-transcriptional mechanism to a greater extent than reported to date.

Genetic determinants of the humoral immune response in MS.

OBJECTIVE: In this observational study, we investigated the impact of genetic factors at the immunoglobulin heavy chain constant locus on chromosome 14 and the major histocompatibility complex region on intrathecal immunoglobulin G, A, and M levels as well as on B cells and plasmablasts in the CSF and blood of patients with multiple sclerosis (MS). METHODS: Using regression analyses, we tested genetic variants on chromosome 14 and imputed human leukocyte antigen (HLA) alleles for associations with intrathecal immunoglobulins in 1,279 patients with MS or clinically isolated syndrome and with blood and CSF B cells and plasmablasts in 301 and 348 patients, respectively. RESULTS: The minor alleles of variants on chromosome 14 were associated with higher intrathecal immunoglobulin G levels (ß = 0.58 [0.47 to 0.68], lowest adjusted p = 2.32 × 10-23), and lower intrathecal immunoglobulin M (ß = -0.56 [-0.67 to -0.46], p = 2.06 × 10-24) and A (ß = -0.42 [-0.54 to -0.31], p = 7.48 × 10-11) levels. Alleles from the HLA-B*07:02-DRB1*15:01-DQA1*01:02-DQB1*06:02 haplotype were associated with higher (lowest p = 2.14 × 10-7) and HLA-B*44:02 with lower (ß = -0.35 [-0.54 to -0.17], p = 1.38 × 10-2) immunoglobulin G levels. Of interest, different HLA alleles were associated with lower intrathecal immunoglobulin M (HLA-C*02:02, ß = -0.45 [-0.61 to -0.28], p = 1.01 × 10-5) and higher immunoglobulin A levels (HLA-DQA1*01:03-DQB1*06:03-DRB1*13:01 haplotype, ß = 0.40 [0.21 to 0.60], p = 4.46 × 10-3). The impact of HLA alleles on intrathecal immunoglobulin G and M levels could mostly be explained by associations with CSF B cells and plasmablasts. CONCLUSION: Although some HLA alleles seem to primarily drive the extent of humoral immune responses in the CNS by increasing CSF B cells and plasmablasts, genetic variants at the immunoglobulin heavy chain constant locus might regulate intrathecal immunoglobulins levels via different mechanisms.

Epidemiology, characteristics and treatment of patients with relapsing remitting multiple sclerosis and incidence of high disease activity: Real world evidence based on German claims data.

BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory, immune mediated disease of the central nervous system, with Relapsing Remitting MS (RRMS) being the most common type. Within the last years, the status of high disease activity (HDA) has become increasingly important for clinical decisions. Nevertheless, little is known about the incidence, the characteristics, and the current treatment of patients with RRMS and HDA in Germany. Therefore, this study aims to estimate the incidence of HDA in a German RRMS patient population, to characterize this population and to describe current drug treatment routines and further healthcare utilization of these patients. METHODS: A claims data analyses has been conducted, using a sample of the InGef Research Database that comprises data of approximately four million insured persons from around 70 German statutory health insurances (SHI). The study was conducted in a retrospective cohort design, including the years 2012-2016. Identification of RRMS population based on ICD-10 code (ICD-10-GM: G35.1). For identification of HDA, criteria from other studies as well as expert opinions have been used. Information on incidence, characteristics and current treatment of patients with RRMS and HDA was considered. RESULTS: The overall HDA incidence within the RRMS population was 8.5% for 2016. It was highest for the age group of 0-19 years (29.4% women, 33.3% men) and lowest for the age group of ≥ 50 years (4.3% women, 5.6% men). Mean age of patients with RRMS and incident HDA was 38.4 years (SD: 11.8) and women accounted for 67.8%. Analyses of drug utilization showed that 82.4% received at least one disease-modifying drug (DMD) in 2016. A percentage of 49.8% of patients received drugs for relapse therapy. A share of 55% of RRMS patients with HDA had at least one hospitalization with a mean length of stay of 13.9 days (SD: 18.3 days) in 2016. The average number of outpatient physician contacts was 28.1 (SD: 14.0). CONCLUSIONS: This study based on representative Germany-wide claims data from the SHI showed a high incidence of HDA especially within the young RRMS population. Future research should consider HDA as an important criterion for the quality of care for MS patients.

Clinical implications of serum neurofilament in newly diagnosed MS patients: A longitudinal multicentre cohort study.

BACKGROUND: We aim to evaluate serum neurofilament light chain (sNfL), indicating neuroaxonal damage, as a biomarker at diagnosis in a large cohort of early multiple sclerosis (MS) patients. METHODS: In a multicentre prospective longitudinal observational cohort, patients with newly diagnosed relapsing-remitting MS (RRMS) or clinically isolated syndrome (CIS) were recruited between August 2010 and November 2015 in 22 centers. Clinical parameters, MRI, and sNfL levels (measured by single molecule array) were assessed at baseline and up to four-year follow-up. FINDINGS: Of 814 patients, 54.7% (445) were diagnosed with RRMS and 45.3% (369) with CIS when applying 2010 McDonald criteria (RRMS[2010] and CIS[2010]). After reclassification of CIS[2010] patients with existing CSF analysis, according to 2017 criteria, sNfL levels were lower in CIS[2017] than RRMS[2017] patients (9.1 pg/ml, IQR 6.2-13.7 pg/ml, n = 45; 10.8 pg/ml, IQR 7.4-20.1 pg/ml, n = 213; p = 0.036). sNfL levels correlated with number of T2 and Gd+ lesions at baseline and future clinical relapses. Patients receiving disease-modifying therapy (DMT) during the first four years had higher baseline sNfL levels than DMT-naïve patients (11.8 pg/ml, IQR 7.5-20.7 pg/ml, n = 726; 9.7 pg/ml, IQR 6.4-15.3 pg/ml, n = 88). Therapy escalation decisions within this period were reflected by longitudinal changes in sNfL levels. INTERPRETATION: Assessment of sNfL increases diagnostic accuracy, is associated with disease course prognosis and may, particularly when measured longitudinally, facilitate therapeutic decisions. FUNDING: Supported the German Federal Ministry for Education and Research, the German Research Council, and Hertie-Stiftung.

Is APOE ε4 associated with cognitive performance in early MS?

OBJECTIVE: To assess the impact of APOE polymorphisms on cognitive performance in patients newly diagnosed with clinically isolated syndrome (CIS) or relapsing-remitting MS (RRMS). METHODS: This multicenter cohort study included 552 untreated patients recently diagnosed with CIS or RRMS according to the 2005 revised McDonald criteria. The single nucleotide polymorphisms rs429358 (ε4) and rs7412 (ε2) of the APOE haplotype were assessed by allelic discrimination assays. Cognitive performance was evaluated using the 3-second paced auditory serial addition test and the Multiple Sclerosis Inventory Cognition (MUSIC). Sum scores were calculated to approximate the overall cognitive performance and memory-centered cognitive functions. The impact of the APOE carrier status on cognitive performance was assessed using multiple linear regression models, also including demographic, clinical, MRI, and lifestyle factors. RESULTS: APOE ε4 homozygosity was associated with lower overall cognitive performance, whereas no relevant association was observed for APOE ε4 heterozygosity or APOE ε2 carrier status. Furthermore, higher disability levels, MRI lesion load, and depressive symptoms were associated with lower cognitive performance. Patients consuming alcohol had higher test scores than patients not consuming alcohol. Female sex, lower disability, and alcohol consumption were associated with better performance in the memory-centered subtests of MUSIC, whereas no relevant association was observed for APOE carrier status. CONCLUSION: Along with parameters of a higher disease burden, APOE ε4 homozygosity was identified as a potential predictor of cognitive performance in this large cohort of patients with CIS and early RRMS.

Complete Epstein-Barr virus seropositivity in a large cohort of patients with early multiple sclerosis.

OBJECTIVE: To determine the prevalence of antibodies to Epstein-Barr virus (EBV) in a large cohort of patients with early multiple sclerosis (MS). METHODS: Serum samples were collected from 901 patients with a clinically isolated syndrome (CIS) or early relapsing-remitting multiple sclerosis (RRMS) participating in the German National MS cohort, a prospective cohort of patients with early MS with stringent inclusion criteria. Epstein-Barr nuclear antigen (EBNA)-1 and viral capsid antigen (VCA) antibodies were measured in diluted sera by chemiluminescence immunoassays (CLIAs). Sera of EBNA-1 and VCA antibody-negative patients were retested undiluted by an EBV IgG immunoblot. For comparison, we retrospectively analysed the EBV seroprevalence across different age cohorts, ranging from 0 to >80 years, in a large hospital population (N=16 163) from Berlin/Northern Germany. RESULTS: EBNA-1 antibodies were detected by CLIA in 839 of 901 patients with CIS/RRMS. Of the 62 patients without EBNA-1 antibodies, 45 had antibodies to VCA as detected by CLIA. In all of the remaining 17 patients, antibodies to EBV were detected by immunoblot. Altogether, 901 of 901 (100%) patients with CIS/RRMS were EBV-seropositive. EBV seropositivity increased with age in the hospital population but did not reach 100% in any of the investigated age cohorts. CONCLUSION: The complete EBV seropositivity in this large cohort of patients with early MS strengthens the evidence for a role of EBV in MS. It also suggests that a negative EBV serology in patients with suspected inflammatory central nervous system disease should alert clinicians to consider diagnoses other than MS.

Longitudinal prevalence and determinants of pain in multiple sclerosis: results from the German National Multiple Sclerosis Cohort study.

Pain is frequent in multiple sclerosis (MS) and includes different types, with neuropathic pain (NP) being most closely related to MS pathology. However, prevalence estimates vary largely, and causal relationships between pain and biopsychosocial factors in MS are largely unknown. Longitudinal studies might help to clarify the prevalence and determinants of pain in MS. To this end, we analyzed data from 410 patients with newly diagnosed clinically isolated syndrome or relapsing-remitting MS participating in the prospective multicenter German National MS Cohort Study (NationMS) at baseline and after 4 years. Pain was assessed by self-report using the PainDETECT Questionnaire. Neuropsychiatric assessment included tests for fatigue, depression, and cognition. In addition, sociodemographic and clinical data were obtained. Prevalence of pain of any type was 40% and 36% at baseline and after 4 years, respectively, whereas prevalence of NP was 2% and 5%. Pain of any type and NP were both strongly linked to fatigue, depression, and disability. This link was even stronger after 4 years than at baseline. Moreover, changes in pain, depression, and fatigue were highly correlated without any of these symptoms preceding the others. Taken together, pain of any type seems to be much more frequent than NP in early nonprogressive MS. Moreover, the close relationship between pain, fatigue, and depression in MS should be considered for treatment decisions and future research on a possible common pathophysiology.

Long-term real-world evidence for sustained clinical benefits of fingolimod following switch from natalizumab.

BACKGROUND: The risk of progressive multifocal leukoencephalopathy limits the duration over which patients can receive natalizumab before requiring a switch to other therapies such as fingolimod. To date, no studies have assessed the long-term real-world effectiveness and safety of fingolimod following a switch from natalizumab. We aimed to investigate the benefit-risk profile of fingolimod over 48 months in patients switching from natalizumab, and the impact of washout duration after natalizumab discontinuation on outcomes during fingolimod treatment. METHODS: This analysis used data from PANGAEA, an ongoing German multicenter, prospective, non-interventional, observational study. In total, 3912 patients were included: 530 had switched from natalizumab (natalizumab subpopulation), and a reference population of 3382 had switched from other treatments or were treatment-naïve (non-natalizumab subpopulation). The natalizumab subpopulation was stratified by washout duration (30-89 days, 90-149 days, and ≥ 150 days) prior to fingolimod initiation. RESULTS: In the natalizumab subpopulation over 48 months of fingolimod treatment, 58.2% (n = 227/390) of patients remained on fingolimod. Over this period, mean annualized relapse rates (ARRs) and proportions of patients who relapsed were similar across washout durations, and ranged from 0.455 (95% confidence interval [CI]: 0.363-0.571) to 0.546 (95% CI: 0.446-0.669) and 54.1% (n = 92/170) to 60.2% (n = 127/211), respectively. Overall, 17.1% (n = 36/211) had 6-month confirmed disability worsening. In the non-natalizumab subpopulation, ARR was 0.300, 40.9% (n = 1325/3237) of patients relapsed, and a similar proportion to the natalizumab subpopulation had 6-month disability worsening (16.6% [n = 232/1394]). In both subpopulations, the safety profile of fingolimod was consistent with that observed in randomized controlled trials. CONCLUSIONS: In patients discontinuing natalizumab, fingolimod has a favorable benefit-risk profile over 48 months. These findings also suggest using a short washout following natalizumab discontinuation, consistent with guidelines and current clinical practice in Germany.

IL-17+ CD8+ T cell suppression by dimethyl fumarate associates with clinical response in multiple sclerosis.

IL-17-producing CD8+ (Tc17) cells are enriched in active lesions of patients with multiple sclerosis (MS), suggesting a role in the pathogenesis of autoimmunity. Here we show that amelioration of MS by dimethyl fumarate (DMF), a mechanistically elusive drug, associates with suppression of Tc17 cells. DMF treatment results in reduced frequency of Tc17, contrary to Th17 cells, and in a decreased ratio of the regulators RORC-to-TBX21, along with a shift towards cytotoxic T lymphocyte gene expression signature in CD8+ T cells from MS patients. Mechanistically, DMF potentiates the PI3K-AKT-FOXO1-T-BET pathway, thereby limiting IL-17 and RORγt expression as well as STAT5-signaling in a glutathione-dependent manner. This results in chromatin remodeling at the Il17 locus. Consequently, T-BET-deficiency in mice or inhibition of PI3K-AKT, STAT5 or reactive oxygen species prevents DMF-mediated Tc17 suppression. Overall, our data disclose a DMF-AKT-T-BET driven immune modulation and suggest putative therapy targets in MS and beyond.

Thymoma-Associated Paraneoplastic Autoimmune Multiorgan Syndrome-From Pemphigus to Lichenoid Dermatitis.

Introduction: Paraneoplastic autoimmune multi-organ syndrome (PAMS) is a rare clinical condition characterized by variable and heterogeneous clinical phenotypes in the presence of neoplasias which largely depend on the activation of humoral and cellular immune responses. Clinically, these patients present with a spectrum of antibody-driven pemphigus-like lesions to graft-vs.-host-disease-like exanthemas with a lichenoid inflammatory infiltrate in the skin. PAMS is occasionally associated with thymoma, in which altered immune surveillance eventually leads to multiorgan autoimmunity which often includes variable cutaneous symptoms. This disorder is associated with a profound disturbance of peripheral immune tolerance against human autoantigens. Objectives: We here present a patient with relapsing thymoma who developed PAMS with several cutaneous and extracutaneous autoimmune disorders. Materials: Peripheral blood mononuclear cells (PBMC), sera, and lesional skin biopsies were obtained at different clinical disease stages. Peripheral T cell subsets were characterized phenotypically and the cytokine profile of the peripheral blood T cellular response against distinct epidermal and dermal autoantigens of the skin was analyzed by ELISpot assay. Serological screening was performed by ELISA and immunoblot analysis. Skin biopsies were subjected to immunohistochemical analysis of distinct T cell subsets. Thymoma tissue was analyzed for the presence of T regulatory cells and compared with adult thymus and indolent thymoma. Results and Conclusions: In the present case, thymoma was the cause of the observed multi-organ autoimmune syndromes as its recurrence and surgical removal was associated with the relapse and regression of the cutaneous symptoms, respectively. Initially, the patient presented with two autoimmune disorders with Th2/Th1 imbalance, myasthenia gravis (MG) and pemphigus foliaceus (PF), which regressed upon immunosuppressive treatment. Months later, the patient developed a lichenoid exanthema with a Th1-dominated skin infiltrate. Further clinical evaluation revealed the recurrence of the thymoma and the lichenoid exanthema gradually regressed upon thymectomy. Our contention that T cell recognition against distinct cutaneous autoantigens, such as desmoglein 1 (Dsg1), shifted from a Th2 to a Th1-dominated immune response could not be fully substantiated as the patient was on a stringent immunosuppressive treatment regimen. We could only observe a decrease of the initially present serum IgG autoantibodies against Dsg1. Phenotypic analysis of the associated thymoma showed a lower number of T regulatory cells compared to adult thymus and indolent thymoma, suggesting that impaired thymus-derived immune surveillance had a direct impact on the outcome of the observed cutaneous autoimmune disorders.