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The multifaceted nature of multiple sclerosis requires quantitative biomarkers that can provide insights related to diverse physiological pathways. To this end, proteomic analysis of deeply-phenotyped serum samples, biological pathway modeling, and network analysis were performed to elucidate inflammatory and neurodegenerative processes, identifying sensitive biomarkers of multiple sclerosis disease activity. Here, we evaluated the concentrations of > 1400 serum proteins in 630 samples from three multiple sclerosis cohorts for association with clinical and radiographic new disease activity. Twenty proteins were associated with increased clinical and radiographic multiple sclerosis disease activity for inclusion in a custom assay panel. Serum neurofilament light chain showed the strongest univariate correlation with gadolinium lesion activity, clinical relapse status, and annualized relapse rate. Multivariate modeling outperformed univariate for all endpoints. A comprehensive biomarker panel including the twenty proteins identified in this study could serve to characterize disease activity for a patient with multiple sclerosis.

While neurodegeneration underlies the pathological basis for permanent disability in multiple sclerosis (MS), predictive biomarkers for progression are lacking. Using an animal model of chronic MS, we find that synaptic injury precedes neuronal loss and identify thinning of the inner plexiform layer (IPL) as an early feature of inflammatory demyelination-prior to symptom onset. As neuronal domains are anatomically segregated in the retina and can be monitored longitudinally, we hypothesize that thinning of the IPL could represent a biomarker for progression in MS. Leveraging our dataset with over 800 participants enrolled for more than 12 years, we find that IPL atrophy directly precedes progression and propose that synaptic loss is predictive of functional decline. Using a blood proteome-wide analysis, we demonstrate a strong correlation between demyelination, glial activation, and synapse loss independent of neuroaxonal injury. In summary, monitoring synaptic injury is a biologically relevant approach that reflects a potential driver of progression.

Open-label extension (OLE) studies help inform long-term safety and efficacy of disease-modifying therapies in multiple sclerosis (MS). We report exploratory analyses from a phase 2 trial on the longest follow-up to date of ocrelizumab-treated patients with relapsing-remitting MS (RRMS). The primary treatment period (PTP) comprised four 24-week treatment cycles; participants were randomized to double-blind ocrelizumab (2000 mg or 600 mg), placebo, or interferon β-1a (open label) for one cycle, then dose-blinded ocrelizumab 1000 mg or 600 mg for the remaining cycles. The PTP was followed by consecutive assessed and unassessed treatment-free periods (TFPs) and then the OLE (ocrelizumab 600 mg every 24 weeks). Safety and efficacy were prospectively assessed. Of 220 participants randomized, 183 (84%) completed the PTP. After the TFP, 103 entered OLE (median OLE ocrelizumab exposure 6.5 years). Most common adverse events across all periods were infusion-related reactions. MRI activity, annualized relapse rate, and confirmed disability progression (CDP) rates remained low throughout. During the assessed TFP, there was a trend toward less and later B-cell repletion, and later CDP, for patients randomized to ocrelizumab; MRI activity was observed in 16.3% of patients, the earliest 24 weeks after the last ocrelizumab dose. This is the longest follow-up of ocrelizumab-treated patients with RRMS, with no new safety signals emerging during an observation period from 2008 to 2020. Results reinforce the sustained efficacy of long-term ocrelizumab. Reduced disease activity was maintained following interruption of 6-month dosing cycles, with no evidence of rebound.

BackgroundPatients with multiple sclerosis (MS) face several challenges in accessing clinical tools to help them monitor, understand, and make meaningful decisions about their disease course. The University of California San Francisco MS BioScreen is a web-based precision medicine tool initially designed to be clinician facing. We aimed to design a second, openly available tool, Open MS BioScreen, that would be accessible, understandable, and actionable by people with MS. ObjectiveThis study aimed to describe the human-centered design and development approach (inspiration, ideation, and implementation) for creating the Open MS BioScreen platform. MethodsWe planned an iterative and cyclical development process that included stakeholder engagement and iterative feedback from users. Stakeholders included patients with MS along with their caregivers and family members, MS experts, generalist clinicians, industry representatives, and advocacy experts. Users consisted of anyone who wants to track MS measurements over time and access openly available tools for people with MS. Phase I (inspiration) consisted of empathizing with users and defining the problem. We sought to understand the main challenges faced by patients and clinicians and what they would want to see in a web-based app. In phase II (ideation), our multidisciplinary team discussed approaches to capture, display, and make sense of user data. Then, we prototyped a series of mock-ups to solicit feedback from clinicians and people with MS. In phase III (implementation), we incorporated all concepts to test and iterate a minimally viable product. We then gathered feedback through an agile development process. The design and development were cyclical—many times throughout the process, we went back to the drawing board. ResultsThis human-centered approach generated an openly available, web-based app through which patients with MS, their clinicians, and their caregivers can access the site and create an account. Users can enter information about their MS (basic level as well as more advanced concepts), visualize their data longitudinally, access a series of algorithms designed to empower them to make decisions about their treatments, and enter data from wearable devices to encourage realistic goal setting about their ambulatory activity. Agile development will allow us to continue to incorporate precision medicine tools, as these are validated in the clinical research arena. ConclusionsAfter engaging intended users into the iterative human-centered design of the Open MS BioScreen, we will now monitor the adaptation and dissemination of the tool as we expand its functionality and reach. The insights generated from this approach can be applied to the development of a number of self-tracking, self-management, and user engagement tools for patients with chronic conditions.

BACKGROUND: Ofatumumab has demonstrated superior efficacy and favorable safety for up to 2.5 years versus teriflunomide in relapsing multiple sclerosis (RMS). OBJECTIVE: Further characterize efficacy and safety of ofatumumab in RMS. METHODS: Efficacy set: patients randomized to ofatumumab/teriflunomide in ASCLEPIOS I/II (core). Safety set: patients who received ⩾ 1 dose of ofatumumab in ASCLEPIOS I/II, APLIOS, APOLITOS (all core), or ALITHIOS (umbrella open-label extension). Patients received continuous ofatumumab or were newly switched from teriflunomide. Data cut-off: 25 September 2021. RESULTS: In the efficacy set (n = 1882), the continuous ofatumumab group had a low annualized relapse rate (ARR 0.05 (95% confidence interval: 0.04-0.07)), low numbers of gadolinium-enhancing (Gd+) T1 lesions (0.01 lesions/scan) and fewer new/enlarging T2 lesions (annualized rate 0.08). Overall, 78.8% met three-parameter no evidence of disease activity criteria through 4 years. Switching from teriflunomide led to reduced ARR, risk of confirmed disability worsening (CDW), new/enlarging T2 lesions, Gd+ T1 lesions, and serum neurofilament light chain. In the continuous and newly switched ofatumumab groups, cumulative 3- and 6-month CDW rates remained low. In the safety set (n = 1969), the most frequently reported adverse events were infections and infestations (58.35%). No new safety signals were identified. CONCLUSION: Ofatumumab has a favorable longer-term benefit-risk profile in RMS. TRIAL REGISTRY: ALITHIOS (NCT03650114): https://clinicaltrials.gov/ct2/show/NCT03650114.

The importance of B cells in multiple sclerosis (MS) has been demonstrated through the advent of B-cell-depleting anti-CD20 antibody therapies. Ofatumumab is the first fully human anti-CD20 monoclonal antibody (mAb) developed and tested for subcutaneous (SC) self-administration at monthly doses of 20 mg, and has been approved in the US, UK, EU, and other regions and countries worldwide for the treatment of relapsing MS. The development goal of ofatumumab was to obtain a highly efficacious anti-CD20 therapy, with a safety and tolerability profile that allows for self-administration by MS patients at home and a positive benefit-risk balance for use in the broad relapsing MS population. This development goal was enabled by the unique binding site, higher affinity to B cells, and higher potency of ofatumumab compared to previous anti-CD20 mAbs; these properties of ofatumumab facilitate rapid B-cell depletion and maintenance with a low dose at a low injection volume (20 mg/0.4 ml). The high potency in turn enables the selective targeting of B cells that reside in the lymphatic system via subcutaneous (SC) administration. Through a comprehensive dose-finding program in two phase 2 studies (one intravenous and one SC) and model simulations, it was found that safety and tolerability can be further improved, and the risk of systemic injection-related reactions (IRRs) minimized, by avoiding doses ≥ 30 mg, and by reaching initial and rapid B-cell depletion via stepwise weekly administration of ofatumumab at Weeks 0, 1, and 2 (instead of a single high dose). Once near-complete B-cell depletion is reached, it can be maintained by monthly doses of 20 mg/0.4 ml. Indeed, in phase 3 trials (ASCLEPIOS I/II), rapid and sustained near-complete B-cell depletion (largely independent of body weight, race and other factors) was observed with this dosing regimen, which resulted in superior efficacy of ofatumumab versus teriflunomide on relapse rates, disability worsening, neuronal injury (serum neurofilament light chain), and imaging outcomes. Likely due to its fully human nature, ofatumumab has a low immunogenic risk profile-only 2 of 914 patients receiving ofatumumab in ASCLEPIOS I/II developed anti-drug antibodies-and this may also underlie the infrequent IRRs (20% with ofatumumab vs. 15% with the placebo injection in the teriflunomide arm) that were mostly (99.8%) mild to moderate in severity. The overall rates of infections and serious infections in patients treated with ofatumumab were similar to those in patients treated with teriflunomide (51.6% vs. 52.7% and 2.5% vs. 1.8%, respectively). The benefit-risk profile of ofatumumab was favorable compared to teriflunomide in the broad RMS population, and also in the predefined subgroups of both recently diagnosed and/or treatment-naïve patients, as well as previously disease-modifying therapy-treated patients. Interim data from the ongoing extension study (ALITHIOS) have shown that long-term treatment with ofatumumab up to 4 years is well-tolerated in RMS patients, with no new safety risks identified. In parallel to the phase 3 trials in which SC administration was carried out with a pre-filled syringe, an autoinjector pen for more convenient self-administration of the ofatumumab 20 mg dose was developed and is available for use in clinical practice.

BackgroundNeurofilament light chain (NfL), a neuronal cytoskeletal protein that is released upon neuroaxonal injury, is associated with multiple sclerosis (MS) relapsing activity and has demonstrated some prognostic ability for future relapse-related disease progression, yet its value in assessing non-relapsing disease progression remains unclear.MethodsWe examined baseline and longitudinal blood NfL levels in 1421 persons with relapsing MS (RMS) and 596 persons with primary progressive MS (PPMS) from the pivotal ocrelizumab MS trials. NfL treatment-response and risk for disease worsening (including disability progression into the open-label extension period and slowly expanding lesions [SELs] on brain MRI) at baseline and following treatment with ocrelizumab were evaluated using time-to-event analysis and linear regression models.FindingsIn persons from the RMS control arms without acute disease activity and in the entire PPMS control arm, higher baseline NfL was prognostic for greater whole brain and thalamic atrophy, greater volume expansion of SELs, and clinical progression. Ocrelizumab reduced NfL levels vs. controls in persons with RMS and those with PPMS, and abrogated the prognostic value of baseline NfL on disability progression. Following effective suppression of relapse activity by ocrelizumab, NfL levels at weeks 24 and 48 were significantly associated with long-term risk for disability progression, including up to 9 years of observation in RMS and PPMS.InterpretationHighly elevated NfL from acute MS disease activity may mask a more subtle NfL abnormality that reflects underlying non-relapsing progressive biology. Ocrelizumab significantly reduced NfL levels, consistent with its effects on acute disease activity and disability progression. Persistently elevated NfL levels, observed in a subgroup of persons under ocrelizumab treatment, demonstrate potential clinical utility as a predictive biomarker of increased risk for clinical progression. Suppression of relapsing biology with high-efficacy immunotherapy provides a window into the relationship between NfL levels and future non-relapsing progression.FundingF. Hoffmann-La Roche Ltd.

BackgroundA study was undertaken to evaluate remote monitoring via smartphone sensor-based tests in people with multiple sclerosis (PwMS). This analysis aimed to explore regional neural correlates of digital measures derived from these tests.MethodsIn a 24-week, non-randomized, interventional, feasibility study (NCT02952911), sensor-based tests on the Floodlight Proof-of-Concept app were used to assess cognition (smartphone-based electronic Symbol Digit Modalities Test), upper extremity function (Draw a Shape Test, Pinching Test), and gait and balance (Static Balance Test, Two-Minute Walk Test, U-Turn Test). In this post-hoc analysis, digital measures and standard clinical measures (e.g., Nine-Hole Peg Test [9HPT]) were correlated against regional structural magnetic resonance imaging outcomes. Seventy-six PwMS aged 18-55 years with an Expanded Disability Status Scale score of 0.0-5.5 were enrolled from two different sites (USA and Spain). Sixty-two PwMS were included in this analysis.ResultsWorse performance on digital and clinical measures was associated with smaller regional brain volumes and larger ventricular volumes. Whereas digital and clinical measures had many neural correlates in common (e.g., putamen, globus pallidus, caudate nucleus, lateral occipital cortex), some were observed only for digital measures. For example, Draw a Shape Test and Pinching Test measures, but not 9HPT score, correlated with volume of the hippocampus (r = 0.37 [drawing accuracy over time on the Draw a Shape Test]/ - 0.45 [touching asynchrony on the Pinching Test]), thalamus (r = 0.38/ - 0.41), and pons (r = 0.35/ - 0.35).ConclusionsMultiple neural correlates were identified for the digital measures in a cohort of people with early MS. Digital measures showed associations with brain regions that clinical measures were unable to demonstrate, thus providing potential novel information on functional ability compared with standard clinical assessments.

BackgroundRemote activity monitoring has the potential to evaluate real-world, motor function, and disability at home. The relationships of daily physical activity with spinal cord white matter and gray matter (GM) areas, multiple sclerosis (MS) disability and leg function, are unknown.ObjectiveEvaluate the association of structural central nervous system pathology with ambulatory disability.MethodsFifty adults with progressive or relapsing MS with motor disability who could walk >2 minutes were assessed using clinician-evaluated, patient-reported outcomes, and quantitative brain and spinal cord magnetic resonance imaging (MRI) measures. Fitbit Flex2, worn on the non-dominant wrist, remotely assessed activity over 30 days. Univariate and multivariate analyses were performed to assess correlations between physical activity and other disability metrics.ResultsMean age was 53.3 years and median Expanded Disability Status Scale (EDSS) was 4.0. Average daily step counts (STEPS) were highly correlated with EDSS and walking measures. Greater STEPS were significantly correlated with greater C2-C3 spinal cord GM areas (ρ = 0.39, p = 0.04), total cord area (TCA; ρ = 0.35, p = 0.04), and cortical GM volume (ρ = 0.32, p = 0.04).ConclusionThese results provide preliminary evidence that spinal cord GM area is a neuroanatomical substrate associated with STEPS. STEPS could serve as a proxy to alert clinicians and researchers to possible changes in structural nervous system pathology.

Background and objectivesProspective, deeply phenotyped research cohorts monitoring individuals with chronic neurologic conditions, such as multiple sclerosis (MS), depend on continued participant engagement. The COVID-19 pandemic restricted in-clinic research activities, threatening this longitudinal engagement, but also forced adoption of televideo-enabled care. This offered a natural experiment in which to analyze key dimensions of remote research: (1) comparison of remote vs in-clinic visit costs from multiple perspectives and (2) comparison of the remote with in-clinic measures in cross-sectional and longitudinal disability evaluations.MethodsBetween March 2020 and December 2021, 207 MS cohort participants underwent hybrid in-clinic and virtual research visits; 96 contributed 100 "matched visits," that is, in-clinic (Neurostatus-Expanded Disability Status Scale [NS-EDSS]) and remote (televideo-enabled EDSS [tele-EDSS]; electronic patient-reported EDSS [ePR-EDSS]) evaluations. Clinical, demographic, and socioeconomic characteristics of participants were collected.ResultsThe costs of remote visits were lower than in-clinic visits for research investigators (facilities, personnel, parking, participant compensation) but also for participants (travel, caregiver time) and carbon footprint (p < 0.05 for each). Median cohort EDSS was similar between the 3 modalities (NS-EDSS: 2, tele-EDSS: 1.5, ePR-EDSS: 2, range 0.6.5); the remote evaluations were each noninferior to the NS-EDSS within ±0.5 EDSS point (TOST for noninferiority, p < 0.01 for each). Furthermore, year to year, the % of participants with worsening/stable/improved EDSS scores was similar, whether each annual evaluation used NS-EDSS or whether it switched from NS-EDSS to tele-EDSS.DiscussionAltogether, the current findings suggest that remote evaluations can reduce the costs of research participation for patients, while providing a reasonable evaluation of disability trajectory longitudinally. This could inform the design of remote research that is more inclusive of diverse participants.

Background and objectivesOcrelizumab improved clinical and MRI measures of disease activity and progression in three phase 3 multiple sclerosis (MS) studies. Post hoc analyses demonstrated a correlation between the ocrelizumab serum concentration and the degree of blood B-cell depletion, and body weight was identified as the most influential covariate on ocrelizumab pharmacokinetics. The magnitude of ocrelizumab treatment benefit on disability progression was greater in lighter vs heavier patients. These observations suggest that higher ocrelizumab serum levels provide more complete B-cell depletion and a greater delay in disability progression. The current post hoc analyses assessed population exposure-efficacy/safety relationships of ocrelizumab in patients with relapsing and primary progressive MS.MethodsPatients in OPERA I/II and ORATORIO were grouped in exposure quartiles based on their observed individual serum ocrelizumab level over the treatment period. Exposure-response relationships were analyzed for clinical efficacy (24-week confirmed disability progression (CDP), annualized relapse rate [ARR], and MRI outcomes) and adverse events.ResultsOcrelizumab reduced new MRI lesion counts to nearly undetectable levels in patients with relapsing or primary progressive MS across all exposure subgroups, and reduced ARR in patients with relapsing MS to very low levels (0.13-0.18). A consistent trend of higher ocrelizumab exposure leading to lower rates of CDP was seen (0%-25% [lowest] to 75%-100% [highest] quartile hazard ratios and 95% confidence intervals; relapsing MS: 0.70 [0.41-1.19], 0.85 [0.52-1.39], 0.47 [0.25-0.87], and 0.34 [0.17-0.70] vs interferon β-1a; primary progressive MS: 0.88 [0.59-1.30], 0.86 [0.60-1.25], 0.77 [0.52-1.14], and 0.55 [0.36-0.83] vs placebo). Infusion-related reactions, serious adverse events, and serious infections were similar across exposure subgroups.DiscussionThe almost complete reduction of ARR and MRI activity already evident in the lowest quartile, and across all ocrelizumab-exposure groups, suggests a ceiling effect. A consistent trend of higher ocrelizumab exposure leading to greater reduction in risk of CDP was observed, particularly in the relapsing MS trials, and was not associated with a higher rate of adverse events. Higher ocrelizumab exposure may provide improved control of disability progression by reducing disease activity below that detectable by ARR and MRI, and/or by attenuating other B-cell-related pathologies responsible for tissue damage.Classification of evidenceThis analysis provides Class III evidence that higher ocrelizumab serum levels are related to greater reduction in risk of disability progression in patients with multiple sclerosis. The study is rated Class III because of the initial treatment randomization disclosure that occurred after inclusion in the open-label extension.Trial registration informationClinicalTrials.gov Identifier: NCT01247324 (OPERA I), NCT01412333 (OPERA II), and NCT01194570 (ORATORIO).

Genome-wide association studies (GWAS) successfully identified multiple sclerosis (MS) susceptibility variants. Despite this notable progress, understanding the biological context of these associations remains challenging, due in part to the complexity of linking GWAS results to causative genes and cell types. Here, we aimed to address this gap by integrating GWAS data with single-cell and bulk chromatin accessibility data and histone modification profiles from immune and nervous systems. MS-GWAS associations are significantly enriched in regulatory regions of microglia and peripheral immune cell subtypes, especially B cells and monocytes. Cell-specific polygenic risk scores were developed to examine the cumulative impact of the susceptibility genes on MS risk and clinical phenotypes, showing significant associations with risk and brain white matter volume. The findings reveal enrichment of GWAS signals in B cell and monocyte/microglial cell-types, consistent with the known pathology and presumed targets of effective MS therapeutics.

Polygenic inheritance plays a pivotal role in driving multiple sclerosis susceptibility, an inflammatory demyelinating disease of the CNS. We developed polygenic risk scores (PRS) of multiple sclerosis and assessed associations with both disease status and severity in cohorts of European descent. The largest genome-wide association dataset for multiple sclerosis to date (n = 41 505) was leveraged to generate PRS scores, serving as an informative susceptibility marker, tested in two independent datasets, UK Biobank [area under the curve (AUC) = 0.73, 95% confidence interval (CI): 0.72-0.74, P = 6.41 × 10-146] and Kaiser Permanente in Northern California (KPNC, AUC = 0.8, 95% CI: 0.76-0.82, P = 1.5 × 10-53). Individuals within the top 10% of PRS were at higher than 5-fold increased risk in UK Biobank (95% CI: 4.7-6, P = 2.8 × 10-45) and 15-fold higher risk in KPNC (95% CI: 10.4-24, P = 3.7 × 10-11), relative to the median decile. The cumulative absolute risk of developing multiple sclerosis from age 20 onwards was significantly higher in genetically predisposed individuals according to PRS. Furthermore, inclusion of PRS in clinical risk models increased the risk discrimination by 13% to 26% over models based only on conventional risk factors in UK Biobank and KPNC, respectively. Stratifying disease risk by gene sets representative of curated cellular signalling cascades, nominated promising genetic candidate programmes for functional characterization. These pathways include inflammatory signalling mediation, response to viral infection, oxidative damage, RNA polymerase transcription, and epigenetic regulation of gene expression to be among significant contributors to multiple sclerosis susceptibility. This study also indicates that PRS is a useful measure for estimating susceptibility within related individuals in multicase families. We show a significant association of genetic predisposition with thalamic atrophy within 10 years of disease progression in the UCSF-EPIC cohort (P < 0.001), consistent with a partial overlap between the genetics of susceptibility and end-organ tissue injury. Mendelian randomization analysis suggested an effect of multiple sclerosis susceptibility on thalamic volume, which was further indicated to be through horizontal pleiotropy rather than a causal effect. In summary, this study indicates important, replicable associations of PRS with enhanced risk assessment and radiographic outcomes of tissue injury, potentially informing targeted screening and prevention strategies.

BACKGROUND: The HLA-DRB1 gene in the major histocompatibility complex (MHC) region in chromosome 6p21 is the strongest genetic factor identified as influencing multiple sclerosis (MS) susceptibility. DNA methylation changes associated with MS have been consistently detected at the MHC region. However, understanding the full scope of epigenetic regulations of the MHC remains incomplete, due in part to the limited coverage of this region by standard whole genome bisulfite sequencing or array-based methods. METHODS: We developed and validated an MHC capture protocol coupled with bisulfite sequencing and conducted a comprehensive analysis of the MHC methylation landscape in blood samples from 147 treatment naïve MS study participants and 129 healthy controls. RESULTS: We identified 132 differentially methylated region (DMRs) within MHC region associated with disease status. The DMRs overlapped with established MS risk loci. Integration of the MHC methylome with human leukocyte antigen (HLA) genetic data indicate that the methylation changes are significantly associated with HLA genotypes. Using DNA methylation quantitative trait loci (mQTL) mapping and the causal inference test (CIT), we identified 643 cis-mQTL-DMRs paired associations, including 71 DMRs possibly mediating causal relationships between 55 single nucleotide polymorphisms (SNPs) and MS risk. RESULTS: The results describe MS-associated methylation changes in MHC region and highlight the association between HLA genotypes and methylation changes. Results from the mQTL and CIT analyses provide evidence linking MHC region variations, methylation changes, and disease risk for MS.

BackgroundIn the phase III ASCLEPIOS I and II trials, participants with relapsing multiple sclerosis receiving ofatumumab had significantly better clinical and magnetic resonance imaging (MRI) outcomes than those receiving teriflunomide.ObjectivesTo assess the efficacy and safety of ofatumumab versus teriflunomide in recently diagnosed, treatment-naive (RDTN) participants from ASCLEPIOS.MethodsParticipants were randomized to receive ofatumumab (20 mg subcutaneously every 4 weeks) or teriflunomide (14 mg orally once daily) for up to 30 months. Endpoints analysed post hoc in the protocol-defined RDTN population included annualized relapse rate (ARR), confirmed disability worsening (CDW), progression independent of relapse activity (PIRA) and adverse events.ResultsData were analysed from 615 RDTN participants (ofatumumab: n = 314; teriflunomide: n = 301). Compared with teriflunomide, ofatumumab reduced ARR by 50% (rate ratio (95% confidence interval (CI)): 0.50 (0.33, 0.74); p

BackgroundOfatumumab is approved for the treatment of relapsing multiple sclerosis (RMS). Ongoing safety reporting is crucial to understand its long-term benefit-risk profile.ObjectiveReport the safety and tolerability of ofatumumab in RMS after extended treatment up to 3.5 years.MethodsPatients completing ASCLEPIOS I/II (phase 3), APLIOS, or APOLITOS (phase 2) trials could enter ALITHIOS, a phase 3b, open-label, long-term safety study. We analyzed cumulative data of continuous ofatumumab treatment and of patients newly switched from teriflunomide.ResultsThe safety population had 1969 patients: 1292 continuously treated with ofatumumab (median time-at-risk 35.5 months, 3253 patient-years) and 677 newly switched (median time-at-risk 18.3 months, 986 patient-years). A total of 1650 patients (83.8%) had ⩾1 adverse events and 191 (9.7%) had ⩾1 serious adverse events. No opportunistic infections or progressive multifocal leukoencephalopathy events were identified; the risk of malignancies was low. Mean serum immunoglobulin (Ig) G levels remained stable. Mean IgM levels decreased but remained above the lower limit of normal in most. Serious infection incidence was low; decreased Ig levels were not associated with serious infections.ConclusionIn patients with up to 3.5 years' exposure, ofatumumab was well tolerated, with no new safety risks identified. These findings, with its established effectiveness, support a favorable benefit-risk profile of ofatumumab in RMS.

In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.

BackgroundSensor-based monitoring tools fill a critical gap in multiple sclerosis (MS) research and clinical care.ObjectiveThe aim of this study is to assess performance characteristics of the Floodlight Proof-of-Concept (PoC) app.MethodsIn a 24-week study (clinicaltrials.gov: NCT02952911), smartphone-based active tests and passive monitoring assessed cognition (electronic Symbol Digit Modalities Test), upper extremity function (Pinching Test, Draw a Shape Test), and gait and balance (Static Balance Test, U-Turn Test, Walk Test, Passive Monitoring). Intraclass correlation coefficients (ICCs) and age- or sex-adjusted Spearman's rank correlation determined test-retest reliability and correlations with clinical and magnetic resonance imaging (MRI) outcome measures, respectively.ResultsSeventy-six people with MS (PwMS) and 25 healthy controls were enrolled. In PwMS, ICCs were moderate-to-good (ICC(2,1) = 0.61-0.85) across tests. Correlations with domain-specific standard clinical disability measures were significant for all tests in the cognitive (r = 0.82, p < 0.001), upper extremity function (|r|= 0.40-0.64, all p < 0.001), and gait and balance domains (r = -0.25 to -0.52, all p < 0.05; except for Static Balance Test: r = -0.20, p > 0.05). Most tests also correlated with Expanded Disability Status Scale, 29-item Multiple Sclerosis Impact Scale items or subscales, and/or normalized brain volume.ConclusionThe Floodlight PoC app captures reliable and clinically relevant measures of functional impairment in MS, supporting its potential use in clinical research and practice.

Background and purposeReaching Expanded Disability Status Scale (EDSS) ≥7.0 represents the requirement for a wheelchair. Here we (i) assess the effect of ocrelizumab on time to EDSS ≥7.0 over the ORATORIO (NCT01194570) double-blind and extended controlled periods (DBP+ECP), (ii) quantify likely long-term benefits by extrapolating results, and (iii) assess the plausibility of extrapolations using an independent real-world cohort (MSBase registry; ACTRN12605000455662).MethodsPost hoc analyses assessing time to 24-week confirmed EDSS ≥7.0 in two cohorts of patients with primary progressive multiple sclerosis (baseline EDSS 3.0-6.5) were investigated in ORATORIO and MSBase.ResultsIn the ORATORIO DBP+ECP, ocrelizumab reduced the risk of 24-week confirmed EDSS ≥7.0 (hazard ratio = 0.54, 95% confidence interval [CI]: 0.31-0.92; p = 0.022). Extrapolated median time to 24-week confirmed EDSS ≥7.0 was 12.1 and 19.2 years for placebo and ocrelizumab, respectively (7.1-year delay [95% CI: -4.3 to 18.4]). In MSBase, the median time to 24-week confirmed EDSS ≥7.0 was 12.4 years.ConclusionsCompared with placebo, ocrelizumab significantly delayed time to 24-week confirmed wheelchair requirement in ORATORIO. The plausibility of the extrapolated median time to reach this milestone in the placebo group was supported by observed real-world data from MSBase. Extrapolated benefits for ocrelizumab over placebo could represent a truly meaningful delay in loss of ambulation and independence.

Background and purposeRequiring a walking aid is a fundamental milestone in multiple sclerosis (MS), represented by an Expanded Disability Status Scale (EDSS) score ≥6.0. In the present study, we assess the effect of ocrelizumab (OCR) on time to EDSS score ≥6.0 in relapsing MS.MethodsTime to EDSS score ≥6.0 confirmed for ≥24 and ≥48 weeks was assessed over the course of 6.5 years (336 weeks) in the double-blind period (DBP) and open-label extension (OLE) period of the OPERA I (NCT01247324) and OPERA II (NCT01412333) studies.ResultsTime to reach EDSS score ≥6.0 was significantly delayed in those initially randomized to OCR versus interferon. Over 6.5 years, the risk of requiring a walking aid confirmed for ≥24 weeks was 34% lower among those who initiated OCR earlier versus delayed treatment (average hazard ratio [HR] DBP + OLE 0.66, 95% confidence interval [CI] 0.45-0.95; p = 0.024); the risk of requiring a walking aid confirmed for ≥48 weeks was 46% lower (average HR DBP+OLE 0.54, 95% CI 0.35-0.83; p = 0.004).ConclusionThe reduced risk of requiring a walking aid in earlier initiators of OCR demonstrates the long-term implications of earlier highly effective treatment.

ObjectiveA major challenge in multiple sclerosis (MS) research is the understanding of silent progression and Progressive MS. Using a novel method to accurately capture upper cervical cord area from legacy brain MRI scans we aimed to study the role of spinal cord and brain atrophy for silent progression and conversion to secondary progressive disease (SPMS).MethodsFrom a single-center observational study, all RRMS (n = 360) and SPMS (n = 47) patients and 80 matched controls were evaluated. RRMS patient subsets who converted to SPMS (n = 54) or silently progressed (n = 159), respectively, during the 12-year observation period were compared to clinically matched RRMS patients remaining RRMS (n = 54) or stable (n = 147), respectively. From brain MRI, we assessed the value of brain and spinal cord measures to predict silent progression and SPMS conversion.ResultsPatients who developed SPMS showed faster cord atrophy rates (-2.19%/yr) at least 4 years before conversion compared to their RRMS matches (-0.88%/yr, p

BackgroundThe ability to assess brain and cord atrophy simultaneously would improve the efficiency of MRI to track disease evolution.ObjectiveTo test a promising tool to simultaneously map the regional distribution of atrophy in multiple sclerosis (MS) patients across the brain and cord.MethodsVoxel-based morphometry combined with a statistical parametric mapping probabilistic brain-spinal cord (SPM-BSC) template was applied to standard T1-weighted magnetic resonance imaging (MRI) scans covering the brain and cervical cord from 37 MS patients and 20 healthy controls (HC). We also measured the cord area at C2-C3 with a semi-automatic segmentation method using (i) the same T1-weighted acquisitions used for the new voxel-based analysis and (ii) dedicated spinal cord phase sensitive inversion recovery (PSIR) acquisitions. Cervical cord findings derived from the three approaches were compared to each other and the goodness to fit to clinical scores was assessed by regression analyses.ResultsThe SPM-BSC approach revealed a severity-dependent pattern of atrophy across the cervical cord and thalamus in MS patients when compared to HCs. The magnitude of cord atrophy was confirmed by the semi-automatic extraction approach at C2-C3 using both standard brain T1-weighted and advanced cord dedicated acquisitions. Associations between atrophy of cord and thalamus with disability and cognition were demonstrated.ConclusionAtrophy in the brain and cervical cord of MS patients can be identified simultaneously and rapidly at the voxel-level. The SPM-BSC approach yields similar results as available standard processing tools with the added advantage of performing the analysis simultaneously and faster.

ObjectiveThis study aims to confirm the prognostic value of baseline serum neurofilament light chain (sNfL) for on-study disease activity and worsening in patients with relapsing MS (RMS).BackgroundPrevious post-hoc studies suggested that sNfL could be a prognostic biomarker in RMS. In the phase 3 ASCLEPIOS I/II trials in which ofatumumab demonstrated better efficacy outcomes than teriflunomide, treatment with ofatumumab also led to significantly reduced sNfL levels compared to teriflunomide treatment.Design/methodsIn this study, we report protocol-planned analyses from the pooled ASCLEPIOS I/II trials (N=1882). Per protocol, patients were stratified by median baseline sNfL levels (9.3 pg/ml) into high (>median) and low (≤median) categories to prognosticate: annualized rate of new/enlarging T2 (neT2) lesions in year 1 and 2, annualized relapse rate, annual percentage change in whole brain (WB) and regional brain volume [thalamus, white matter (WM), cortical gray matter (cGM)], and disability outcomes. Similar analyses were performed for the recently diagnosed (within 3 years), treatment-naive patients (no prior disease-modifying therapy) subgroup.ResultsHigh versus low sNfL at baseline was prognostic of increased on-study T2 lesion formation at year 1 (relative increase: ofatumumab +158%; teriflunomide +69%, both p

Epigenetic changes have been consistently detected in different cell types in multiple sclerosis (MS). However, their contribution to MS pathogenesis remains poorly understood partly because of sample heterogeneity and limited coverage of array-based methods. To fill this gap, we conducted a comprehensive analysis of genome-wide DNA methylation patterns in four peripheral immune cell populations isolated from 29 MS patients at clinical disease onset and 24 healthy controls. We show that B cells from new-onset untreated MS cases display more significant methylation changes than other disease-implicated immune cell types, consisting of a global DNA hypomethylation signature. Importantly, 4,933 MS-associated differentially methylated regions in B cells were identified, and this epigenetic signature underlies specific genetic programs involved in B cell differentiation and activation. Integration of the methylome to changes in gene expression and susceptibility-associated regions further indicates that hypomethylated regions are significantly associated with the up-regulation of cell activation transcriptional programs. Altogether, these findings implicate aberrant B cell function in MS etiology.

Background and objectivesTo report safety of ocrelizumab (OCR) up to 7 years in patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS) enrolled in clinical trials or treated in real-world postmarketing settings.MethodsSafety analyses are based on integrated clinical and laboratory data for all patients who received OCR in 11 clinical trials, including the controlled treatment and open-label extension (OLE) periods of the phase 2 and 3 trials, plus the phase 3b trials VELOCE, CHORDS, CASTING, OBOE, ENSEMBLE, CONSONANCE, and LIBERTO. For selected adverse events (AEs), additional postmarketing data were used. Incidence rates of serious infections (SIs) and malignancies were contextualized using multiple epidemiologic sources.ResultsAt data cutoff (January 2020), 5,680 patients with multiple sclerosis (MS) received OCR (18,218 patient-years [PY] of exposure) in clinical trials. Rates per 100 PY (95% confidence interval) of AEs (248; 246-251), serious AEs (7.3; 7.0-7.7), infusion-related reactions (25.9; 25.1-26.6), and infections (76.2; 74.9-77.4) were similar to those within the controlled treatment period of the phase 3 trials. Rates of the most common serious AEs, including SIs (2.01; 1.81-2.23) and malignancies (0.46; 0.37-0.57), were consistent with the ranges reported in epidemiologic data.DiscussionContinuous administration of OCR for up to 7 years in clinical trials, as well as its broader use for more than 3 years in the real-world setting, are associated with a favorable and manageable safety profile, without emerging safety concerns, in a heterogeneous MS population.Classification of evidenceThis analysis provides Class III evidence that long-term, continuous treatment with OCR has a consistent and favorable safety profile in patients with RMS and PPMS. This study is rated Class III because of the use of OLE data and historical controls.

ObjectiveAdvances in medical discoveries have bolstered expectations of precise and complete care, but delivering on such a promise for complex, chronic neurologic care delivery requires solving last-mile challenges. We describe the iterative human-centered design and pilot process for multiple sclerosis (MS) NeuroShare, a digital health solution that brings practical information to the point of care so that clinicians and patients with MS can view, discuss, and make informed decisions together.MethodsWe initiated a comprehensive human-centered process to iteratively design, develop, and implement a digital health solution for managing MS in the routine outpatient setting of the nonprofit Sutter Health system in Northern California. The human-centered codesign process included 3 phases: discovery and design, development, and implementation and pilot. Stakeholders included Sutter Health's Research Development and Dissemination team, academic domain experts, neurologists, patients with MS, and an advisory group.ResultsMS NeuroShare went live in November 2018. It included a patient- and clinician-facing web application that launches from the electronic health record, visually displays a patient's data relevant to MS, and prompts the clinician to comprehensively evaluate and treat the patient. Both patients and clinicians valued the ability to jointly view patient-generated and other data. Preliminary results suggest that MS NeuroShare promotes patient-clinician communication and more active patient participation in decision-making.ConclusionsLessons learned in the design and implementation of MS NeuroShare are broadly applicable to the design and implementation of digital tools aiming to improve the experience of delivering and receiving high-quality care for complex, neurologic conditions across large health systems.

BackgroundIn persons with multiple sclerosis (MS), the Expanded Disability Status Scale (EDSS) is the criterion standard for assessing disability, but its in-person nature constrains patient participation in research and clinical assessments.ObjectiveThe aim of this study was to develop and validate a scalable, electronic, unsupervised patient-reported EDSS (ePR-EDSS) that would capture MS-related disability across the spectrum of severity.MethodsWe enrolled 136 adult MS patients, split into a preliminary testing Cohort 1 (n = 50), and a validation Cohort 2 (n = 86), which was evenly distributed across EDSS groups. Each patient completed an ePR-EDSS either immediately before or after a MS clinician's Neurostatus EDSS evaluation.ResultsIn Cohort 2, mean age was 50.6 years (range = 26-80) and median EDSS was 3.5 (interquartile range (IQR) = [1.5, 5.5]). The ePR-EDSS and EDSS agreed within 1-point for 86% of examinations; kappa for agreement within 1-point was 0.85 (p

AimsOcrelizumab is a humanized monoclonal antibody that selectively targets CD20-positive B cells and is indicated for treatment of patients with relapsing forms of multiple sclerosis (RMS) or primary progressive multiple sclerosis (PPMS). The pharmacokinetics and pharmacodynamics of ocrelizumab in patients with RMS or PPMS were assessed.MethodsA population pharmacokinetic model was developed based on data from the Phase II study and the Phase III studies OPERA I and OPERA II in patients with RMS. Data from the ORATORIO Phase III study in patients with PPMS became available after model finalization and was used for external model evaluation.ResultsThe ocrelizumab serum concentration vs time course was accurately described by a 2-compartment model with time-dependent clearance. Body weight was found to be the main covariate. The area under the concentration-time curve over the dosing interval was estimated to be 26% higher for patients with RMS weighing 90 kg when compared with the 60-90 kg group. The terminal half-life of ocrelizumab was estimated as 26 days. The extent of B-cell depletion in blood, as the pharmacodynamic marker, was greater with increasing ocrelizumab exposure.ConclusionThe pharmacokinetics of ocrelizumab was described with pharmacokinetic parameters typical for an immunoglobulin G1 monoclonal antibody, with body weight as the main covariate. The pharmacokinetics and B-cell depletion in blood were comparable across the RMS and PPMS trials, and the extent of blood B-cell depletion was greater with higher exposure.

The success of clinical trials of selective B-cell depletion in patients with relapsing multiple sclerosis (MS) and primary progressive MS has led to a conceptual shift in the understanding of MS pathogenesis, away from the classical model in which T cells were the sole central actors and toward a more complex paradigm with B cells having an essential role in both the inflammatory and neurodegenerative components of the disease process. The role of B cells in MS was selected as the topic of the 27th Annual Meeting of the European Charcot Foundation. Results of the meeting are presented in this concise review, which recaps current concepts underlying the biology and therapeutic rationale behind B-cell-directed therapeutics in MS, and proposes strategies to optimize the use of existing anti-B-cell treatments and provide future directions for research in this area. ANN NEUROL 2021;89:13-23.

Multiple sclerosis (MS) susceptibility shows strong genetic associations with HLA alleles and haplotypes. We genotyped 11 HLA genes in 477 non-Hispanic European MS patients and their 954 unaffected parents using a validated next-generation sequencing (NGS) methodology. HLA haplotypes were assigned unequivocally by tracing HLA allele transmissions. We explored HLA haplotype/allele associations with MS using the genotypic transmission disequilibrium test (gTDT) and multiallelic TDT (mTDT). We also conducted a case-control (CC) study with all patients and 2029 healthy unrelated ethnically matched controls. We performed separate analyses of 54 extended multi-case families by reviewing transmission of haplotype blocks. The haplotype fragment including DRB5*01:01:01~DRB1*15:01:01:01 was significantly associated with predisposition (gTDT: p < 2.20e-16; mTDT: p =1.61e-07; CC: p < 2.22e-16) as reported previously. A second risk allele, DPB1*104:01 (gTDT: p = 3.69e-03; mTDT: p = 2.99e-03; CC: p = 1.00e-02), independent from the haplotype bearing DRB1*15:01 was newly identified. The allele DRB1*01:01:01 showed significant protection (gTDT: p = 8.68e-06; mTDT: p = 4.50e-03; CC: p = 1.96e-06). Two DQB1 alleles, DQB1*03:01 (gTDT: p = 2.86e-03; mTDT: p = 5.56e-02; CC: p = 4.08e-05) and DQB1*03:03 (gTDT: p = 1.17e-02; mTDT: p = 1.16e-02; CC: p = 1.21e-02), defined at two-field level also showed protective effects. The HLA class I block, A*02:01:01:01~C*03:04:01:01~B*40:01:02 (gTDT: p = 5.86e-03; mTDT: p = 3.65e-02; CC: p = 9.69e-03) and the alleles B*27:05 (gTDT: p = 6.28e-04; mTDT: p = 2.15e-03; CC: p = 1.47e-02) and B*38:01 (gTDT: p = 3.20e-03; mTDT: p = 6.14e-03; CC: p = 1.70e-02) showed moderately protective effects independently from each other and from the class II associated factors. By comparing statistical significance of 11 HLA loci and 19 haplotype segments with both untruncated and two-field allele names, we precisely mapped MS candidate alleles/haplotypes while eliminating false signals resulting from 'hitchhiking' alleles. We assessed genetic burden for the HLA allele/haplotype identified in this study. This family-based study including the highest-resolution of HLA alleles proved to be powerful and efficient for precise identification of HLA genotypes associated with both, susceptibility and protection to development of MS.

Multiple sclerosis (MS) is an autoimmune demyelinating and neurodegenerative disease of the central nervous system, and the leading cause of nontraumatic neurological disability in young adults. Effective management requires a multifaceted approach to control acute attacks, manage progressive worsening, and remediate bothersome or disabling symptoms associated with this illness. Remarkable advances in treatment of all forms of MS, and especially for relapsing disease, have favorably changed the long-term outlook for many patients. There also has been a conceptual shift in understanding the immune pathology of MS, away from a purely T-cell-mediated model to recognition that B cells have a key role in pathogenesis. The emergence of higher-efficacy drugs requiring less frequent administration have made these preferred options in terms of tolerability and adherence. Many experts now recommend use of these as first-line treatment for many patients with early disease, before permanent disability is evident.

Changes in gut microbiota composition and a diverse role of B cells have recently been implicated in multiple sclerosis (MS), a central nervous system (CNS) autoimmune disease. Immunoglobulin A (IgA) is a key regulator at the mucosal interface. However, whether gut microbiota shape IgA responses and what role IgA+ cells have in neuroinflammation are unknown. Here, we identify IgA-bound taxa in MS and show that IgA-producing cells specific for MS-associated taxa traffic to the inflamed CNS, resulting in a strong, compartmentalized IgA enrichment in active MS and other neuroinflammatory diseases. Unlike previously characterized polyreactive anti-commensal IgA responses, CNS IgA cross-reacts with surface structures on specific bacterial strains but not with brain tissue. These findings establish gut microbiota-specific IgA+ cells as a systemic mediator in MS and suggest a critical role of mucosal B cells during active neuroinflammation with broad implications for IgA as an informative biomarker and IgA-producing cells as an immune subset to harness for therapeutic interventions.

Epidemiological studies have suggested differences in the rate of multiple sclerosis (MS) in individuals of European ancestry compared to African ancestry, motivating genetic scans to identify variants that could contribute to such patterns. In a whole-genome scan in 899 African-American cases and 1155 African-American controls, we confirm that African-Americans who inherit segments of the genome of European ancestry at a chromosome 1 locus are at increased risk for MS [logarithm of odds (LOD) = 9.8], although the signal weakens when adding an additional 406 cases, reflecting heterogeneity in the two sets of cases [logarithm of odds (LOD) = 2.7]. The association in the 899 individuals can be fully explained by two variants previously associated with MS in European ancestry individuals. These variants tag a MS susceptibility haplotype associated with decreased CD58 gene expression (odds ratio of 1.37; frequency of 84% in Europeans and 22% in West Africans for the tagging variant) as well as another haplotype near the FCRL3 gene (odds ratio of 1.07; frequency of 49% in Europeans and 8% in West Africans). Controlling for all other genetic and environmental factors, the two variants predict a 1.44-fold higher rate of MS in European-Americans compared to African-Americans.

ObjectiveTo assess over 3 years of follow-up the effects of maintaining or switching to ocrelizumab (OCR) therapy on clinical and MRI outcomes and safety measures in the open-label extension (OLE) phase of the pooled OPERA: I/II studies in relapsing multiple sclerosis.MethodsAfter 2 years of double-blind, controlled treatment, patients continued OCR (600 mg infusions every 24 weeks) or switched from interferon (IFN)-β-1a (44 μg 3 times weekly) to OCR when entering the OLE phase (3 years). Adjusted annualized relapse rate, time to onset of 24-week confirmed disability progression (CDP)/improvement (CDP), brain MRI activity (gadolinium-enhanced and new/enlarging T2 lesions), and percentage brain volume change were analyzed.ResultsOf patients entering the OLE phase, 88.6% completed year 5. The cumulative proportion with 24-week CDP was lower in patients who initiated OCR earlier vs patients initially receiving IFN-β-1a (16.1% vs 21.3% at year 5; p = 0.014). Patients continuing OCR maintained and those switching from IFN-β-1a to OCR attained near complete and sustained suppression of new brain MRI lesion activity from years 3-5. Over the OLE phase, patients continuing OCR exhibited less whole brain volume loss from double-blind study baseline vs those switching from IFN-β-1a (-1.87% vs -2.15% at year 5; p < 0.01). Adverse events were consistent with past reports and no new safety signals emerged with prolonged treatment.ConclusionCompared with patients switching from IFN-β-1a, earlier and continuous OCR treatment up to 5 years provided sustained benefit on clinical and MRI measures of disease progression.Classification of evidenceThis study provides Class III evidence that earlier and continuous treatment with OCR provided sustained benefit on clinical and MRI outcomes of disease activity and progression compared with patients switching from IFN-β-1a. The study is rated Class III because of the initial treatment randomization disclosure that occurred after inclusion in OLE.Clinical trial identifiersNCT01247324/NCT01412333.

Central nervous system B cells have several potential roles in multiple sclerosis (MS): secretors of proinflammatory cytokines and chemokines, presenters of autoantigens to T cells, producers of pathogenic antibodies, and reservoirs for viruses that trigger demyelination. To interrogate these roles, single-cell RNA sequencing (scRNA-Seq) was performed on paired cerebrospinal fluid (CSF) and blood from subjects with relapsing-remitting MS (RRMS; n = 12), other neurologic diseases (ONDs; n = 1), and healthy controls (HCs; n = 3). Single-cell immunoglobulin sequencing (scIg-Seq) was performed on a subset of these subjects and additional RRMS (n = 4), clinically isolated syndrome (n = 2), and OND (n = 2) subjects. Further, paired CSF and blood B cell subsets (RRMS; n = 7) were isolated using fluorescence activated cell sorting for bulk RNA sequencing (RNA-Seq). Independent analyses across technologies demonstrated that nuclear factor kappa B (NF-κB) and cholesterol biosynthesis pathways were activated, and specific cytokine and chemokine receptors were up-regulated in CSF memory B cells. Further, SMAD/TGF-β1 signaling was down-regulated in CSF plasmablasts/plasma cells. Clonally expanded, somatically hypermutated IgM+ and IgG1+ CSF B cells were associated with inflammation, blood-brain barrier breakdown, and intrathecal Ig synthesis. While we identified memory B cells and plasmablast/plasma cells with highly similar Ig heavy-chain sequences across MS subjects, similarities were also identified with ONDs and HCs. No viral transcripts, including from Epstein-Barr virus, were detected. Our findings support the hypothesis that in MS, CSF B cells are driven to an inflammatory and clonally expanded memory and plasmablast/plasma cell phenotype.

Ataxin-1 (ATXN1) is a ubiquitous polyglutamine protein expressed primarily in the nucleus where it binds chromatin and functions as a transcriptional repressor. Mutant forms of ataxin-1 containing expanded glutamine stretches cause the movement disorder spinocerebellar ataxia type 1 (SCA1) through a toxic gain-of-function mechanism in the cerebellum. Conversely, ATXN1 loss-of-function is implicated in cancer development and Alzheimer's disease (AD) pathogenesis. ATXN1 was recently nominated as a susceptibility locus for multiple sclerosis (MS). Here, we show that Atxn1-null mice develop a more severe experimental autoimmune encephalomyelitis (EAE) course compared to wildtype mice. The aggravated phenotype is mediated by increased T helper type 1 (Th1) cell polarization, which in turn results from the dysregulation of B cell activity. Ataxin-1 ablation in B cells leads to aberrant expression of key costimulatory molecules involved in proinflammatory T cell differentiation, including cluster of differentiation (CD)44 and CD80. In addition, comprehensive phosphoflow cytometry and transcriptional profiling link the exaggerated proliferation of ataxin-1 deficient B cells to the activation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription (STAT) pathways. Lastly, selective deletion of the physiological binding partner capicua (CIC) demonstrates the importance of ATXN1 native interactions for correct B cell functioning. Altogether, we report a immunomodulatory role for ataxin-1 and provide a functional description of the ATXN1 locus genetic association with MS risk.

Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

To evaluate the value of serum immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies reactive with phosphatidylcholine (PC) and lactosylceramide (LC) as biomarkers in MS. We developed an ultrasensitive ELISA technique to analyze serum IgG and IgM antibodies to LC and PC, which we used to analyze samples from 362 patients with MS, 10 patients with non-MS myelin diseases (Non-MSMYDs), 11 patients with nonmyelin neurologic diseases (Non-MYNDs), and 80 controls. MS serum samples included clinically isolated syndrome (CIS, n = 17), relapsing-remitting MS (RRMS, n = 62), secondary progressive MS (SPMS, n = 50), primary progressive MS (PPMS, n = 37), and benign MS (BENMS, n = 36). We detected higher levels of serum IgM antibodies to PC (IgM-PC) in MS than control samples; patients with CIS and RRMS showed higher IgM-PC levels than patients with SPMS, PPMS, and BENMS and controls. MS and control samples did not differ in serum levels of IgM antibodies reactive with LC, nor in IgG antibodies reactive with LC or PC. Serum IgM-PC antibodies are elevated in patients with MS, particularly during the CIS and RRMS phases of the disease. Thus, serum IgM-PC is a candidate biomarker for early inflammatory stages of MS. This study provides Class III evidence that serum antibodies to PC are elevated in patients with MS. The study is rated Class III because of the case control design and the risk of spectrum bias: antibody levels in patients with MS were compared with healthy controls.

Background and purposeBrain MRI-derived lesions and atrophy are related to multiple sclerosis (MS) disability. In the Serially Unified Multicenter MS Investigation (SUMMIT), from Brigham and Women's Hospital (BWH) and University of California, San Francisco (UCSF), we assessed whether MRI methodologic heterogeneity may limit the ability to pool multisite data sets to assess 5-year clinical-MRI associations.MethodsPatients with relapsing-remitting (RR) MS (n = 100 from each site) underwent baseline brain MRI and baseline and 5-year clinical evaluations. Patients were matched on sex (74 women each), age, disease duration, and Expanded Disability Status Scale (EDSS) score. MRI was performed with differences between sites in both acquisition (field strength, voxel size, pulse sequences), and postprocessing pipeline to assess brain parenchymal fraction (BPF) and T2 lesion volume (T2LV).ResultsThe UCSF cohort showed higher correlation than the BWH cohort between T2LV and disease duration. UCSF showed a higher inverse correlation between BPF and age than BWH. UCSF showed a higher inverse correlation than BWH between BPF and 5-year EDSS score. Both cohorts showed inverse correlations between BPF and T2LV, with no between-site difference. The pooled but not individual cohort data showed a link between a lower baseline BPF and the subsequent 5-year worsening in disability in addition to other stronger relationships in the data.ConclusionsMRI acquisition and processing differences may result in some degree of heterogeneity in assessing brain lesion and atrophy measures in patients with MS. Pooling of data across sites is beneficial to correct for potential biases in individual data sets.

Background and purposeThe quantification of spinal cord (SC) atrophy by MRI has assumed an important role in assessment of neuroinflammatory/neurodegenerative diseases and traumatic SC injury. Recent technical advances make possible the quantification of gray matter (GM) and white matter tissues in clinical settings. However, the goal of a reliable diagnostic, prognostic or predictive marker is still elusive, in part due to large intersubject variability of SC areas. Here, we investigated the sources of this variability and explored effective strategies to reduce it.MethodsOne hundred twenty-nine healthy subjects (mean age: 41.0 ± 15.9) underwent MRI on a Siemens 3T Skyra scanner. Two-dimensional PSIR at the C2-C3 vertebral level and a sagittal 1 mm3 3D T1-weighted brain acquisition extended to the upper cervical cord were acquired. Total cross-sectional area and GM area were measured at C2-C3, as well as measures of the vertebra, spinal canal and the skull. Correlations between the different metrics were explored using Pearson product-moment coefficients. The most promising metrics were used to normalize cord areas using multiple regression analyses.ResultsThe most effective normalization metrics were the V-scale (from SienaX) and the product of the C2-C3 spinal canal diameters. Normalization methods based on these metrics reduced the intersubject variability of cord areas of up to 17.74%. The measured cord areas had a statistically significant sex difference, while the effect of age was moderate.ConclusionsThe present work explored in a large cohort of healthy subjects the source of intersubject variability of SC areas and proposes effective normalization methods for its reduction.

No single neuroimaging technique or sequence is capable of reflecting the functional deficits manifest in MS. Given the interest in imaging biomarkers for short- to medium-term studies, we aimed to assess which imaging metrics might best represent functional impairment for monitoring in clinical trials. Given the complexity of functional impairment in MS, however, it is useful to isolate a particular functionally relevant pathway to understand the relationship between imaging and neurological function. We therefore analyzed existing data, combining multiparametric MRI and OCT to describe MS associated visual impairment. We assessed baseline data from fifty MS patients enrolled in ReBUILD, a prospective trial assessing the effect of a remyelinating drug (clemastine). Subjects underwent 3T MRI imaging, including Neurite Orientation Dispersion and Density Imaging (NODDI), myelin content quantification, and retinal imaging, using OCT. Visual function was assessed, using low-contrast letter acuity. MRI and OCT data were studied to model visual function in MS, using a partial, least-squares, regression analysis. Measures of neurodegeneration along the entire visual pathway, described most of the observed variance in visual disability, measured by low contrast letter acuity. In those patients with an identified history of ON, however, putative myelin measures also showed correlation with visual performance. In the absence of clinically identifiable inflammatory episodes, residual disability correlates with neurodegeneration, whereas after an identifiable exacerbation, putative measures of myelin content are additionally informative.

Paraneoplastic neurological disorders are immune-mediated diseases understood to manifest as part of a misdirected anti-tumor immune response. Paraneoplastic neurological disorder-associated autoantibodies can assist with diagnosis and enhance our understanding of tumor-associated immune processes. We designed a comprehensive library of 49-amino-acid overlapping peptides spanning the entire human proteome, including all splicing isoforms and computationally predicted coding regions. Using this library, we optimized a phage immunoprecipitation and sequencing protocol with multiple rounds of enrichment to create high-resolution epitope profiles in serum and cerebrospinal fluid (CSF) samples from patients suffering from two common paraneoplastic neurological disorders, the anti-Yo (n = 36 patients) and anti-Hu (n = 44 patients) syndromes. All (100%) anti-Yo patient samples yielded enrichment of peptides from the canonical anti-Yo (CDR2 and CDR2L) antigens, while 38% of anti-Hu patients enriched peptides deriving from the nELAVL (neuronal embryonic lethal abnormal vision like) family of proteins, the anti-Hu autoantigenic target. Among the anti-Hu patient samples that were positive for nELAVL, we noted a restricted region of immunoreactivity. To achieve single amino acid resolution, we designed a novel deep mutational scanning phage library encoding all possible single-point mutants targeting the reactive nELAVL region. This analysis revealed a distinct preference for the degenerate motif, RLDxLL, shared by ELAVL2, 3 and 4. Lastly, phage immunoprecipitation sequencing identified several known autoantigens in these same patient samples, including peptides deriving from the cancer-associated antigens ZIC and SOX families of transcription factors. Overall, this optimized phage immunoprecipitation sequencing library and protocol yielded the high-resolution epitope mapping of the autoantigens targeted in anti-Yo and anti-Hu encephalitis patients to date. The results presented here further demonstrate the utility and high-resolution capability of phage immunoprecipitation sequencing for both basic science and clinical applications and for better understanding the antigenic targets and triggers of paraneoplastic neurological disorders.

CD8+ T cells are believed to play an important role in multiple sclerosis (MS), yet their role in MS pathogenesis remains poorly defined. Although myelin proteins are considered potential autoantigenic targets, prior studies of myelin-reactive CD8+ T cells in MS have relied on in vitro stimulation, thereby limiting accurate measurement of their ex vivo precursor frequencies and phenotypes. Peptide:MHC I tetramers were used to identify and validate 5 myelin CD8+ T cell epitopes, including 2 newly described determinants in humans. The validated tetramers were used to measure the ex vivo precursor frequencies and phenotypes of myelin-specific CD8+ T cells in the peripheral blood of untreated MS patients and HLA allele-matched healthy controls. In parallel, CD8+ T cell responses against immunodominant influenza epitopes were also measured. There were no differences in ex vivo frequencies of tetramer-positive myelin-specific CD8+ T cells between MS patients and control subjects. An increased proportion of myelin-specific CD8+ T cells in MS patients exhibited a memory phenotype and expressed CD20 compared to control subjects, while there were no phenotypic differences observed among influenza-specific CD8+ T cells. Longitudinal assessments were also measured in a subset of MS patients subsequently treated with anti-CD20 monoclonal antibody therapy. The proportion of memory and CD20+ CD8+ T cells specific for certain myelin but not influenza epitopes was significantly reduced following anti-CD20 treatment. This study, representing a characterization of unmanipulated myelin-reactive CD8+ T cells in MS, indicates these cells may be attractive targets in MS therapy.

BACKGROUND:Chronic lesion activity driven by smoldering inflammation is a pathological hallmark of progressive forms of multiple sclerosis (MS). OBJECTIVE:To develop a method for automatic detection of slowly expanding/evolving lesions (SELs) on conventional brain magnetic resonance imaging (MRI) and characterize such SELs in primary progressive MS (PPMS) and relapsing MS (RMS) populations. METHODS:We defined SELs as contiguous regions of existing T2 lesions showing local expansion assessed by the Jacobian determinant of the deformation between reference and follow-up scans. SEL candidates were assigned a heuristic score based on concentricity and constancy of change in T2- and T1-weighted MRIs. SELs were examined in 1334 RMS patients and 555 PPMS patients. RESULTS:Compared with RMS patients, PPMS patients had higher numbers of SELs (p = 0.002) and higher T2 volumes of SELs (p

OBJECTIVE:To assess the onset of ocrelizumab efficacy on brain MRI measures of disease activity in the phase II study in relapsing-remitting multiple sclerosis (RRMS), and relapse rate in the pooled phase III studies in relapsing multiple sclerosis (RMS). METHODS:Brain MRI activity was determined in the phase II trial at monthly intervals in patients with RRMS receiving placebo, ocrelizumab (600 mg), or intramuscular interferon (IFN) β-1a (30 μg). Annualized relapse rate (ARR; over various epochs) and time to first relapse were analyzed in the pooled population of the phase III OPERA (A Study of Ocrelizumab in Comparison With Interferon Beta-1a [Rebif] in Participants With Relapsing Multiple Sclerosis) I and OPERA II trials in patients with RMS receiving ocrelizumab (600 mg) or subcutaneous IFN-β-1a (44 μg). RESULTS:In patients with RRMS, ocrelizumab reduced the number of new T1 gadolinium-enhancing lesions by week 4 vs placebo (p = 0.042) and by week 8 vs intramuscular IFN-β-1a (p < 0.001). Ocrelizumab also reduced the number of new or enlarging T2 lesions appearing between weeks 4 and 8 vs both placebo and IFN-β-1a (both p < 0.001). In patients with RMS, ocrelizumab significantly reduced ARR (p = 0.005) and the probability of time to first protocol-defined relapse (p = 0.014) vs subcutaneous IFN-β-1a within the first 8 weeks. CONCLUSION:Epoch analysis of MRI-measured lesion activity in the phase II study and relapse rate in the phase III studies consistently revealed a rapid suppression of acute MRI and clinical disease activity following treatment initiation with ocrelizumab in patients with RRMS and RMS, respectively. CLASSIFICATION OF EVIDENCE:This study provides Class II evidence that for patients with RRMS and RMS, ocrelizumab suppressed MRI activity within 4 weeks and clinical disease activity within 8 weeks.