Untreated Multiple Sclerosis Patients Research Articles

Effects of Natalizumab Therapy on Intrathecal Immunoglobulin G Production Indicate Targeting of Plasmablasts.

ObjectivesTo evaluate the long-term effects of natalizumab (NTZ) on different features of intrathecal immunoglobulin (Ig) synthesis in patients with multiple sclerosis (MS) and to quantify the expression of α4-integrin in stages of B-cell maturation.MethodsWe combined a cross-sectional (49 NTZ-treated MS patients, mean treatment duration 5.1 years, and 47 untreated MS patients) and a longitudinal study (33 patients with MS before and during NTZ, mean treatment duration: 4.8 years), analyzing paired serum and CSF samples for IgG, IgA, and IgM levels, reactivity against selected viruses (measles virus, rubella virus, and varicella zoster virus [MRZ] reaction), and oligoclonal bands (OCBs). Banding patterns before and after therapy were directly compared by isoelectric focusing in 1 patient. In addition, we determined the expression of α4-integrin by FACS analysis on blood-derived B-cell subsets (plasmablasts, memory B cells, and naive B cells) of healthy controls.ResultsIn serum, NTZ decreased IgM and IgG, but not IgA, levels. IgM hypogammaglobulinemia occurred in 28% of NTZ-treated patients. In CSF, NTZ treatment resulted in a strong reduction of intrathecally produced IgG and, to a lesser extent, IgA, whereas IgM indices [(Ig CSF/Serum)/(Albumin CSF/Serum)] remained largely unchanged. Reduction of the IgG index correlated with NTZ treatment duration, as did serum IgM and IgA levels. MRZ reaction was unchanged and OCB persisted. Direct comparison of OCB pattern before and after NTZ revealed the persistence of individual bands. α4-Integrin expression was highest on plasmablasts (CD19+CD38+CD27+).ConclusionOur data indicate that NTZ reduces short-lived plasmablasts in the CNS compartment but has little effect on locally persisting long-lived plasma cells.

Serum neurofilament light chain levels in healthy individuals: A proposal of cut-off values for use in multiple sclerosis clinical practice

BackgroundSerum Neurofilament Light (sNFL) is the most promising marker for patient's monitoring in Multiple Sclerosis (MS). However, operating reference values for use in clinical practice are still lacking. Here, we defined sNFL reference cut-off values in a cohort of healthy controls (HC) and assessed their performance in Multiple Sclerosis (MS) patients, as well as the intra-individual sNFL variability. MethodsWe measured sNFL by single molecule array (Simoa) assay in 79 HC assessing their correlation with age. Changes of sNFL levels were evaluated during a short-term follow-up (median 67 days between consecutive samples) in a subgroup of 27 participants. sNFL were tested in 23 untreated MS patients, at both diagnostic time and start of therapy (median 80 days after), considering disease activity. ResultsFindings confirmed a correlation between sNFL levels and age in HC, thus cut-off values specific for age decades were calculated. sNFL did not vary significantly with time during short-term follow-up (median CV 13%).sNFL levels in MS patients were higher and demonstrated a higher variability between diagnostic time and treatment start (median CV 39%). According to cut-off values, “pathologic” sNFL levels were found in 57% of MS patients at diagnostic time, and in 30% of samples at treatment start. In particular, “pathologic” sNFL levels were found in 80% of samples (16/20) obtained during a phase of disease activity, while a total of 85% of samples (22/26) associated with inactive disease showed sNFL in the normal range. ConclusionThis study demonstrates an overall intra-individual stability of sNFL values in the short-term in HC and suggests age-dependent reference cut-off values that could be beneficial for sNFL implementation in clinical practice.

Pregnancy-Induced Changes in microRNA Expression in Multiple Sclerosis.

Pregnancy affects the disease course in multiple sclerosis (MS), particularly in the third trimester, where the relapse rate is reduced by as much as two thirds. This study aimed at identifying changes in microRNA (miRNA) and immune cell phenotypes in pregnant MS patients. Discovery and validation studies to detect differentially expressed miRNAs were performed with quantitative real-time PCR on peripheral blood mononuclear cells (PBMC). Flow cytometry analysis was performed on PBMC stained with antibodies directed against surface markers of antigen presenting cells (APCs), NK-cells, NKT cells, CD4+ and CD8+ T cells and subsets of these cell types, including PDL1 and PDL2 expressing subsets. RNA was extracted from whole blood, monocytes, and NK-cells to investigate expression and correlation between regulated miRNAs and mRNAs. In total, 15 miRNAs were validated to be differentially expressed between third trimester pregnant and postpartum MS patients (Benjamini-Hochberg false discovery rate from p = 0.03–0.00004). Of these, 12 miRNAs were downregulated in pregnancy and 6 of the 15 miRNAs were altered by more than ±2-fold (+2.99- to -6.38-fold). Pregnant MS patients had a highly significant increase in the percentage of monocytes and a decrease of NK-cells and myeloid dendritic cells compared to non-pregnant MS patients. We confirm previous reports of a relative increase in CD56-bright NK-cells and a decrease in CD56-dim NK-cells in third trimester of pregnancy and report an increase in non-committed follicular helper cells. PDL1 and PDL2 expression was increased in pregnant patients together with IL10. Also, in monocytes IL10, PDL1, and PDL2 were upregulated whereas miR-1, miR-20a, miR-28, miR-95, miR-146a, miR-335, and miR-625 were downregulated between pregnant and untreated MS patients. IL10, PDL1, and PDL2 were predicted targets of MS pregnancy-changed miRNAs, further supported by their negative correlations. Additionally, previously identified pregnancy-regulated mRNAs were identified as predicted targets of the miRNAs. PDL1 and PDL2 bind PD-1 expressed on T cells with an inhibitory effect on T-cell proliferation and increase in IL10 production. These results indicate that some of the effects behind the disease-ameliorating third trimester of pregnancy might be caused by changed expression of miRNAs and immunoregulatory molecules in monocytes.

Herpesvirus Antibodies, Vitamin D and Short-Chain Fatty Acids: Their Correlation with Cell Subsets in Multiple Sclerosis Patients and Healthy Controls.

Although the etiology of multiple sclerosis (MS) is still unknown, it is commonly accepted that environmental factors could contribute to the disease. The objective of this study was to analyze the humoral response to Epstein-Barr virus, human herpesvirus 6A/B and cytomegalovirus, and the levels of 25-hydroxyvitamin D (25(OH)D) and the three main short-chain fatty acids (SCFA), propionate (PA), butyrate (BA) and acetate (AA), in MS patients and healthy controls (HC) to understand how they could contribute to the pathogenesis of the disease. With this purpose, we analyzed the correlations among them and with different clinical variables and a wide panel of cell subsets. We found statistically significant differences for most of the environmental factors analyzed when we compared MS patients and HC, supporting their possible involvement in the disease. The strongest correlations with the clinical variables and the cell subsets analyzed were found for 25(OH)D and SCFAs levels. A correlation was also found between 25(OH)D and PA/AA ratio, and the interaction between these factors negatively correlated with interleukin 17 (IL-17)-producing CD4+ and CD8+ T cells in untreated MS patients. Therapies that simultaneously increase vitamin D levels and modify the proportion of SCFA could be evaluated in the future.

Humoral immune response to COVID-19 mRNA vaccine in patients with multiple sclerosis treated with high-efficacy disease-modifying therapies.

Background and Aims:The National Multiple Sclerosis Society and other expert organizations recommended that all patients with multiple sclerosis (MS) should be vaccinated against COVID-19. However, the effect of disease-modifying therapies (DMTs) on the efficacy to mount an appropriate immune response is unknown. We aimed to characterize humoral immunity in mRNA-COVID-19 MS vaccinees treated with high-efficacy DMTs.Methods:We measured SARS-CoV-2 IgG response using anti-spike protein-based serology (EUROIMMUN) in 125 MS patients vaccinated with BNT162b2-COVID-19 vaccine 1 month after the second dose. Patients were either untreated or under treatment with fingolimod, cladribine, or ocrelizumab. A group of healthy subjects similarly vaccinated served as control. The percent of subjects that developed protective antibodies, the titer, and the time from the last dosing were evaluated.Results:Protective humoral immunity of 97.9%, 100%, 100%, 22.7%, and 3.8%, was observed in COVID-19 vaccinated healthy subjects (N = 47), untreated MS patients (N = 32), and MS patients treated with cladribine (N = 23), ocrelizumab (N = 44), and fingolimod (N = 26), respectively. SARS-CoV-2 IgG antibody titer was high in healthy subjects, untreated MS patients, and MS patients under cladribine treatment, within 29.5–55 days after the second vaccine dose. Only 22.7% of patients treated with ocrelizumab developed humoral IgG response irrespective to normal absolute lymphocyte count. Most fingolimod-treated MS patients had very low lymphocyte count and failed to develop SARS-COV-2 antibodies. Age, disease duration, and time from the last dosing did not affect humoral response to COVID-19 vaccination.Conclusions:Cladribine treatment does not impair humoral response to COVID-19 vaccination. We recommend postponing ocrelizumab treatment in MS patients willing to be vaccinated as a protective humoral response can be expected only in some. We do not recommend vaccinating MS patients treated with fingolimod as a protective humoral response is not expected.

Chitinase 3-like 1 is not a target antigen in patients with multiple sclerosis

Autoimmunity to chitinase 3-like 1 (CHI3L1) has recently been reported in hepatic and bowel inflammatory conditions. Considering that CHI3L1 plays a role as prognostic biomarker in multiple sclerosis (MS), here we investigated CHI3L1 as potential autoantigenic target in the disease. We determined serum CHI3L1 autoantibodies by enzyme-linked immunosorbent assay (ELISA) in a cohort of 60 untreated MS patients with different clinical forms of the disease and 20 healthy controls (HC). IgG levels to CHI3L1 were similar between patients with relapsing-remitting MS, primary and secondary progressive MS, and HC. These findings do not support a role for CHI3L1 autoantibodies in MS pathogenesis.

Identification of regulatory T cell molecules associated with severity of multiple sclerosis

Background: Regulatory CD4+ T cells (Tregs) exhibit functional alterations in patients with multiple sclerosis (MS). Transforming growth factor (TGF)-β is a key regulator of Treg development and function. Objective: The objective of this study is to determine whether the expression of functionally relevant TGF-β-regulated molecules is altered in Tregs from patients with MS. Methods: Expression of nine Treg markers was analyzed by multi-color flow cytometry in CD4+ T cells and Treg subpopulations of 31 untreated MS patients and age- and sex-matched healthy donors (HDs). Correlations between Treg marker expression and clinical variables were sought. Results: Expression of the transcription factor Helios, which defines thymic-derived Tregs, was decreased in this Treg subpopulation. The frequency of peripherally generated Tregs was increased in patients with MS, particularly in patients with progressive MS. Low frequencies of thymic-derived Tregs were associated with magnetic resonance imaging (MRI) lesion-burden and a high relapse rate. Four surface markers associated with TGF-β signaling (ABCA1, BTLA, DNAM-1, and GARP) were differentially expressed on Tregs from patients with MS and HDs. Expression levels of CD73, CD103, ABCA1, and PAR2 showed strong correlations with disease severity. Conclusion: We have identified novel markers abnormally expressed on Tregs from patients with MS that could detect patients with severe disease.

Acetate correlates with disability and immune response in multiple sclerosis.

BackgroundGut microbiota has been related to multiple sclerosis (MS) etiopathogenesis. Short-chain fatty acids (SCFA) are compounds derived from microbial metabolism that have a role in gut-brain axis.ObjectivesTo analyse SCFA levels in plasma of MS patients and healthy donors (HD), and the possible link between these levels and both clinical data and immune cell populations.MethodsNinety-five MS patients and 54 HD were recruited. Patients were selected according to their score in the Expanded Disability Status Scale (EDSS) (49 EDSS ≤ 1.5, 46 EDSS ≥ 5.0). SCFA were studied in plasma samples by liquid chromatography-mass spectrometry. Peripheral blood mononuclear cells were studied by flow cytometry. Gender, age, treatments, EDSS and Multiple Sclerosis Severity Score (MSSS) were evaluated at the recruitment.ResultsPlasma acetate levels were higher in patients than in HD (p = 0.003). Patients with EDSS ≥ 5.0 had higher acetate levels than those with EDSS≤ 1.5 (p = 0.029), and HD (p = 2.97e–4). Acetate levels correlated with EDSS (r = 0.387; p = 1.08e–4) and MSSS (r = 0.265; p = 0.011). In untreated MS patients, acetate levels correlated inversely with CD4+ naïve T cells (r = − 0.550, p = 0.001) and directly with CD8+ IL-17+ cells (r = 0.557; p = 0.001).ConclusionsPlasma acetate levels are higher in MS patients than in HD. In MS there exists a correlation between plasma acetate levels, EDSS and increased IL-17+ T cells. Future studies will elucidate the role of SCFA in the disease.

Selective modulation of trans-endothelial migration of lymphocyte subsets in multiple sclerosis patients under fingolimod treatment

Multiple sclerosis (MS) is an autoimmune disorder where auto-aggressive T cells target the central nervous system (CNS), causing demyelination. The trans-endothelial migration of leucocytes across the blood-brain barrier (BBB) is one of the earliest CNS events in MS pathogenesis. We examined the effect of the disease state and treatment with fingolimod on the transmigration of peripheral blood mononuclear cells (PBMCs) in an in vitro BBB model. Patients' leucocyte numbers, subsets and phenotypes were assessed by flow cytometry. As expected, fingolimod treatment induced a significant reduction in T cell and B cell numbers compared to untreated MS patients and healthy controls. Interestingly fingolimod led to a marked reduction of CD4+ and a significant increase in CD8+ cell numbers. In migrated cells, only CD3+ cell numbers were reduced in fingolimod-treated, compared to untreated patients; it had no effect on B cell or monocyte transmigration. T cells were then differentiated into naïve, effector and memory subsets based on their expression of CCR7. This showed that MS patients had increased numbers of effector memory CD4+ cells re-expressing CD45RA (TEMRA) and a decrease in central memory (CM) CD8+ cells. The former was corrected by fingolimod, while the latter was not. CM CD4+ and CD8+ cells migrated across BBB more efficiently in fingolimod-treated patients. We found that while fingolimod reduced the proportions of naïve CD19+ B cells, it significantly increased the proportions of these cells which migrated. When B cells were further stratified based on CD24, CD27 and CD38 expression, the only effect of fingolimod was an enhancement of CD24hiCD27+ B cell migration, compared to untreated MS patients. The migratory capacities of CD8hi Natural Killer (NK), CD8dim NK and NK-T cells were also reduced by fingolimod. While the disease-modifying effects of fingolimod are currently explained by its effect on reducing circulating auto-aggressive lymphocytes, our data suggests that fingolimod may also have a direct though differential effect on the trans-endothelial migration of circulating lymphocyte populations.

Immune signatures of prodromal multiple sclerosis in monozygotic twins.

The tremendous heterogeneity of the human population presents a major obstacle in understanding how autoimmune diseases like multiple sclerosis (MS) contribute to variations in human peripheral immune signatures. To minimize heterogeneity, we made use of a unique cohort of 43 monozygotic twin pairs clinically discordant for MS and searched for disease-related peripheral immune signatures in a systems biology approach covering a broad range of adaptive and innate immune populations on the protein level. Despite disease discordance, the immune signatures of MS-affected and unaffected cotwins were remarkably similar. Twinship alone contributed 56% of the immune variation, whereas MS explained 1 to 2% of the immune variance. Notably, distinct traits in CD4+ effector T cell subsets emerged when we focused on a subgroup of twins with signs of subclinical, prodromal MS in the clinically healthy cotwin. Some of these early-disease immune traits were confirmed in a second independent cohort of untreated early relapsing-remitting MS patients. Early involvement of effector T cell subsets thus points to a key role of T cells in MS disease initiation.

IFI35 as a biomolecular marker of neuroinflammation and treatment response in multiple sclerosis

Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) with unpredictable clinical outcome. As such, there is an urgent need to identify biomarkers that can predict the treatment response. Therefore, in an open-label, clinical, paraclinical and molecular prospective study, we assessed 50 interferon (IFN) treated MS patients for Rio Score (RS)/Modified Rio Score (MRS) and densitometric expression of the interferon-induced protein 35 (IFI35), a signal-protein with potential to be clinically relevant in the management of the disease. We found 4.92-fold upregulated IFI35 in IFN-treated MS group respect to healthy controls (p < .0001) and 2.31-fold respect to untreated MS group (p < .0001). Moreover, IFI35 expression profile correlated with RS and MRS rank values (r = −0.6018, p < .0001; r = −0.620, p < .0001), white matter volume (r = −0.5041; p = .0017) and cerebral lesion load (r = −0.5075; p = .0026). Finally, the main proportion of IFN-treated MS patients non-reaching the 65% threshold in IFI35 expression leaved the RS/MRS rank value 0 in a period ranging from 5 to 15 months (p < .0001) from the study entry; instead, all patients that reaching this threshold maintained the RS/MRS value 0 until the study end (p < .0001). In conclusion, the expression level of IFI35 in untreated MS patients highlights a correlation with neuroinflammation. Furthermore, IFI35 expression in IFN-treated MS patients shows a modular correlation between dosing regimes, which is predictive for long-term clinical outcome and drug efficacy.

Differential effects of disease modifying drugs on peripheral blood B cell subsets: A cross sectional study in multiple sclerosis patients treated with interferon-β, glatiramer acetate, dimethyl fumarate, fingolimod or natalizumab.

BackgroundSeveral disease modifying drugs (DMDs) have been approved for the treatment of multiple sclerosis (MS), however, little is known about their differential impact on peripheral blood (PB) B cell subsets.MethodsWe performed a cross sectional study on PB B cells in MS patients treated with interferon-β (n = 25), glatiramer acetate (n = 19), dimethyl fumarate (n = 15), fingolimod (n = 16) or natalizumab (n = 22), untreated MS patients (n = 20), and in patients with non-inflammatory neurological diseases (n = 12). Besides analyzing routine laboratory data, flow cytometry was performed to analyze naïve B cells (CD19+CD20+CD27-IgD+), non-class switched (CD19+CD20+CD27+IgD+) and class-switched memory B cells (CD19+CD20+CD27+IgD-), double negative B cells (CD19+CD20lowCD27-IgD-) and plasmablasts (CD19+CD20lowCD27+CD38++).ResultsTreatment associated changes were found for the overall B cell pool as well as for all B cell subsets. Natalizumab increased absolute numbers and percentage of all B cells mainly by expanding the memory B cell pool. Fingolimod decreased absolute numbers of all B cell subsets and the percentage of total B cells. Fingolimod, dimethyl fumarate and interferon-β treatments were associated with an increase in the fraction of naïve B cells while class switched and non-class switched memory B cells showed decreased percentages.ConclusionOur results highlight differential effects of DMDs on the PB B cell compartment. Across the examined treatments, a decreased percentage of memory B cells was found in dimethyl fumarate, interferon-β and fingolimod treated patients which might contribute to the drugs’ mode of action in MS. Further studies are necessary to decipher the exact role of B cell subsets during MS pathogenesis.

Multiplex assessment of cerebrospinal fluid biomarkers in multiple sclerosis

BackgroundSeveral roles for biomarkers in multiple sclerosis (MS) exist, including aiding in the diagnosis of MS, predicting disease activity or progression, and defining individuals who may be responsive to specific treatments. Cerebrospinal fluid (CSF) concentrations of soluble B cell maturation antigen (sBCMA) and soluble CD27 (sCD27) have been shown to be sensitive biomarkers of inflammation in MS and are thought to reflect B and T cell activity, respectively. Furthermore, chitinase 3-like 1 (CHI3L1) and soluble CD14 (sCD14) have been suggested as measures of innate immune cell activity in MS. In this study we sought to validate measurements of these CSF biomarkers of inflammation using multiplex bead-based immunoassays. MethodsBy using commercially available multiplex bead-based assays, concentrations of sBCMA, sCD27, sCD14 and CHI3L1 were determined in CSF from 22 patients with either untreated clinically isolated syndromes (CIS) or relapsing-remitting MS (RRMS), 13 patients with RRMS treated with either natalizumab or alemtuzumab, and 35 symptomatic controls (SC). ResultsIncreased CSF concentrations of sBCMA, sCD27 and CHI3L1 were observed in untreated MS patients compared to symptomatic controls (all p < 0.001). Concentrations of sBCMA (p = 0.02) and sCD27 (p = 0.0003) were higher in treated MS patients than in SC, and levels of sBCMA (p = 0.02) and sCD27 (p = 0.01) were even higher in untreated compared to treated patients. sCD14 levels did not differ between the groups. Levels of sBCMA and sCD27 correlated strongly with each other (Spearman's rho: 0.98, p < 0.0001) as well as with the IgG index (Spearman's rho: 0.91, p < 0.0001 and 0.90, p < 0.0001, respectively). ROC curve analysis showed a high discriminatory potential for sBCMA and sCD27 with areas under the curve of 0.88 and 0.93, respectively. ConclusionWe confirm reports of elevated concentrations of sBCMA, sCD27 and CHI3L1 in CSF from untreated MS patients compared to SC. sBCMA and sCD27 levels were elevated in both treated and untreated MS patients compared to SC, but highest in untreated patients. Finally, CSF concentrations of sBCMA, sCD27 and the IgG index correlated strongly, suggesting that the cellular source of sCD27 and sBCMA includes memory B cells, plasmablasts and plasma cells.

Impact of cytomegalovirus infection on B cell differentiation and cytokine production in multiple sclerosis

BackgroundHuman cytomegalovirus (HCMV) infection has been recently associated with a low risk of multiple sclerosis (MS), yet the basis behind this observation remains uncertain. In this study, we aimed to determine in MS patients whether HCMV induces modifications in the peripheral B cell compartment.MethodsHCMV serostatus was determined in 73 MS patients (55 relapsing-remitting MS (RRMS); 18 progressive MS (PMS)) and 30 healthy controls, assessing their B cell immunophenotype and cytokine production (GM-CSF, IL-6, IL-10, and TNFα) by flow cytometry.ResultsHCMV seropositivity in untreated MS patients (n = 45) was associated with reduced switched memory B cells, contrasting with an opposite effect in PMS. Expansions of transitional B cells were observed in HCMV(+) IFNβ-treated RRMS patients but not in HCMV(−) cases (p < 0.01), suggesting that HCMV may influence the distribution of B cell subsets modulating the effects of IFNβ. Considering the B cell functional profile, HCMV(−) PMS displayed an increased secretion of proinflammatory cytokines (IL-6, TNFα) as compared to HCMV(+) PMS and RRMS cases (p < 0.001).ConclusionsOur study reveals an influence of HCMV infection on the phenotype and function of B cells, promoting early differentiation stages in RRMS and reducing the proinflammatory cytokine profile in advanced MS forms, which might be related with the putative protective role of this virus in MS.

Oral Administration of miR-30d from Feces of MS Patients Suppresses MS-like Symptoms in Mice by Expanding Akkermansia muciniphila

Fecal transfer from healthy donors is being explored as a microbiome modality. MicroRNAs (miRNAs) have been found to affect the microbiome. Multiple sclerosis (MS) patients have been shown to have an altered gut microbiome. Here, we unexpectedly found that transfer of feces harvested at peak disease from the experimental autoimmune encephalomyelitis (EAE) model of MS ameliorates disease in recipients in a miRNA-dependent manner. Specifically, we show that miR-30d is enriched in the feces of peak EAE and untreated MS patients. Synthetic miR-30d given orally ameliorates EAE through expansion of regulatory Tcells (Tregs). Mechanistically, miR-30d regulates the expression of a lactase in Akkermansia muciniphila, which increases Akkermansia abundance in the gut. The expanded Akkermansia in turn increases Tregs to suppress EAE symptoms. Our findings report the mechanistic underpinnings of a miRNA-microbiome axis and suggest that the feces of diseased subjects might be enriched with miRNAs with therapeutic properties.

Intrathecal, Not Systemic Inflammation Is Correlated With Multiple Sclerosis Severity, Especially in Progressive Multiple Sclerosis.

Objective: To test the hypothesis that Multiple Sclerosis (MS) patients have increased peripheral inflammation compared to healthy donors and that this systemic activation of the immune system, reflected by acute phase reactants (APRs) measured in the blood, contributes to intrathecal inflammation, which in turn contributes to the development of disability in MS.Methods: Eight serum APRs measured in a prospectively-collected cross-sectional cohort with a total of 51 healthy donors and 291 untreated MS patients were standardized and assembled into related biomarker clusters to derive global measures of systemic inflammation. The resulting APR clusters were compared between diagnostic categories and correlated to equivalently-derived cerebrospinal fluid (CSF) biomarkers of innate and adaptive immunity. Finally, correlations were calculated between biomarkers of systemic and intrathecal inflammation and MS severity measures, which predict future rates of disability progression.Results: While two blood APR clusters were elevated in MS patients, only one exhibited a weak correlation with MS severity. All CSF inflammation clusters, except CSF albumin, correlated with at least one measure of MS severity, with biomarkers of humoral adaptive immunity exhibiting the strongest correlations, especially in Progressive MS.Conclusion: Systemic inflammation does not appear to be strongly associated with intrathecal inflammation in MS. Positive correlations between markers of intrathecal inflammation, especially of humoral immunity, with MS severity measures support a pathogenic role of intrathecal (compartmentalized) inflammation in central nervous system tissue destruction, including in Progressive MS.

Anti-CD20 therapy depletes activated myelin-specific CD8+ T cells in multiple sclerosis

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.

Blood transcriptome profiling captures dysregulated pathways and response to treatment in neuroimmunological disease.

Blood transcriptome profiling captures dysregulated pathways and response to treatment in neuroimmunological disease.

Long-term use of interferon-\u03b2 in multiple sclerosis increases V\u03b41\u2212V\u03b42\u2212V\u03b39\u2212 \u03b3\u03b4 T cells that are associated with a better outcome

BackgroundWe previously reported that Vδ2+Vγ9+ γδ T cells were significantly decreased in multiple sclerosis (MS) patients without disease-modifying therapies (untreated MS) and were negatively correlated with Expanded Disability Status Scale (EDSS) scores, suggesting protective roles of Vδ2+Vγ9+ γδ T cells. Interferon-β (IFN-β) is one of the first-line disease-modifying drugs for MS. However, no previous studies have reported changes in γδ T cell subsets under IFN-β treatment. Therefore, we aimed to clarify the effects of the long-term usage of IFN-β on γδ T cell subsets in MS patients.MethodsComprehensive flow cytometric immunophenotyping was performed in 35 untreated MS and 21 MS patients on IFN-β for more than 2 years (IFN-β-treated MS) including eight super-responders fulfilling no evidence of disease activity criteria, and 44 healthy controls (HCs).ResultsThe percentages of Vδ2+Vγ9+ cells in γδ T cells were significantly lower in untreated and IFN-β-treated MS patients than in HCs. By contrast, the percentages of Vδ1−Vδ2−Vγ9− cells in γδ T cells were markedly higher in IFN-β-treated MS patients than in HCs and untreated MS patients (both p < 0.001). A significant negative correlation between the percentages of Vδ2+Vγ9+ cells in γδ T cells and EDSS scores was confirmed in untreated MS but not evident in IFN-β-treated MS. Moreover, class-switched memory B cells were decreased in IFN-β-treated MS compared with HCs (p < 0.001) and untreated MS patients (p = 0.006). Interestingly, the percentages of Vδ1−Vδ2−Vγ9− cells in γδ T cells were negatively correlated with class-switched memory B cell percentages in all MS patients (r = − 0.369, p = 0.005), and the percentages of Vδ1−Vδ2−Vγ9− cells in Vδ1−Vδ2− γδ T cells were negatively correlated with EDSS scores only in IFN-β super-responders (r = − 0.976, p < 0.001).ConclusionsThe present study suggests that long-term usage of IFN-β increases Vδ1−Vδ2−Vγ9− γδ T cells, which are associated with a better outcome, especially in IFN-β super-responders. Thus, increased Vδ1−Vδ2−Vγ9− cells together with decreased class-switched memory B cells may contribute to the suppression of disease activity in MS patients under IFN-β treatment.

Epstein Barr virus nuclear antigen 1 (EBNA-1) peptides recognized by adult multiple sclerosis patient sera induce neurologic symptoms in a murine model

Multiple sclerosis (MS) is an autoimmune demyelinating disease with progressive neurodegeneration and complex etiology likely involving genetic and environmental factors. MS has been associated with Epstein Barr virus (EBV) infection, with patients often showing enhanced responses to EBV antigens. To determine whether abnormal EBV nuclear antigen-1 (EBNA-1) humoral immunity can serve as an initiator of autoimmune responses in MS, we investigated the fine specificities of the humoral immune response against EBNA-1 in MS patients using solid phase epitope mapping. Antibodies from MS patients recognized an EBNA-1 epitope spanning amino acids 411–426, previously unknown to be recognized specifically by untreated MS patients. Antibodies against this epitope cross-reacted to myelin basic protein (MBP). Furthermore, animals immunized with this EBNA-1 polypeptide mounted a response against MBP and developed signs of experimental autoimmune encephalitis (EAE). These data support a link between MS and EBV through antibodies that cross-react between EBV proteins and the MBP autoantigen.