Ocrelizumab Alters Cytotoxic Lymphocyte Function While Reducing EBV-Specific CD8+ T-Cell Proliferation in Patients With Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: The role of B cells in the pathogenic events leading to relapsing multiple sclerosis (R-MS) has only been recently elucidated. A pivotal step in defining this role has been provided by therapeutic efficacy of anti-CD20 monoclonal antibodies. Indeed, treatment with anti-CD20 can also alter number and function of other immune cells not directly expressing CD20 on their cell surface, whose activities can contribute to unknown aspects influencing therapeutic efficacy. We examined the phenotype and function of cytotoxic lymphocytes and Epstein-Barr virus (EBV)-specific immune responses in people with R-MS before and after ocrelizumab treatment. METHODS: In this prospective study, we collected blood samples from people with R-MS (n = 41) before and 6 and 12 months after initiating ocrelizumab to assess the immune phenotype and the indirect impact on cytotoxic functions of CD8+ T and NK cells. In addition, we evaluated the specific anti-EBV proliferative responses of both CD8+ T and NK lymphocytes as surrogate markers of anti-EBV activity. RESULTS: We observed that while ocrelizumab depleted circulating B cells, it also reduced the expression of activation and migratory markers on both CD8+ T and NK cells as well as their in vitro cytotoxic activity. A comparable pattern in the modulation of immune molecules by ocrelizumab was observed in cytotoxic cells even when patients with R-MS were divided into groups based on their prior disease-modifying treatment. These effects were accompanied by a significant and selective reduction of CD8+ T-cell proliferation in response to EBV antigenic peptides. DISCUSSION: Taken together, our findings suggest that ocrelizumab-while depleting B cells-affects the cytotoxic function of CD8+ and NK cells, whose reduced cross-activity against myelin antigens might also contribute to its therapeutic efficacy during MS.

Predicting clinical progression and cognitive decline in patients with relapsing-remitting multiple sclerosis: a 6-year follow-up study.

INTRODUCTION: Cognitive impairment occurs from the earliest stages of multiple sclerosis (MS) and progresses over time. The introduction of disease modifying therapies (DMTs) has changed the prognosis for MS patients, offering a potential opportunity for improvement in the cognitive arena as well. MATERIAL AND METHODS: 41 patients with relapsing-remitting multiple sclerosis (MS) were recruited to the study. Thirty patients were available for final follow-up and were included in the analysis. Baseline (BL) brain MRI including volumetry and neuropsychological tests were performed. Blood samples were collected at BL and follow-up (FU) and were tested for: vascular endothelial growth factor (VEGF), soluble vascular cell adhesion molecule-1 (sVCAM1), soluble platelet-endothelial CAM-1 (sPECAM1), and soluble intercellular CAM-1 (sICAM-1). Patients were invited for a final neuropsychological follow-up after a median of 6 years. Disease activity (relapses, EDSS increase, new/active brain lesions on MRI) was analysed between BL and FU. RESULTS: The study group deteriorated in the Rey-Osterrieth Complex Figure (ROCF) test (p = 0.001), but improved significantly in three other tests, i.e. semantic fluency test (p = 0.013), California Verbal Learning Test (CVLT, p = 0.016), and Word Comprehension Test (WCT, p < 0.001). EDSS increase correlated negatively with semantic fluency and WCT scores (r = -0.579, p = 0.001 and r = -0.391, p = 0.033, respectively). Improvements in semantic fluency test and WCT correlated positively with baseline deep grey matter, grey matter, and cortical volumes (p < 0.05, r > 0). Higher EDSS on FU correlated significantly negatively with baseline left and right pallidum, right caudate, right putamen, right accumbens, and cortical volume (p < 0.05, r < 0). No significant relationship was found between the number of relapses and EDSS on FU or neuropsychological deteriorations. Improvements in WCT and CVLT correlated positively with baseline sPECAM1 and sVCAM1 results, respectively (r > 0, p < 0.05). Deterioration in ROCF test correlated significantly with higher levels of baseline VEGF and sVCAM1 (p < 0.05). CONCLUSIONS: Brain volume is an important predictor of future EDSS and cognitive functions outcome. MS patients have a potential for improving in neuropsychological tests over time. It remains to be established whether this is related to successful disease modification with immunotherapy. Baseline volumetric measures are stronger predictors of cognitive performance than relapse activity, which yet again highlights the importance of atrophy in MS prognosis.

The alterations of cerebrospinal fluid TNF-alpha and TGF-beta2 levels in early relapsing-remitting multiple sclerosis.

AIM OF THE STUDY: This study aimed to analyze serum and cerebrospinal fluid (CSF) concentrations of proinflammatory and anti-inflammatory cytokines produced by T regulatory (Treg) cells in early RRMS according to the 2017 McDonald criteria. CLINICAL RATIONALE FOR THE STUDY: Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS) with the cytokine network playing an important role. However, there is a continual lack of data regarding the immunopathogenesis of early RRMS, especially according to the 2017 McDonald criteria. MATERIALS AND METHODS: The study groups included early RRMS patients during relapse (n = 18), remission (n = 14), and the control group. The MS diagnosis was established according to the 2017 McDonald criteria. Patients were studied up to 1 year after diagnosis was made. A quantitative test kit based on ELISA was used for cytokine measurement in the serum and CSF. Comparative and correlation analyses between the levels of TNF-α, TGF-ß2, IgG index, and relapse duration were performed. RESULTS: Significantly higher CSF concentrations of TNF-α in both RRMS-relapse and RRMS-remission groups were found compared to the controls (p < 0.01). The CSF levels of TGF-ß2 in the RRMS-relapse group were significantly lower in comparison to the control group (p = 0.01). CONCLUSIONS AND CLINICAL IMPLICATIONS: An inappropriate inflammatory response seems to occur in early RRMS and includes the production of TNF-α and a decrease in TGF-ß2 release suggesting a significant Treg cells role. Further studies on the topic may contribute to developing new disease-modifying drugs and biochemical markers of the disorder.

Concomitant Expression of IL-6 and TGF-ß Cytokines and their Receptors in Peripheral Blood of Patients with Multiple Sclerosis: The Effects of INFß Drugs.

BACKGROUND: Concomitant signals from IL-6 and TGF-ß have a central role in the Th17 cells development and differentiation, and these cells are the main promoters of demyelinating inflammation in the central nervous system (CNS) resulting in multiple sclerosis (MS). OBJECTIVES: To evaluate the simultaneous IL-6 and TGF-ß gene and their receptor protein expression in patients with Relapsing-Remitting (RR)-MS. MATERIALS AND METHODS: IL-6 and TGF-ß mRNA and their receptor expression on the surface of CD4+T cells were evaluated using real-time PCR (RT-PCR) and flow cytometry, respectively. RESULTS: The IL-6 mRNA expression in patients with RRMS was significantly higher than in the controls (p= 0.019). When patients who did not receive any other treatment were compared with the controls, the significant difference was substantial (p=0.006). The TGF-ß mRNA expression in patients was lower than in the controls (p = 0.03). However, in patients receiving IFNß, it increased compared with the other patients (p= 0.036). There was no difference in cytokine receptor expression between patients and the control group. CONCLUSION: Our data conclude an increase and decrease in mRNA expression levels of IL-6 and TGF-ß in patients with RRMS, respectively. Moreover, there were no significant differences in receptor expression of either cytokines. Based on our data the balance of TGF and IL-6 appears to have a positive impact on the disease control.

Serum levels of lipocalin-2 are elevated at early times in African American relapsing remitting multiple sclerosis patients.

Previous studies showed that depleting Liver Kinase-B1 (LKB1) from astrocytes increased inflammatory factors lipocalin-2 (LCN2) and osteopontin (OPN) in EAE. A single nucleotide polymorphism (SNP) in STK11 (encoding LKB1) is a risk factor for MS, suggesting increased LCN2 or OPN contributes to risk. Serum LCN2 and OPN levels in African American female MS patients were higher than healthy controls, and while levels increased with disease duration in cases without the SNP, levels decreased with duration in cases with the SNP. Increased MS risk associated with the STK11 SNP may be due to higher LCN2 or OPN levels at early times.

Melanoma Cell Adhesion Molecule Expressing Helper T Cells in CNS Inflammatory Demyelinating Diseases.

BACKGROUND AND OBJECTIVE: To elucidate the relationship between melanoma cell adhesion molecule (MCAM)-expressing lymphocytes and pathogenesis of CNS inflammatory demyelinating diseases (IDDs). METHODS: Patients with multiple sclerosis (MS) (n = 72) and neuromyelitis optica spectrum disorder (NMOSD, n = 29) were included. We analyzed the frequency and absolute numbers of MCAM+ lymphocytes (memory helper T [mTh] cells, naive helper T cells, CD8+ T cells, and B cells) in the peripheral blood (PB) and the CSF of patients with MS and NMOSD, treated with/without disease-modifying drugs (DMDs) or steroids, using flow cytometry. RESULTS: The frequency of MCAM+ cells was higher in the mTh cell subset than that in other lymphocyte subsets. A significant increase in the frequency and the absolute number of MCAM+ mTh cells was observed in the PB of patients with NMOSD, whereas no increase was observed in the PB of patients with MS. The frequency of CSF MCAM+ mTh cells was higher in relapsing patients with MS and NMOSD than that in the control group. Although there was no difference in the frequencies of MCAM+ lymphocytes among the DMD-treated groups, fingolimod decreased the absolute number of MCAM+ lymphocytes. DISCUSSION: MCAM+ mTh cells were elevated in the CSF of relapsing patients with MS and in both the PB and CSF of patients with NMOSD. These results indicate that MCAM contributes to the pathogenesis of MS and NMOSD through different mechanisms. MCAM could be a therapeutic target of CNS IDDs, and further study is needed to elucidate the underlying mechanism of MCAM in CNS IDD pathogenesis.

Transcriptome and Function of Novel Immunosuppressive Autoreactive Invariant Natural Killer T Cells That Are Absent in Progressive Multiple Sclerosis.

BACKGROUND AND OBJECTIVE: The aim of this study was to determine whether natural killer T (NKT) cells, including invariant (i) NKT cells, have clinical value in preventing the progression of multiple sclerosis (MS) by examining the mechanisms by which a distinct self-peptide induces a novel, protective invariant natural killer T cell (iNKT cell) subset. METHODS: We performed a transcriptomic and functional analysis of iNKT cells that were reactive to a human collagen type II self-peptide, hCII707-721, measuring differentially induced genes, cytokines, and suppressive capacity. RESULTS: We report the first transcriptomic profile of human conventional vs novel hCII707-721-reactive iNKT cells. We determined that hCII707-721 induces protective iNKT cells that are found in the blood of healthy individuals but not progressive patients with MS (PMS). By transcriptomic analysis, we observed that hCII707-721 promotes their development and proliferation, favoring the splicing of full-length AKT serine/threonine kinase 1 (AKT1) and effector function of this unique lineage by upregulating tumor necrosis factor (TNF)-related genes. Furthermore, hCII707-721-reactive iNKT cells did not upregulate interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-13, or IL-17 by RNA-seq or at the protein level, unlike the response to the glycolipid alpha-galactosylceramide. hCII707-721-reactive iNKT cells increased TNFα only at the protein level and suppressed autologous-activated T cells through FAS-FAS ligand (FAS-FASL) and TNFα-TNF receptor I signaling but not TNF receptor II. DISCUSSION: Based on their immunomodulatory properties, NKT cells have a potential value in the treatment of autoimmune diseases, such as MS. These significant findings suggest that endogenous peptide ligands can be used to expand iNKT cells, without causing a cytokine storm, constituting a potential immunotherapy for autoimmune conditions, including PMS.

Fingolimod Therapy in Multiple Sclerosis Leads to the Enrichment of a Subpopulation of Aged NK Cells.

Fingolimod is an approved oral treatment for relapsing-remitting multiple sclerosis (RRMS) that modulates agonistically the sphingosin-1-phosphate receptor (S1PR), inhibiting thereby the egress of lymphocytes from the lymph nodes. In this interventional prospective clinical phase IV trial, we longitudinally investigated the impact of fingolimod on frequencies of NK cell subpopulations by flow cytometry in 17 RRMS patients at baseline and 1, 3, 6, and 12 months after treatment initiation. Clinical outcome was assessed by the Expanded Disability Status Scale (EDSS) and annualized relapse rates (ARR). Over the study period, median EDSS remained stable from month 3 to month 12, and ARR decreased compared to ARR in the 24 months prior treatment. Treatment was paralleled by an increased frequency of circulating NK cells, due primarily to an increase in CD56dimCD94low mature NK cells, while the CD56bright fraction and CD127+ innate lymphoid cells (ILCs) decreased over time. An unsupervised clustering algorithm further revealed that a particular fraction of NK cells defined by the expression of CD56dimCD16++KIR+/-NKG2A-CD94-CCR7+/-CX3CR1+/-NKG2C-NKG2D+NKp46-DNAM1++CD127+ increased during treatment. This specific phenotype might reflect a status of aged, fully differentiated, and less functional NK cells. Our study confirms that fingolimod treatment affects both NK cells and ILC. In addition, our study suggests that treatment leads to the enrichment of a specific NK cell subset characterized by an aged phenotype. This might limit the anti-microbial and anti-tumour NK cell activity in fingolimod-treated patients.

Serum endocan levels in multiple sclerosis relapse and remission.

OBJECTIVE: Endocan has been defined as an important marker of inflammatory diseases, vascular and endothelial injury, tumour progression, cell adhesion and angiogenesis. In our study, we compared the serum endocan, C-reactive protein (CRP) and neutrophil-lymphocyte ratio (NLR) levels of relapsing-remitting multiple sclerosis (RRMS) patients in remission and in relapse. PATIENTS AND METHODS: This study included 53 RRMS remission patients, 30 RRMS relapse/post-relapse patients and 44 healthy volunteers. Blood samples were collected once from RRMS patients in remission and from the control group, and twice from RRMS relapse patients: once when relapsing and another 1 month after relapse. The endocan, CRP and NLR levels of the RRMS patients measured while in relapse, 1 month after relapse and while in remission were compared to those of the control group. The studied parameters were compared with the disease duration, relapse frequency, Expanded Disability Status Scale (EDSS) score, applied treatment and lesion burden assessed using magnetic resonance imaging (MRI). RESULTS: The endocan, CRP and NLR levels were significantly higher in the RRMS group than in the control group (p < 0.05). The serum endocan levels were found to be significantly higher in the RRMS relapse group than in the post-relapse and control groups (p < 0.05). There were no significant correlations between the disease duration, EDSS score, relapse frequency and lesion burden on MRI and the endocan, CRP and NLR values (p > 0.05). According to the correlation analysis, there was a statistically strong positive relationship between the MRI lesion localisation and the EDSS score, disease duration and relapse frequency (p < 0.001). CONCLUSIONS: Endocan increase is a marker of the endothelial injury that develops secondary to the inflammatory process in MS patients. It can thus be considered a moderately good indicator of relapse.

Exosomes From Subjects With Multiple Sclerosis Express EBV-Derived Proteins and Activate Monocyte-Derived Macrophages.

OBJECTIVE: To investigate in a cross-sectional study the effect of serum-derived exosomes on primary human blood monocyte-derived macrophages (MDMs) comparing exosomes from healthy donors vs patients with relapsing-remitting multiple sclerosis in remission and in relapse and to assess whether the response correlates with exosomal Epstein-Barr virus (EBV) protein expression. METHODS: A total of 45 serum-derived exosome preparations were isolated from patients and healthy controls and verified for the expression of exosomal and EBV markers. MDMs were differentiated from monocytes for 7 days and incubated for 24 hours with exosomes, and then, cell supernatants were collected for cytokine measurement by cytometric bead array. Cells were immunophenotyped before and after differentiation. RESULTS: Serum-derived exosomes of patients with multiple sclerosis (MS) expressed higher levels of EBV proteins than healthy controls. Of interest, expression of EBV nuclear antigen EBNA1 and latent membrane proteins LMP1 and 2A was higher on exosomes derived from patients with active RRMS compared with healthy controls and stable patients. After data normalization, we observed that incubation with EBV(+) exosomes induced CXCL10 and CCL2 secretion by MDMs. MDMs differentiated from patients with active disease were better secretors of CXCL10 and other interferon-γ-inducible chemokines, including CCL2 and CXCL9, than MDMs from healthy and stable MS groups. MDMs from active patients had a higher frequency of a CD14(++) subset that correlated with the secreted CXCL10. CONCLUSION: Exosomes expressing EBV proteins correlate with disease activity and induce an inflammatory response in MDMs that is compounded by the origin of the responder cells.

Serum Neurofilament Levels and PML Risk in Patients With Multiple Sclerosis Treated With Natalizumab.

OBJECTIVES: The study aimed to assess the potential for serum neurofilament light chain (NFL) levels to predict the risk of progressive multifocal leukoencephalopathy (PML) in natalizumab (NTZ)-treated patients with multiple sclerosis (MS) and to discriminate PML from MS relapses. METHODS: NFL levels were measured with single molecule array (Simoa) in 4 cohorts: (1) a prospective cohort of patients with MS who developed PML under NTZ therapy (pre-PML) and non-PML NTZ-treated patients (NTZ-ctr); (2) a cohort of patients whose blood was collected during PML; (3) an independent cohort of non-PML NTZ-treated patients with serum NFL determinations at 2 years (replication cohort); and (4) a cohort of patients whose blood was collected during exacerbations. RESULTS: Serum NFL levels were significantly increased after 2 years of NTZ treatment in pre-PML patients compared with NTZ-ctr. The prognostic performance of serum NFL levels to predict PML development at 2 years was similar in the NTZ-ctr group and replication cohort. Serum NFL levels also distinguished PML from MS relapses and were 8-fold higher during PML compared with relapses. CONCLUSIONS: These results support the use of serum NFL levels in clinical practice to identify patients with relapsing-remitting MS at higher PML risk and to differentiate PML from clinical relapses in NTZ-treated patients. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that serum NFL levels can identify NTZ-treated patients with MS who will develop PML with a sensitivity of 67% and specificity of 80%.

Differential Function of a Novel Population of the CD19+CD24hiCD38hi Bregs in Psoriasis and Multiple Myeloma.

Psoriasis (Ps), an autoimmune disease, and multiple myeloma (MM), a blood neoplasm, are characterized by immune dysregulation resulting from the imbalance between the effector and regulatory cells, including B regulatory (Breg) lymphocytes. Peripheral blood samples from 80 Ps patients, 17 relapsed/refractory MM patients before and after daratumumab (anti-CD38 monoclonal antibody) treatment, 23 healthy volunteers (HVs), and bone marrow samples from 59 MM patients were used in the study. Bregs were determined by flow cytometry using CD19, CD24, and CD38. Intracellular production of interleukin-10 (IL-10) was assessed by flow cytometry after CD40L, LPS, and CpG stimulation. IL-10 serum or plasma concentrations were tested using ELISA method. The percentage of CD19+CD24hiCD38hi Bregs was not different whereas the production of IL-10 in Bregs was significantly higher in Ps patients in comparison with HVs. The percentage of CD19+CD24hiCD38hi Bregs in MM patients was significantly higher than in HVs (p < 0.0001). The percentage of CD19+CD24hiCD38hi Bregs was significantly higher in MM patients with the ISS stage I (p = 0.0233) while IL-10 production in Bregs was significantly higher in ISS stage III (p = 0.0165). IL-10 serum or plasma concentration was significantly higher in Ps and MM patients when compared to HVs (p < 0.0001). Following the treatment with daratumumab the percentages of CD19+CD24hiCD38hi Bregs significantly decreased (p < 0.0003). Here, in the two opposite immune conditions, despite the differences in percentages of Bregs in Ps and MM we have identified some similarities in the IL-10 producing Bregs. Effective treatment of daratumumab besides the anti-myeloma effect was accompanied by the eradication of Bregs.

Urokinase, CX3CL1, CCL2, TRAIL and IL-18 induced by interferon-ß treatment.

OBJECTIVE: To identify serum proteins associated with MS and affected by interferon beta treatment. METHODS: Plasma samples from 29 untreated relapsing-remitting MS patients and 15 healthy controls were investigated with a multiplexed panel containing 92 proteins related to inflammation. Follow-up samples were available from 13 patients at 1 and 3 months after initiation of treatment with interferon beta-1a. RESULTS: Ten proteins were differentially expressed in MS patients. Five of these were altered by treatment with IFN-ß 1a: uPA, CX3CL1, CCL2, TRAIL and IL18. CONCLUSION: CCL2 and TRAIL were confirmed to be modulated with interferon beta treatment in MS. As novel findings, we now report that uPA and CX3CL1 were differentially expressed in MS and increased after IFN-beta-1a treatment. Conflicting results have been reported on how interferon beta affects IL-18.

Association of polymorphism -308G/A in tumor necrosis factor-alpha gene (TNF-α) and TNF-α serum levels in patients with relapsing-remitting multiple sclerosis.

TNF-α is an important cytokine of the inflammatory response involved in the pathogenesis of relapsing-remitting multiple sclerosis (RRMS). The aim of this study is to explore the association between the promoter polymorphism -308G/A in the TNF-α gene (rs1800629) with genetic susceptibility to RRMS.Methods: A group of 183 RRMS patients and 169 age and gender-matched healthy controls were enrolled in the study. Genotyping of the polymorphism was performed by PCR-restriction fragment length polymorphism and quantification of TNF-α serum levels was conducted by ELISA.Results: The genotype distribution in female patients showed a significantly elevated frequency of heterozygotes (AG) (23.5% vs. 12.8%, OR = 2.072, p = 0.029) in comparison with the healthy women. Substantially higher TNF-α serum levels were observed in females compared to males, in both patients and healthy controls (p < 0.05). According to the genotype, TNF-α levels in the RRMS group were calculated in the following order: for GA/AA genotypes (5.67pg/ml vs. 3.48pg/ml, p = 0.0031) and for GG genotypes (4.58pg/ml vs. 3.52pg/ml, p = 0.00043). Moreover, the carriers of at least one A-allele of -308G/A TNF-α polymorphism (GA+AA) are significantly associated with two fold increased risk for RRMS development (OR = 1.950; p = 0.042) in women in contrast to men as well as associated with early onset of the disease (OR = 2.400; p = 0.021).Conclusion: Our study showed that the level of TNF-α in the serum of patients with RRMS showed a significant association with the -308G/A TNF-α polymorphism and gender dependency.

Guidelines for analysis of low-frequency antigen-specific T cell results: Dye-based proliferation assay vs 3H-thymidine incorporation.

It is generally recognized that dysregulation of the immune system plays a critical role in many diseases, including autoimmune diseases and cancer. T cells play a crucial role in maintaining self-tolerance, while loss of immune tolerance and T cell activation can lead to severe inflammation and tissue damage. T cell responses have a key role in the effectiveness of vaccination strategies and immunomodulating therapies. Immunomonitoring methods have the ability to elucidate immunological processes, monitor the development of disease and assess therapeutic effects. In this respect, it is of particular interest to evaluate antigen (Ag)-specific T cells by determining their frequency, type and functionality in cellular assays. Nevertheless, Ag-specific T cells are detected infrequently in most diseases using current techniques. Many efforts have been made to develop more sensitive, reproducible, and reliable methods for Ag-specific T cell detection. It has been found that analysis of cellular proliferation can be a useful tool to determine the presence and frequency of Ag-specific T cell and to provides insight into modulation of the T cell response by a specific antigen or therapy. However, the selection of a cut-off value for a positive response and therefore a more accurate interpretation of the data, continues to be a major concern. Here, we provide guidelines to select a proper cut-off for monitoring of Ag-specific CD4+ T cell responses. In vitro Ag-stimulation has been assessed with two methods; a dye-based proliferation assay and 3H-thymidine-based assay. Two cut-off approaches are compared; mean and variance of control wells, and the stimulation index. By evaluating the proliferative response to the in vitro Ag-stimulation using these two methods, we demonstrate the importance of taking into consideration the variability of the control wells to distinguish a positive from a false positive response.

Effects of Alemtuzumab on (Auto)antigen-Specific Immune Responses.

Alemtuzumab (anti-CD52 mAb) leads to a long-lasting disease activity suppression in patients with relapsing forms of multiple sclerosis (MS). In this study, we examined the change of the immune cell repertoire and the cellular reactivity after treatment with alemtuzumab. We analyzed the number of IFN-γ-secreting cells in presence of several peptides which had been eluted from the central nervous system (CNS) of MS patients and are possible targets of autoreactive T cells in MS. The patients showed a stabilized disease activity measured in clinical parameters and lesion formation after the treatment. We detected a reduction of the number of IFN-γ-secreting cells in the presence of every tested self-antigen. The number of IFN-γ-secreting cells was also reduced in the presence of non-self-antigens. We also found a clear change in the immune cell repertoire. After an almost complete depletion of all lymphocytes, the cell specificities showed different reconstitution patterns, resulting in different cell fractions. The percentage of CD4+ T cells was clearly reduced after therapy, whereas the fractions of B and NK cells were elevated. When we evaluated the number of IFN-γ-secreting cells in relation to the number of present CD4+ T cells, we still found a significant reduction. We conclude that the reduction of IFN-γ-secreting cells by alemtuzumab is not only due to a reduction of the CD4+ T cell fraction within the peripheral blood mononuclear cell (PBMC) compartment but might also be caused by functional changes or a shift in the distribution of different subtypes in the CD4+ T cell pool.

Adenosine-to-Inosine RNA Editing of Alu Double-Stranded (ds)RNAs Is Markedly Decreased in Multiple Sclerosis and Unedited Alu dsRNAs Are Potent Activators of Proinflammatory Transcriptional Responses.

Sensors that detect dsRNA stimulate IFN responses as a defense against viral infection. IFN responses are also well documented in a variety of human autoimmune diseases, including relapsing-remitting multiple sclerosis (MS), in which increased IFN responses result from increased levels of double-stranded endogenous Alu RNAs. Mechanisms underlying increases in double-stranded Alu RNAs in MS are obscure. We find widespread loss of adenosine-to-inosine editing of Alu RNAs in MS. Unedited Alu RNAs are potent activators of both IFN and NF-κB responses via the dsRNA sensors, RIG-I, and TLR3. Minor editing of highly active Alu elements abrogates the ability to activate both transcriptional responses. Thus, adenosine-to-inosine editing may also represent an important defense against autoimmune diseases such as MS.

Association between TNFA, IL10 and IL18 promoter gene variants and cognitive functions in patients with relapsing-remitting multiple sclerosis.

OBJECTIVES: To investigate the relationship between TNFA-308G > A, IL10-1082A > G, IL18-607C > A, and cognitive functioning in relapsing-remitting multiple sclerosis (RRMS). RESULTS: In the patients' group: AG genotype of TNFA-308G > A was associated with higher serum tumor necrosis factor-alpha (TNF-alpha) than GG genotype, and higher TNF-alpha levels correlated with poorer results on Symbol Digit Modalities Test; CC genotype of IL18-607C > A was related to lower score on Isaacs test, compared to AC variant; AA genotype of IL10-1082A > G was associated with abnormally low results on Paced Auditory Series Addition Test. CONCLUSIONS: TNFA-308G > A, IL10-1082A > G and IL18-607C > A gene variants may be associated with impaired cognitive functions in RRMS patients.

EBV-specific CD8 T lymphocytes and B cells during glatiramer acetate therapy in patients with MS.

OBJECTIVE: Infection with Epstein-Barr virus (EBV) has been associated with clinical activity and risk of developing MS. The purpose of this study is to investigate the impact of glatiramer acetate (GA) therapy on EBV-specific immune responses and disease course. METHODS: We characterized EBV-specific CD8 T lymphocytes and B cells during disease-modifying treatments in 2 groups of patients with MS. We designed a 2-pronged approach consisting of a cross-sectional study (39 untreated patients, 38 patients who had undergone 12 months of GA treatment, and 48 healthy donors compatible for age and sex with the patients with MS) and a 12-month longitudinal study (35 patients treated with GA). CD8 EBV-specific T cells and B lymphocytes were studied using pentamers and multiparametric flow cytometry. RESULTS: We find that treatment with GA enhances viral recognition by inducing an increased number of circulating virus-specific CD8 T cells (p = 0.0043) and by relieving their features of exhaustion (p = 0.0053) and senescence (p < 0.0001, p = 0.0001). B cells, phenotypically and numerically tracked along the 1-year follow-up study, show a steady decrease in memory B-cell frequencies (p = 0.025), paralleled by an increase of the naive B subset. CONCLUSION: GA therapy acts as a disease-modifying therapy restoring homeostasis in the immune system, including anti-EBV responses.

Effect of combined exercise training on pentraxins and pro- inflammatory cytokines in people with multiple sclerosis as a function of disability status.

OBJECTIVES: There is some evidence for beneficial effects of exercise on cytokines in people with multiple sclerosis (MS), but it is unclear if such effects differ by disability status (i.e., stage of the disease). This study investigated the effect of combined exercise training on pentraxins and pro- inflammatory cytokines in people with multiple sclerosis as a function of disability status. METHODS: This randomized control trial included 94 women with MS who were randomly assigned into exercise or control conditions with randomization stratified by Expanded Disability Status Scale (EDSS) scores of low (EDSS < 4.5), moderate (4.5 ≤ EDSS ≤ 6), or high (EDSS ≥ 6.5) disability. The exercise program lasted 12 weeks and comprised resistance, endurance, Pilates, balance and stretch exercises performed? days/week; the control condition involved a waitlist control. We measured resting levels of inflammatory factors, functional capacity, and lipid profile before and after the 12-week intervention period. RESULTS: Combined exercise training significantly decreased hs-CRP (p = 0.029) and IL-6 (p = 0.001) and increased PTX-3 (p = 0.001) and IFN-Æ´ (p = 0.001), but there was no significant change in Fibrin D-dimer (FDD) (p = 0.876) compared with control, and those effects were independent of disability status. 1RM for lat pull-down, knee extension, and seated row and 6MWT (i.e., walking further) significantly increased and TUG performance significantly decreased (i.e., faster performance) (all, p < 0.001) after combined exercise compared with control, and this too was independent of disability status. CONCLUSIONS: Exercise may stimulate anti-inflammatory effects in MS, and this is generally not influenced by disability status. Exercise training may be an adjuvant for disease-modifying therapy among people with MS, and such effects might not be moderated by disability status.