To identify differentially expressed genes in multiple sclerosis, microarrays were used in a stringent experimental setting—leukapheresis from disease-discordant monozygotic twins and gene expression profiling in CD4⁺ and CD8⁺ T-cell subsets. Disease-related differences emerged only in the CD8⁺ T-cell subset. The five differentially expressed genes identified included killer cell lectin-like receptor subfamily B, member 1, also known as natural killer receptor protein 1a/CD161, presented by the International Multiple Sclerosis Genetics Consortium as one of the non-MHC candidate loci. Flow cytometric analysis on peripheral blood of healthy donors and patients with multiple sclerosis and rheumatoid arthritis confirmed an upregulation of CD161 at the protein level, showing also a significant excess of CD161^highCD8⁺ T cells in multiple sclerosis. This subset prevalently included chemokine (C-C motif) receptor 6⁺, cytokine-producing, effector-memory T cells with proinflammatory profiles. It also included all circulating interleukin-17⁺CD8⁺ T cells. In the CD161^highCD8⁺ subset, interleukin-12 facilitated proliferation and interferon-γ production, with CD161 acting as a co-stimulatory receptor. CD161⁺CD8⁺CD3⁺ T cells producing interferon-γ were part of intralesional immune infiltrates and ectopic B cell follicles in autopsy multiple sclerosis brains. Variations of CD161 expression on CD8⁺ T cells identify a subset of lymphocytes with proinflammatory characteristics that have not been previously reported in multiple sclerosis and are likely to contribute to disease immunopathology.

It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse. There is general consensus that multiple sclerosis (MS) is associated with Epstein-Barr virus (EBV) infection but the mechanistic links are still debated. EBV is a B-lymphotropic herpesvirus widespread in the human population and normally contained as a persistent, asymptomatic infection by immune surveillance. However, EBV can cause infectious mononucleosis, is associated with numerous human malignancies, and is implicated in some common autoimmune diseases. While EBV infection alone cannot explain MS development, it has been postulated that in susceptible individuals alterations in the mechanisms regulating the immune response to the virus may contribute to MS pathogenesis. Here, we show that MS patients with inactive disease exhibit a lower CD8+ T-cell response to EBV when compared to healthy donors and active MS patients while the latter have a higher frequency of CD8+ T cells specific for EBV lytic antigens. Therapy with interferon-β and natalizumab, two treatments for relapsing-remitting MS, was associated with marked changes in the EBV specific CD8+ T cell response. We also demonstrate that one of the EBV lytic antigens recognized by CD8+ T cells expanding in the blood during active MS is expressed in the inflamed MS brain. Our results support a model of MS pathogenesis in which EBV infection and reactivation in the CNS stimulates an immunopathological response and suggest that antiviral or immunomodulatory therapies aimed at restoring the host-EBV balance could be beneficial to MS patients.

Alterations of intestinal permeability (IP) may contribute to the pathophysiology of immune-mediated diseases. We investigated the possible association between IP changes and multiple sclerosis (MS). We studied 22 patients with relapsing–remitting multiple sclerosis (RRMS) and 18 age- and sex-matched healthy donors (HDs), including five twin pairs (one concordant, and four discordant for disease). Measurement of lactulose (L) and mannitol (M; two non-metabolized sugars) levels in urine samples, after an oral load, allowed to quantify gut dysfunction. The proportion of participants with increased IP was significantly higher in patients than in HDs (16/22 (73%) versus 5/18 (28%); p = 0.001). Accordingly, the L/M urinary ratio showed significantly higher values in patients than in controls (p = 0.0284). Urinary mannitol concentration was significantly lower in patients than in controls (p = 0.022), suggesting a deficit of absorption from intestinal lumen. Such changes did not appear related to patients’ clinical–radiological features. The relatively high proportion of IP changes in RR-MS patients seems to confirm our work hypothesis and warrants more work to confirm the result on a larger sample, and to understand the implications for related immunological disturbances and intestinal microbiota alterations. Our finding may also have relevance for oral treatments, recently introduced in clinical practice.

Background: Peptidylarginine deiminase 2 (PAD2) and peptidylarginine deiminase 4 (PAD4) are two members of PAD family which are over-expressed in the multiple sclerosis (MS) brain. Through its enzymatic activity PAD2 converts myelin basic protein (MBP) arginines into citrullines – an event that may favour autoimmunity – while peptidylarginine deiminase 4 (PAD4) is involved in chromatin remodelling.