Epstein-Barr virus (EBV) infection shows the strongest causative association with multiple sclerosis (MS), but its contribution to disease progression and the mechanisms allowing for viral persistence in the MS brain are still elusive. Studies in post-mortem MS brain tissue indicate an ongoing yet ineffective antiviral immune reaction in advanced stages of the disease. EBV has evolved strategies to evade immune recognition and clearance by the host immune system during both the latency and lytic phase of its life cycle. Recent evidence demonstrates that cells expressing EBV latent membrane protein (LMP) 2A exploit the PD-1/PDL1 inhibitory immune checkpoint to escape immune surveillance and maintain a persistent latent infection in the MS brain. This study investigated whether the virus also utilizes this inhibitory mechanism during other phases of the viral life cycle. By using multiple immunostainings on highly inflamed MS brain tissues containing meningeal tertiary lymphoid structures (TLSs), we analyzed PD-L1 expression on EBV-infected cells expressing EBNA2, five EBV lytic gene products, BZLF1, BHRF1, BMRF1, BALF2, and gp350/220, as well as on follicular dendritic cells within the TLSs. This is the first study describing in secondary progressive MS brain tissue the expression and the cellular and tissue distribution of PD-L1 on EBV-infected cells being in different stages of the viral life cycle, and confirms the meningeal TLSs as immune-permissive habitats favoring the maintenance of an intracerebral EBV reservoir.

Regulatory T-cells (Tregs) maintain immune homeostasis, but antigens activating adaptive Tregs in human pathologies are ill-defined. EOMES+type-1 regulatory (EOMES+Tr1-like) T-cells had a dysregulated homeostasis in multiple sclerosis (MS), which was related to their activation in the central nervous system (CNS). EOMES+Tr1-like cells were strongly enriched in patient's cerebrospinal fluid (CSF) and were the main IL-10-producing T-cells in the CSF. IL-10+EOMES+Tr1-like were present in MS brain lesions, and some were found close to Epstein-Barr Virus (EBV)-infected B-cells. EOMES+Tr1-like cells and FOXP3+Tregs produced IL-10 with EBV-derived antigens, but not with major myelin antigens. EOMES+Tr1-like cells responded selectively to the latency-associated antigen EBNA1, whereas FOXP3+Tregs responded also to lytic antigens. EBNA1-specific EOMES+Tr1-like cells were associated with anti-EBNA1 IgG, disappeared upon therapeutic B-cell depletion and were relatively abundant in patients with the HLA-DRB1*15 risk haplotype. The aberrant anti-viral specificities of Tregs in MS could undermine their protective functions and anti-EBV immune surveillance. ### Competing Interest Statement The authors have declared no competing interest.

The presence of EBV infected B cells in postmortem multiple sclerosis (MS) brain tissue suggests immune evasion strategies. Using immunohistochemical techniques we analysed the expression of the immune checkpoint molecule PD-L1 and its receptor PD-1 in MS brains containing B cell-enriched perivascular infiltrates and meningeal follicles, a major EBV reservoir. PD-1 and PD-L1 immunoreactivities were restricted to CNS-infiltrating immune cells. PD-L1 was expressed on B cells, including EBV infected B cells, while PD-1 was expressed on many CD8+ T cells, including EBV-specific CD8+ T-cells, and fewer CD4+ T cells. PD-L1+ cells and EBV infected cells were in close contact with PD-1+ T cells. PD-L1 expressed by EBV infected B cells could favour local immune evasion leading to EBV persistence and immunopathology in the MS brain.

Presence of EBV infected B cells and EBV-specific CD8 T cells in the multiple sclerosis (MS) brain suggests a role for virus-driven immunopathology in brain inflammation. Tissue-resident memory (Trm) T cells differentiating in MS lesions could provide local protection against EBV reactivation. Using immunohistochemical techniques to analyse canonical tissue residency markers in postmortem brains from control and MS cases, we report that CD103 and/or CD69 are mainly expressed in a subset of CD8+ T cells that intermingle with and contact EBV infected B cells in the infiltrated MS white matter and meninges, including B-cell follicles. Some Trm-like cells were found to express granzyme B and PD-1, mainly in white matter lesions. In the MS brain, Trm cells could fail to constrain EBV infection while contributing to sustain inflammation.

The EBV as the 'gluten of MS' hypothesis discussed by Drosu et al. in a recent Editorial envisages the existence of similar mechanisms leading to celiac disease and multiple sclerosis, such as induction of immunity against an ubiquitous exogenous antigen - gluten and EBV, respectively - and subsequent development of autoimmunity that is maintained by persistence of the initial trigger. While this hypothesis provides the rationale for treating MS with antivirals to lower EBV load, it can be misleading when trying to translate concepts of T cell-B cell interaction and autoimmunity development in celiac disease to multiple sclerosis. Here, we propose that EBV might act as the driver of multiple sclerosis without involving autoimmunity.

Signaling from central nervous system (CNS)-infiltrating lymphocytes and macrophages is critical to activate microglia and cause tissue damage in multiple sclerosis (MS). We combined laser microdissection with high-throughput real time RT-PCR to investigate separately the CNS exogenous and endogenous inflammatory components in postmortem brain tissue of progressive MS cases. A previous analysis of immune infiltrates isolated from the white matter (WM) and the meninges revealed predominant expression of genes involved in antiviral and cytotoxic immunity, including IFNγ and TNF. Here, we assessed the expression of 71 genes linked to IFN and TNF signaling and microglia/macrophage activation in the parenchyma surrounding perivascular cuffs at different stages of WM lesion evolution and in gray matter (GM) lesions underlying meningeal infiltrates. WM and GM from non-neurological subjects were used as controls. Transcriptional changes in the WM indicate activation of a classical IFNγ-induced macrophage defense response already in the normal-appearing WM, amplification of detrimental (proinflammatory/pro-oxidant) and protective (anti-inflammatory/anti-oxidant) responses in actively demyelinating WM lesions and persistence of these dual features at the border of chronic active WM lesions. Transcriptional changes in chronic subpial GM lesions indicate skewing toward a proinflammatory microglia phenotype. TNF receptor 2 (TNFR2) mediating TNF neuroprotective functions was one of the genes upregulated in the MS WM. Using immunohistochemistry we show that TNFR2 is highly expressed in activated microglia in the normal-appearing WM, at the border of chronic active WM lesions, and in foamy macrophages in actively demyelinating WM and GM lesions. In lysolecithin-treated mouse cerebellar slices, a model of demyelination and remyelination, TNFR2 RNA and soluble protein increased immediately after toxin-induced demyelination along with transcripts for microglia/macrophage-derived pro- and anti-inflammatory cytokines. TNFR2 and IL10 RNA and soluble TNFR2 protein remained elevated during remyelination. Furthermore, myelin basic protein expression was increased after selective activation of TNFR2 with an agonistic antibody. This study highlights the key role of cytotoxic adaptive immunity in driving detrimental microglia activation and the concomitant healing response. It also shows that TNFR2 is an early marker of microglia activation and promotes myelin synthesis, suggesting that microglial TNFR2 activation can be exploited therapeutically to stimulate CNS repair.

EBV establishes a lifelong and asymptomatic infection in most individuals and more rarely causes infectious mononucleosis and malignancies, like lymphomas. The virus is also strongly associated with MS, a chronic neuroinflammatory disease with unknown etiology. Infectious mononucleosis increases the risk of developing MS, and immune reactivity toward EBV is higher in persons with MS, indicating inadequate control of the virus. Previous studies have suggested that persistent EBV infection in the CNS stimulates an immunopathological response, causing bystander neural cell damage. To verify this, we need to identify the immune culprits responsible for the detrimental antiviral response in the CNS. In this study, we analyzed postmortem brains donated by persons with MS and show that CD8 cytotoxic T cells recognizing EBV enter the brain and interact locally with the virus-infected cells. This antiviral CD8 T cell-mediated immune response likely contributes to MS pathology.

Megalencephalic leukoencephalopathy with subcortical cysts protein-1 (MLC1) is a membrane protein expressed by perivascular astrocytes. MLC1 mutations cause MLC, an incurable leukodystrophy characterized by macrocephaly, brain edema, cysts, myelin vacuolation, and astrocytosis, leading to cognitive/motor impairment and epilepsy. Although its function is unknown, MLC1 favors regulatory volume decrease after astrocyte osmotic swelling and down-regulates intracellular signaling pathways controlling astrocyte activation and proliferation. By combining analysis of human brain tissues with in vitro experiments, here we investigated MLC1 role in astrocyte activation during neuroinflammation, a pathological condition exacerbating patient symptoms. MLC1 upregulation was observed in brain tissues from multiple sclerosis, Alzheimer’s, and Creutzfeld-Jacob disease, all pathologies characterized by strong astrocytosis and release of inflammatory cytokines, particularly IL-1β. Using astrocytoma lines overexpressing wild-type (WT) or mutated MLC1 and astrocytes from control and Mlc1 knock-out (KO) mice, we found that IL-1β stimulated WT-MLC1 plasma membrane expression in astrocytoma cells and control primary astrocytes. In astrocytoma, WT-MLC1 inhibited the activation of IL-1β–induced inflammatory signals (pERK, pNF-kB) that, conversely, were constitutively activated in mutant expressing cells or abnormally upregulated in KO astrocytes. WT-MLC1+ cells also expressed reduced levels of the astrogliosis marker pSTAT3. We then monitored MLC1 expression timing in a demyelinating/remyelinating murine cerebellar organotypic culture model where, after the demyelination and release of inflammatory cytokines, recovery processes occur, revealing MLC1 upregulation in these latter phases. Altogether, these findings suggest that by modulating specific pathways, MLC1 contributes to restore astrocyte homeostasis after inflammation, providing the opportunity to identify drug target molecules to slow down disease progression.

Studying rebound mechanisms in MS patients after interruption of natalizumab may advance our understanding of MS pathogenesis. To verify the role of Epstein-Barr virus (EBV) infection and anti-EBV immunity in post-natalizumab rebound, we analyzed postmortem brain tissue of a case of fatal MS relapse. Using immunohistochemistry, EBV latent, early and late lytic proteins and CD8+ T cells sticking to EBV lytically-infected cells were detected in multiple inflammatory white matter lesions. Using the pentamer technology on brain sections, EBV-specific CD8+ T cells were observed perivascularly. Cell-free EBV DNA was detected in cerebrospinal fluid. These findings confirm and extend the results obtained in another case of post-natalizumab fatal MS relapse, suggesting that this condition represents an EBV-associated immune reconstitution inflammatory syndrome.

BACKGROUND:It is debated whether multiple sclerosis (MS) might result from an immunopathological response toward an active Epstein-Barr virus (EBV) infection brought into the central nervous system (CNS) by immigrating B cells. Based on this model, a relationship should exist between the local immune milieu and EBV infection status in the MS brain. To test this hypothesis, we analyzed expression of viral and cellular genes in brain-infiltrating immune cells.METHODS:Twenty-three postmortem snap-frozen brain tissue blocks from 11 patients with progressive MS were selected based on good RNA quality and prominent immune cell infiltration. White matter perivascular and intrameningeal immune infiltrates, including B cell follicle-like structures, were isolated from brain sections using laser capture microdissection. Enhanced PCR-based methods were used to investigate expression of 75 immune-related genes and 6 EBV genes associated with latent and lytic infection. Data were analyzed using univariate and multivariate statistical methods.RESULTS:Genes related to T cell activation, cytotoxic cell-mediated (or type 1) immunity, B cell growth and differentiation, pathogen recognition, myeloid cell function, type I interferon pathway activation, and leukocyte recruitment were found expressed at different levels in most or all MS brain immune infiltrates. EBV genes were detected in brain samples from 9 of 11 MS patients with expression patterns suggestive of in situ activation of latent infection and, less frequently, entry into the lytic cycle. Comparison of data obtained in meningeal and white matter infiltrates revealed higher expression of genes related to interferonγ production, B cell differentiation, cell proliferation, lipid antigen presentation, and T cell and myeloid cell recruitment, as well as more widespread EBV infection in the meningeal samples. Multivariate analysis grouped genes expressed in meningeal and white matter immune infiltrates into artificial factors that were characterized primarily by genes involved in type 1 immunity effector mechanisms and type I interferon pathway activation.CONCLUSION:These results confirm profound in situ EBV deregulation and suggest orchestration of local antiviral function in the MS brain, lending support to a model of MS pathogenesis that involves EBV as possible antigenic stimulus of the persistent immune response in the central nervous system.

The composition of the intestinal microbiota plays a critical role in shaping the immune system. Modern lifestyle, the inappropriate use of antibiotics, and exposure to pollution have significantly affected the composition of commensal microorganisms. The intestinal microbiota has been shown to sustain inappropriate autoimmune responses at distant sites in animal models of disease, and may also have a role in immune-mediated central nervous system (CNS) diseases such as multiple sclerosis (MS). We studied the composition of the gut mycobiota in fecal samples from 27 persons with MS (pwMS) and in 18 healthy donors (HD), including 5 pairs of homozygous twins discordant for MS. We found a tendency towards higher fungal abundance and richness in the MS group, and we observed that MS twins showed a higher rate of food-associated strains, such as Saccharomyces cerevisiae . We then found that in pwMS, a distinct population of cells with antibacterial and antifungal activity is expanded during the remitting phase and markedly decreases during clinically and/or radiologically active disease. These cells, named MAIT (mucosal-associated invariant T cells) lymphocytes, were significantly more activated in pwMS compared to HD in response to S. cerevisiae and Candida albicans strains isolated from fecal samples. This activation was also mediated by fungal-induced IL-23 secretion by innate immune cells. Finally, immunofluorescent stainings of MS post-mortem brain tissues from persons with the secondary progressive form of the disease showed that MAIT cells cross the blood–brain barrier (BBB) and produce pro-inflammatory cytokines in the brain. These results were in agreement with the hypothesis that dysbiosis of the gut microbiota might determine the inappropriate response of a subset of pathogenic mucosal T cells and favor the development of systemic inflammatory and autoimmune diseases.

Rebound of disease activity in multiple sclerosis patients after natalizumab withdrawal is a potentially life-threatening event. To verify whether highly destructive inflammation after natalizumab withdrawal is associated with Epstein-Barr virus (EBV) reactivation in central nervous system infiltrating B-lineage cells and cytotoxic immunity, we analyzed post-mortem brain tissue from a patient who died during a fulminating MS relapse following natalizumab withdrawal. Numerous EBV infected B cells/plasma cells and CD8+ T cells infiltrated all white matter lesions; the highest frequency of EBV lytically infected cells and granzyme B+ CD8+ T cells was observed in actively demyelinating lesions. These results may encourage switching to B-cell depleting therapy after natalizumab discontinuation.

Ectopic B-cell follicle-like structures (ELS) are found in the meninges of patients with secondary progressive multiple sclerosis (SPMS). Because cells expressing the transcriptional regulator retinoic acid receptor-related orphan receptor-γt (RORγt) and producing interleukin 17 (IL17), e.g. T helper 17 cells and lymphoid tissue inducer (LTi) cells, have been implicated in the formation of ELS, we studied RORγt and IL17 expression in brain tissue from patients with SPMS an assessed their relationships to immune infiltrates and meningeal ELS. By immunohistochemistry, small numbers of RORγt-positive cells were detected in the meninges of 6 of 12 SPMS cases analyzed. RORγt-positive cells were localized in B-cell follicles or aggregates and nearby diffuse meningeal infiltrates, and predominantly co-expressed CD3. Only a few RORγt-positive, CD3-negative cells were observed, suggesting the presence of group 3 innate lymphoid cells, which comprise the LTi cell subset. Some IL17-positive cells, co-expressing in part RORγt and predominantly CD3, were found in meningeal B-cell follicles from 4 SPMS cases. Rare RORγt-positive and IL17-positive cells were detected in white matter. Gene expression analysis of laser dissected meningeal infiltrates and white matter lesions confirmed low frequencies and virtual absence of RORγt and IL17 signals, respectively. Thus, there is selective migration or survival of RORγt-positive cells in MS patient meninges and an association of these cells with ELS.

Objective: We analyzed the Epstein-Barr nuclear antigen 2 (EBNA2) gene, which contains the most variable region of the viral genome, in persons with multiple sclerosis (MS) and control subjects to verify whether virus genetic variants are involved in disease development.Methods: A seminested PCR approach and Sanger sequencing were used to analyze EBNA2 in 53 patients and 38 matched healthy donors (HDs). High-throughput sequencing by Illumina MiSeq was also applied in a subgroup of donors (17 patients and 17 HDs). Patients underwent gadolinium-enhanced MRI and human leucocyte antigen typing.Results: MS risk significantly correlated with an excess of 1.2 allele (odds ratio [OR] = 5.13; 95% confidence interval [CI] 1.84-14.32; p = 0.016) and underrepresentation of 1.3B allele (OR = 0.23; 95% CI 0.08-0.51; p = 0.0006). We identified new genetic variants, mostly 1.2 allele-and MS-associated (especially amino acid variation at position 245; OR = 9.4; 95% CI 1.19-78.72; p = 0.0123). In all cases, the consensus sequence from deep sequencing confirmed Sanger sequencing (including the cosegregation of newly identified variants with known EBNA2 alleles) and showed that the extent of genotype intraindividual variability was higher than expected: rare EBNA2 variants were detected in all HDs and patients with MS (range 1-17 and 3-19, respectively). EBNA2 variants did not seem to correlate with human leucocyte antigen typing or clinical/MRI features.Conclusions: Our study unveils a strong association between Epstein-Barr virus genomic variants and MS, reinforcing the idea that Epstein-Barr virus contributes to disease development.

Although Epstein-Barr virus (EBV) has strong associations with multiple sclerosis (MS), the mechanisms linking EBV infection to CNS pathology remain elusive (1). Selective EBV deregulation in MS is manifested by elevated antibody titers and T-cell responses to EBV but not to other herpes viruses (1, 2). The absence of EBV in the cerebrospinal fluid of most MS patients and the lack of significant differences in blood EBV load between patients and healthy subjects point to a secluded low-grade active EBV infection (2). Our previous studies suggest that EBV may contribute to MS pathology by establishing a persistent infection in the CNS through infiltrating B cells harboring latent and reactivated virus and by stimulating an immunopathologic response (3, 4). Whereas this model remains controversial (5), no data are available on the EBV status of MS patient lymphoid tissue, where most of the viral life cycle occurs. Using in situ hybridization and immunohistochemistry, we have analyzed for the first time EBV in both postmortem CNS and lymph nodes from a patient with primary progressive MS. A 43-year-old woman with primary progressive MS (10-year disease duration) and severe disability (Expanded Disability Status Scale 7.0) died in 2002 because of an ischemic stroke; she underwent autopsy with a postmortem delay of less than 24 hours at the Pathologic Anatomy Institute, Agostino Gemelli Hospital, Rome, Italy. No therapy or comorbidities were reported. Paraffin-embedded tissue blocks from the right cerebral hemisphere, brainstem, and cervical spinal cord, a deep lateral cervical lymph node, and a pulmonary hilar lymph node were analyzed. …