Epstein-Barr Virus-Specific CD8 T Cells Selectively Infiltrate the Brain in Multiple Sclerosis and Interact Locally with Virus-Infected Cells: Clue for a Virus-Driven Immunopathological Mechanism.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus strongly associated with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). However, the mechanisms linking EBV infection to MS pathology are uncertain. Neuropathological and immunological studies suggest that a persistent EBV infection in the CNS can stimulate a CD8 T-cell response aimed at clearing the virus but inadvertently causing CNS injury. Inasmuch as in situ demonstration of EBV-specific CD8 T cells and their effector function is missing, we searched for EBV-specific CD8 T cells in MS brain tissue using the pentamer technique. Postmortem brain samples from 12 donors with progressive MS and known HLA class I genotype were analyzed. Brain sections were stained with HLA-matched pentamers coupled with immunogenic peptides from EBV-encoded proteins, control virus (cytomegalovirus and influenza A virus) proteins, and myelin basic protein. CD8 T cells recognizing proteins expressed in the latent and lytic phases of the EBV life cycle were visualized in white matter lesions and/or meninges of 11/12 MS donors. The fraction (median value) of CD8 T cells recognizing individual EBV epitopes ranged from 0.5 to 2.5% of CNS-infiltrating CD8 T cells. Cytomegalovirus-specific CD8 T cells were detected at a lower frequency (≤0.3%) in brain sections from 4/12 MS donors. CNS-infiltrating EBV-specific CD8 T cells were CD107a positive, suggesting a cytotoxic phenotype, and stuck to EBV-infected cells. Together with local EBV dysregulation, selective enrichment of EBV-specific CD8 T cells in the MS brain supports the notion that skewed immune responses toward EBV contribute to inflammation causing CNS injury.IMPORTANCE 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.

Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis.

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.

Increased CD8+ T cell response to Epstein-Barr virus lytic antigens in the active phase of multiple sclerosis.

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-ß 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.

EBV infected cells in the multiple sclerosis brain express PD-L1: How the virus and its niche may escape immune surveillance.

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.

Tissue-resident memory T cells in the multiple sclerosis brain and their relationship to Epstein-Barr virus infected B cells.

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.

Proinflammatory mucosal-associated invariant CD8+ T cells react to gut flora yeasts and infiltrate multiple sclerosis brain.

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.

Epstein-Barr virus persistence and reactivation in myasthenia gravis thymus.

OBJECTIVE: Increasing evidence supports a link between Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic human herpesvirus, and common B-cell-related autoimmune diseases. We sought evidence of EBV infection in thymuses from patients with myasthenia gravis (MG), an autoimmune disease characterized by intrathymic B-cell activation. METHODS: Seventeen MG thymuses (6 follicular hyperplastic, 6 diffuse hyperplastic, 5 involuted) and 6 control thymuses were analyzed using in situ hybridization for EBV-encoded small RNAs (EBERs), immunohistochemistry for EBV latent and lytic proteins, and polymerase chain reaction for EBV DNA and mRNA. RESULTS: All 17 MG thymuses showed evidence of active EBV infection, whereas none of the control thymuses were infected. Cells expressing EBERs (12 of 17) and EBV latency proteins (EBNA2, LMP1, and LMP2A) (16 of 17) were detected in medullary infiltrates and in germinal centers. Cells expressing early (BFRF1, BMRF1) and late (p160, gp350/220) lytic phase EBV proteins were present in 16 MG thymuses. Latency (EBNA1, LMP2A) or lytic (BZLF1) transcripts (often both) were present in all MG thymuses, and EBV DNA (LMP1 gene) was detected in 13 MG thymuses. We also found CD8+ T cells, CD56 + CD3-natural killer cells, and BDCA-2+ plasmacytoid dendritic cells in immune infiltrates of MG thymuses, but not germinal centers, suggesting an attempt of the immune system to counteract EBV infection. INTERPRETATION: Dysregulated EBV infection in the pathological thymus appears common in MG and may contribute to the immunological alterations initiating and/or perpetuating the disease.

Connecting Immune Cell Infiltration to the Multitasking Microglia Response and TNF Receptor 2 Induction in the Multiple Sclerosis Brain.

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.

DEHP effects on histology and cell proliferation in lung of newborn rats.

Di-(2-ethylhexyl)-phthalate (DEHP), the plasticizer employed in the fabrication of polyvinyl chloride, is known to be released by many medical devices, namely endotracheal tubes currently utilised for pulmonary ventilation of pre-term newborns. When experimentally administered, especially to rodents, the phthalate reportedly causes alterations to several tissues, immature animals being even more responsive targets than adult ones. In the present research, female rats were fed with DEHP in the last week of pregnancy and after delivery, and lung of their pups was morphologically and immunohistochemically analysed. We detected significant alveolar simplification (larger but fewer alveoli with decreased septation), with consequent sensible reduction of gas-exchange surface, at several stages of postnatal development, in distal lung parenchyma of DEHP-treated rats. Moreover, the quantification of PCNA-expressing cells demonstrates that in treated pups the proliferation rates of epithelial and mesenchymal cells progressively increased during the first two postnatal weeks, at difference with controls animals, where the highest proliferation levels were reached at postnatal day 7. The obtained results strongly support the hypothesis that DEHP profoundly affects the alveolarization process in mammalian lung.

Expression of TWEAK and its receptor Fn14 in the multiple sclerosis brain: implications for inflammatory tissue injury.

The expression patterns of tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a pleiotropic cytokine with proinflammatory and cell death-inducing activities, and its receptor, fibroblast growth factor-inducible 14 (Fn14), were examined in postmortem brain tissue samples from patients with multiple sclerosis (MS) and controls. Immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction demonstrated that both TWEAK and Fn14 were upregulated in the MS compared with control unaffected brain samples. Perivascular and meningeal macrophages and astrocytes and microglia associated with lesions were identified as the main sources of TWEAK in the MS brains. The highest frequency of TWEAK+ cells was found at edges of chronic active white matter lesions and in subpial cortical lesions inMS cases with abundant meningeal inflammation and ectopic B-cell follicles. Neurons and reactive astrocytes expressing Fn14 were mainly localized in the cerebral cortex in highly infiltrated MS brains. Numerous TWEAK-expressing microglia were associated with the extensive loss of myelin and astrocytosis, neuronal damage, and vascular abnormalities in subpial cortical lesions; this suggests that TWEAK could synergize with other cytotoxic factors diffusing from the inflamed meninges to promote cortical injury. Taken together, these findings indicate that the TWEAK/Fn14 pathway contributes to inflammation and tissue injury and is, therefore, a potential therapeutic target in MS.

Lymphoid chemokines in chronic neuroinflammation.

Lymphoid chemokines play an essential role in the establishment and maintenance of lymphoid tissue microarchitecture and have been implicated in the formation of tertiary (or ectopic) lymphoid tissue in chronic inflammatory conditions. Here, we review recent advances in lymphoid chemokine research in central nervous system inflammation, focusing on multiple sclerosis and the animal model experimental autoimmune encephalomyelitis. We also highlight how the study of lymphoid chemokines, particularly CXCL13, has led to the identification of intrameningeal B-cell follicles in the multiple sclerosis brain paving the way to the discovery that these abnormal structures are highly enriched in Epstein-Barr virus-infected B cells and plasma cells.

Massive intracerebral Epstein-Barr virus reactivation in lethal multiple sclerosis relapse after natalizumab withdrawal.

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.

Dendritic cells in multiple sclerosis lesions: maturation stage, myelin uptake, and interaction with proliferating T cells.

In multiple sclerosis (MS), dendritic cells (DCs) recruited to the central nervous system (CNS) are thought to be involved in the regulation of autoimmune responses directed against myelin antigens. To better understand the role of DCs in CNS inflammation, we performed a detailed immunohistochemical analysis of DC maturation markers and of DC relationship to CNS-infiltrating T cells in autopsy brain tissue of patients with MS. We also investigated the presence of DCs containing myelin debris in MS lesions. Myeloid DC subsets were identified using the following markers: CD1a for immature DCs; DC-SIGN for immature and mature DCs; and fascin, CD83, DC-LAMP, and CCR7 for mature DCs. The most common finding was the presence of cells expressing DC-SIGN and containing myelin components in the perivascular cuffs of early active and chronic (both active and inactive) MS lesions. Perivascular CD1a DCs were detected in active lesions in only one of 10 patients with MS who were examined. Although less numerous than DC-SIGN DCs, cells expressing mature DC markers were consistently detected in the inflamed meninges and perivascular cuffs of most active lesions examined. CCR7 immunostaining was predominantly confined to activated microglia at the lesion edges. Some perivascular DC-SIGN cells were found in close proximity to or contacting rare proliferating lymphocytes, most of which expressed the DC-SIGN ligand ICAM-3 and CD8. These data suggest that DCs recruited and maturing in MS lesions, where self-antigens are made available by continuous myelin destruction, may contribute to the local activation and expansion of presumably pathogenic T cells.

Suppression of established experimental autoimmune encephalomyelitis and formation of meningeal lymphoid follicles by lymphotoxin beta receptor-Ig fusion protein.

We have recently shown that de novo formation of lymphoid structures resembling B-cell follicles occurs in the inflamed central nervous system (CNS) meninges in a subset of patients with secondary progressive multiple sclerosis and in SJL mice with relapsing-remitting experimental autoimmune encephalomyelitis (EAE). Because lymphotoxin (LT) alpha(1)beta(2) is essential for lymphoid tissue organization, we used real-time PCR to examine LTbeta and LTbeta receptor (LTbetaR) gene expression in the CNS of SJL mice immunized with PLP 139-151 peptide. Moreover, we used the decoy receptor LTbetaR-immunoglobulin fusion protein to block the interaction of lymphotoxin (LT) alpha(1)beta(2) with the LTbeta receptor (LTbetaR) in mice with established EAE and evaluate the effect of systemic and local treatments with the fusion protein on disease progression, CNS lymphocytic infiltration and formation of meningeal B-cell follicles. The present findings indicate that both LTbeta and LTbetaR are upregulated at EAE onset and during subsequent relapses and that systemic and local blockade of the LT pathway with LTbetaR-Ig results in protracted and transient inhibition of EAE clinical signs, respectively. LTbetaR-Ig treatment also reduces T- and B-cell infiltration and prevents the induction of the chemokines CXCL10 and CXCL13 and the formation of organized ectopic follicles in the EAE-affected CNS. Targeting of molecules involved in lymphoid organogenesis could represent a valid strategy to inhibit CNS inflammation and formation of ectopic follicles, which may play a role in maintaining an abnormal, intrathecal humoral immune response in CNS autoimmune disease.

RORγt Expression and Lymphoid Neogenesis in the Brain of Patients with Secondary Progressive Multiple Sclerosis.

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.

Astrocytes produce dendritic cell-attracting chemokines in vitro and in multiple sclerosis lesions.

As a result of their close association with the blood-brain barrier, astrocytes play an important role in regulating the homing of different leukocyte subsets to the inflamed central nervous system (CNS). In this study, we investigated whether human astrocytes produce chemokines that promote the migration of myeloid dendritic cells (DCs). By reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, we show that cultured human astrocytes stimulated with interleukin-1beta and tumor necrosis factor produce CCL2, CCL3, CCL4, CCL5, CCL20, and CXCL12 that act on immature DCs, but not CCL19 and CCL21, 2 chemokines specific for mature DCs. Compared with controls, supernatants of cytokine-stimulated astrocytes are more effective in promoting the migration of immature monocyte-derived DCs (iMDDCs). Desensitization of CXCR4 (receptor for CXCL12), CCR1-3-5 (shared receptors for CCL3-4-5), and CCR6 (receptor for CCL20) on iMDDC reduces cell migration toward astrocyte supernatants, indicating that astrocytes release biologically relevant amounts of iMDDC-attracting chemokines. By immunohistochemistry, we show that CXCL12 and, to a lesser extent, CCL20 are expressed by reactive astrocytes in multiple sclerosis lesions. These data lend support to the idea that astrocyte-derived chemokines may contribute to immature DC recruitment to the inflamed CNS.

Migration of dendritic cells into the brain in a mouse model of prion disease.

The immune system plays a key role in the dissemination of prion infections from the periphery to the central nervous system (CNS). While follicular dendritic cells are critical for prion replication in lymphoid tissue and subsequent neuroinvasion, myeloid dendritic cells (DCs) have been implicated in both the clearance and propagation of pathological prion protein. Since nothing is known on the ability of DCs to migrate to the CNS during prion diseases, we investigated the immunohistochemical localization of CD205(+) DCs in the brain of C57BL/6 mice intraperitoneally infected with the mouse-adapted KFu strain of Gerstmann-Sträussler-Scheinker syndrome, a human genetic prion disorder. In normal brain, CD205(+) cells were present in the meninges and choroid plexus, whereas in the majority of mice sacrificed between 120 and 300 days post infection, CD205(+) DCs were also detected in the cerebral cortex, subcortical white matter, thalamus and medulla oblongata. These findings demonstrate that DCs can enter the CNS of prion-infected mice, suggesting a possible role for these cells in the pathogenesis of prion disorders.

Epstein-Barr virus genetic variants are associated with multiple sclerosis.

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.

Detection of ectopic B-cell follicles with germinal centers in the meninges of patients with secondary progressive multiple sclerosis.

Multiple sclerosis (MS) is characterized by synthesis of oligoclonal immunoglobulins and the presence of B-cell clonal expansions in the central nervous system (CNS). Because ectopic lymphoid tissue generated at sites of chronic inflammation is thought to be important in sustaining immunopathological processes, we have investigated whether structures resembling lymphoid follicles could be identified in the CNS of MS patients. Sections from post-mortem MS brains and spinal cords were screened using immunohistochemistry for the presence of CD20+ B-cells, CD3+ T-cells, CD138+ plasma cells and CD21+, CD35+ follicular dendritic cells, and for the expression of lymphoid chemokines (CXCL 13, CCL21) and peripheral node addressin (PNAd). Lymphoid follicle-like structures containing B-cells, T-cells and plasma cells, and a network of follicular dendritic cells producing CXCL13 were observed in the cerebral meninges of 2 out of 3 patients with secondary progressive MS, but not in relapsing remitting and primary progressive MS. We also show that proliferating B-cells are present in intrameningeal follicles, a finding which is suggestive of germinal center formation. No follicle-like structures were detected in parenchymal lesions. The formation of ectopic lymphoid follicies in the meninges of patients with MS could represent a critical step in maintaining humoral autoimmunity and in disease exacerbation.