Samantha Jilek

Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis

Epstein-Barr virus (EBV) has been associated with multiple sclerosis (MS), however, most studies examining the relationship between the virus and the disease have been based on serologies, and if EBV is linked to MS, CD8+ T cells are likely to be involved as they are important both in MS pathogenesis and in controlling viruses. We hypothesized that valuable information on the link between MS and EBV would be ascertained from the study of frequency and activation levels of EBV-specific CD8+ T cells in different categories of MS patients and control subjects. We investigated EBV-specific cellular immune responses using proliferation and enzyme linked immunospot assays, and humoral immune responses by analysis of anti-EBV antibodies, in a cohort of 164 subjects, including 108 patients with different stages of MS, 35 with other neurological diseases and 21 healthy control subjects. Additionally, the cohort were all tested against cytomegalovirus (CMV), another neurotropic herpes virus not convincingly associated with MS, nor thought to be deleterious to the disease. We corrected all data for age using linear regression analysis over the total cohorts of EBV- and CMV-infected subjects. In the whole cohort, the rate of EBV and CMV infections were 99% and 51%, respectively. The frequency of IFN-gamma secreting EBV-specific CD8+ T cells in patients with clinically isolated syndrome (CIS) was significantly higher than that found in patients with relapsing-remitting MS (RR-MS), secondary-progressive MS, primary-progressive MS, patients with other neurological diseases and healthy controls. The shorter the interval between MS onset and our assays, the more intense was the EBV-specific CD8+ T-cell response. Confirming the above results, we found that EBV-specific CD8+ T-cell responses decreased in 12/13 patients with CIS followed prospectively for 1.0 +/- 0.2 years. In contrast, there was no difference between categories for EBV-specific CD4+ T cell, or for CMV-specific CD4+ and CD8+ T-cell responses. Anti-EBV-encoded nuclear antigen-1 (EBNA-1)-specific antibodies correlated with EBV-specific CD8+ T cells in patients with CIS and RR-MS. However, whereas EBV-specific CD8+ T cells were increased the most in early MS, EBNA-1-specific antibodies were increased in early as well as in progressive forms of MS. Our data show high levels of CD8+ T-cell activation against EBV--but not CMV--early in the course of MS, which support the hypothesis that EBV might be associated with the onset of this disease.

Immune responses to JC virus in patients with multiple sclerosis treated with natalizumab: a cross-sectional and longitudinal study

BACKGROUND Natalizumab is used to prevent relapses and progression of disability in patients with multiple sclerosis but has been associated with progressive multifocal leukoencephalopathy (PML). We aimed to better understand the associations between JC virus, which causes PML, and natalizumab treatment. METHODS We prospectively assessed patients with multiple sclerosis who started treatment with natalizumab. Blood and urine samples were tested for the presence of JC virus DNA with quantitative real-time PCR before treatment and at regular intervals after treatment onset for up to 18 months. At the same timepoints, by use of proliferation and enzyme-linked immunospot assays, the cellular immune responses against JC virus, Epstein-Barr virus, cytomegalovirus, myelin oligodendrocyte glycoprotein, and myelin oligodendrocyte basic protein (MOBP) were assessed. Humoral immune response specific to JC virus was assessed with an enzyme immunoassay. The same experiments were done on blood samples from patients with multiple sclerosis before and 10 months after the start of interferon beta treatment. FINDINGS We assessed 24 patients with multiple sclerosis who received natalizumab and 16 who received interferon beta. In patients treated with natalizumab, JC virus DNA was not detected in the blood at any timepoint. However, JC virus DNA was present in the urine of six patients and in most of these patients the concentrations of JC virus DNA were stable over time. Compared with pretreatment values, the cellular immune response was increased to cytomegalovirus at 6 months, to JC virus at 1, 9, and 12 months, and to Epstein-Barr virus and MOBP at 12 months. Humoral responses remained stable. There were no increases in cellular immune responses specific to the viruses or myelin proteins in the 16 patients treated with interferon beta. INTERPRETATION Natalizumab increases cellular immune responses specific to viruses and myelin proteins in the peripheral blood after 1 year, without evidence of viral reactivation. FUNDING Swiss National Foundation, Swiss Society for Multiple Sclerosis, and Biogen Dompé.

Intrathecal immune responses to EBV in early MS

EBV has been consistently associated with MS, but its signature in the CNS has rarely been examined. In this study, we assessed EBV‐specific humoral and cellular immune responses in the cerebrospinal fluid (CSF) of patients with early MS, other inflammatory neurological diseases (OIND) and non‐inflammatory neurological diseases (NIND). The neurotropic herpesvirus CMV served as a control. Virus‐specific humoral immune responses were assessed in 123 consecutive patients and the intrathecal recruitment of virus‐specific antibodies was expressed as antibody indexes. Cellular immune responses tested in the blood of 55/123 patients were positive in 46/55. The CD8+ CTL responses of these 46 patients were assessed in the blood and CSF using a CFSE‐based CTL assay. We found that viral capsid antigen and EBV‐encoded nuclear antigen‐1, but not CMV IgG antibody indexes, were increased in early MS as compared with OIND and NIND patients. There was also intrathecal enrichment in EBV‐, but not CMV‐specific, CD8+ CTL in early MS patients. By contrast, OIND and NIND patients did not recruit EBV‐ nor CMV‐specific CD8+ CTL in the CSF. Our data, showing a high EBV‐, but not CMV‐specific intrathecal immune response, strengthen the association between EBV and MS, in particular at the onset of the disease.

Impairment of JCV-specific T-cell response by corticotherapy Effect on PML-IRIS management?

ABSTRACT Objective: To investigate the impact of corticosteroids (CS) on the viral-specific T-cell response, in particular the JC virus (JCV)–specific one, in an attempt to determine the optimal timing of CS in the management of progressive multifocal leukoencephalopathy–immune reconstitution inflammatory syndrome (PML-IRIS). Methods: A blood draw was performed before and 7 days after the administration of IV CS to 24 patients with relapsing multiple sclerosis (MS). The phenotypic pattern of T cells was determined by CCR7 and CD45RA. To assess the impact of CS treatment on proliferative response of JCV-, influenza-, and Epstein-Barr virus (EBV)–specific T cells, a thymidine incorporation proliferation assay was performed. An intracellular cytokine staining assay was performed to determine the effect of CS treatment on the production of cytokine by virus-specific T cells. JCV T-cell assays were performed only in JCV-infected patients with MS as detected by serologies (Stratify) or detection of JCV DNA in the urine by PCR. Results: CS led T cells, CD4+ and CD8+, toward a less differentiated phenotype. There was a significant decrease of EBV-, influenza-, and JCV-specific T-cell proliferative response upon CS treatment. There was a significant decrease in the frequency of interferon (IFN) γ– and tumor necrosis factor (TNF) α–producing JCV-specific CD8+ T cells, but not EBV- or influenza-specific CD4+ or CD8+ T cells. Conclusions: CS have a profound impact on the virus-specific T-cell response, especially on JCV, suggesting that when CS are considered, they should not be given before the onset of clinical or radiologic signs of IRIS. Studies addressing directly patients with MS with natalizumab-caused PML are warranted. Classification of evidence: This study provides Class III evidence that methylprednisolone treatment decreases the frequency of JCV-specific CD8+ T cells producing IFN-γ and TNFα, impairing control of JCV, suggesting this should be used to treat but not to prevent PML-IRIS. No clinical outcomes were measured.

Vitamin D has a direct immunomodulatory effect on CD8+ T cells of patients with early multiple sclerosis and healthy control subjects

Little is known on a putative effect of vitamin D on CD8+ T cells. Yet, these cells are involved in the immmunopathogenesis of MS. We assessed the cytokine profile of EBV-specific CD8+ T cells of 10 early MS patients and 10 healthy control subjects with or without 1,25(OH)(2)D(3) and found that, with 1,25(OH)(2)D(3), these cells secreted less IFN-γ and TNF-α and more IL-5 and TGF-β. CD4+ T cell depletion or even culture with CD8+ T cells only did not abolish the immunomodulatory effect of 1,25(OH)(2)D(3) on CD8+ T cells, suggesting that 1,25(OH)(2)D(3) can act directly on CD8+ T cells.

HLA-B7–Restricted EBV-Specific CD8+ T Cells Are Dysregulated in Multiple Sclerosis

It was hypothesized that the EBV-specific CD8+ T cell response may be dysregulated in multiple sclerosis (MS) patients, possibly leading to a suboptimal control of this virus. To examine the CD8+ T cell response in greater detail, we analyzed the HLA-A2–, HLA-B7–, and HLA-B8–restricted EBV- and CMV-specific CD8+ T cell responses in a high number of MS patients and control subjects using tetramers. Content in cytolytic granules, as well as cytotoxic activity, of EBV- and CMV-specific CD8+ T cells was assessed. We found that MS patients had a lower or a higher prevalence of HLA-A2 and HLA-B7, respectively. Using HLA class I tetramers in HLA-B7+ MS patients, there was a higher prevalence of MS patients with HLA-B*0702/EBVRPP-specific CD8+ T cells ex vivo. However, the magnitude of the HLA-B*0702/EBVRPP-specific and HLA-B*0702/CMVTPR-specific CD8+ T cell response (i.e., the percentage of tetramer+ CD8+ T cells in a study subject harboring CD8+ T cells specific for the given epitope) was lower in MS patients. No differences were found using other tetramers. After stimulation with the HLA-B*0702/EBVRPP peptide, the production of IL-2, perforin, and granzyme B and the cytotoxicity of HLA-B*0702/EBVRPP-specific CD8+ T cells were decreased. Altogether, our findings suggest that the HLA-B*0702–restricted viral (in particular the EBV one)-specific CD8+ T cell response is dysregulated in MS patients. This observation is particularly interesting knowing that the HLA-B7 allele is more frequently expressed in MS patients and considering that EBV is associated with MS.

Rivastigmine for HIV-associated neurocognitive disorders A randomized crossover pilot study

Objective: To assess the efficacy and safety of rivastigmine for the treatment of HIV-associated neurocognitive disorders (HAND) in a cohort of long-lasting aviremic HIV+ patients. Methods: Seventeen aviremic HIV+ patients with HAND were enrolled in a randomized, double-blind, placebo-controlled, crossover study to receive either oral rivastigmine (up to 12 mg/day for 20 weeks) followed by placebo (20 weeks) or placebo followed by rivastigmine. Efficacy endpoints were improvement on rivastigmine in the Alzheimers Disease Assessment Scale–Cognitive subscale (ADAS-Cog) and individual neuropsychological scores of information processing speed, attention/working memory, executive functioning, and motor skills. Measures of safety included frequency and nature of adverse events and abnormalities on laboratory tests and on plasma concentrations of antiretroviral drugs. Analyses of variance with repeated measures were computed to look for treatment effects. Results: There was no change on the primary outcome ADAS-Cog on drug. For secondary outcomes, processing speed improved on rivastigmine (Trail Making Test A: F1,13 = 5.57, p = 0.03). One measure of executive functioning just failed to reach significance (CANTAB Spatial Working Memory [strategy]: F1,13 = 3.94, p = 0.069). No other change was observed. Adverse events were frequent, but not different from those observed in other populations treated with rivastigmine. No safety issues were recorded. Conclusions: Rivastigmine in aviremic HIV+ patients with HAND seemed to improve psychomotor speed. A larger trial with the better tolerated transdermal form of rivastigmine is warranted. Classification of evidence: This study provides Class III evidence that rivastigmine is ineffective for improving ADAS-Cog scores, but is effective in improving some secondary outcome measures in aviremic HIV+ patients with HAND.

Marked Increase of the Astrocytic Marker S100B in the Cerebrospinal Fluid of HIV-infected Patients on LPV/r-Monotherapy

Objective:To determine changes of cerebrospinal fluid (CSF) biomarkers of patients on monotherapy with lopinavir/ritonavir. Design:The Monotherapy Switzerland/Thailand study (MOST) trial compared monotherapy with ritonavir-boosted lopinavir with continued therapy. The trial was prematurely stopped due to virological failure in six patients on monotherapy. It, thus, offers a unique opportunity to assess brain markers in the early stage of HIV virological escape. Methods:Sixty-five CSF samples (34 on continued therapy and 31 on monotherapy) from 49 HIV-positive patients enrolled in MOST. Using enzyme-linked immunosorbent assay, we determined the CSF concentration of S100B (astrocytosis), neopterin (inflammation), total Tau (tTau), phosphorylated Tau (pTau), and amyloid-&bgr; 1–42 (A&bgr;), the latter three indicating neuronal damage. Controls were CSF samples of 29 HIV-negative patients with Alzheimer dementia. Results:In the CSF of monotherapy, concentrations of S100B and neopterin were significantly higher than in continued therapy (P = 0.006 and P = 0.013, respectively) and Alzheimer dementia patients (P < 0.0001 and P = 0.0005, respectively). In Alzheimer dementia, concentration of A&bgr; was lower than in monotherapy (P = 0.005) and continued therapy (P = 0.016) and concentrations of tTau were higher than in monotherapy (P = 0.019) and continued therapy (P = 0.001). There was no difference in pTau among the three groups. After removal of the 16 CSF with detectable viral load in the blood and/or CSF, only S100B remained significantly higher in monotherapy than in the two other groups. Conclusion:Despite full viral load-suppression in blood and CSF, antiretroviral monotherapy with lopinavir/ritonavir can raise CSF levels of S100B, suggesting astrocytic damage.

Natalizumab treatment alters the expression of T-cell trafficking marker LFA-1 α-chain (CD11a) in MS patients.

Objective: To determine the long-term effect of natalizumab (NTZ) treatment on the expression of integrins and chemokine receptors involved in the migration of T cells towards the central nervous system (CNS). Methods: We drew the blood of 23 patients just before starting NTZ therapy and every 12 months thereafter, for up to 48 months of treatment. We assessed the ex-vivo expression of phenotype markers (CCR7 and CD45RA), CNS-addressing integrins (CD11a, CD49d and CD29) and chemokine receptors (CXCR3 and CCR6) in CD4+ or CD8+ T-cell subsets by flow cytometry. Results: As compared to the pre-NTZ values, there was a marked increase in central memory (CCR7+/CD45RA-) CD4+ T cells and in effector memory (CCR7-/CD45RA-) CD8+ T cells at 12 and 24 months. In addition to an expected downregulation of both VLA-4 subunits (CD49d/CD29), we also found decreased T-cell expression of CXCR3 at 12 months, and of CD11a (LFA-1 αL subunit) at 12 months, but mostly at 24 months of NTZ treatment. Conclusion: Our data show a nadir of CD11a expression at 2 years of NTZ treatment, at the peak of incidence of progressive multifocal leukoencephalopathy (PML), indirectly suggesting that a lack of these molecules may play a role in the onset of PML in NTZ-treated patients.

Severe post-EBV encephalopathy associated with myelin oligodendrocyte glycoprotein-specific immune response

The mechanisms leading to CNS disorders after EBV infections are unclear. We report the case of a patient who developed a severe, but reversible, encephalopathy following an infectious mononucleosis. We detected no EBV DNA in the blood or in the cerebrospinal fluid (CSF) and no EBV-specific antibodies in the CSF. However, we found a potent MOG-specific cellular and humoral immune response. Interestingly, MOG-specific cellular immune response rapidly decreased, paralleling the improvement of clinical condition. In conclusion, this detailed study shows that acute EBV infection can trigger a potent auto-inflammatory response in the CNS, without evidence of an overt infection.