Robert Medaer

Compromised CD4+ CD25high regulatory T‐cell function in patients with relapsing‐remitting multiple sclerosis is correlated with a reduced frequency of FOXP3‐positive cells and reduced FOXP3 expression at the single‐cell level

CD4+ CD25high regulatory T cells (Tregs) of patients with relapsing‐remitting (RR) multiple sclerosis (MS), in contrast to those of patients with secondary progressive (SP) MS, show a reduced suppressive function. In this study, we analysed forkhead box P3 (FOXP3) at the single‐cell level in MS patients and controls (healthy individuals and patients with other neurological diseases) by means of intracellular flow cytometry. Our data revealed a reduced number of peripheral blood CD4+ CD25high FOXP3+ T cells and lower FOXP3 protein expression per cell in RR‐MS patients than in SP‐MS patients and control individuals, which was correlated with the suppressive capacity of Tregs in these patients. Interestingly, interferon (IFN)‐β‐treated RR‐MS patients showed restored numbers of FOXP3+ Tregs. Furthermore, a higher percentage of CD4+ CD25high FOXP3+ Tregs in RR‐MS patients, as compared with controls and SP‐MS patients, expressed CD103 and CD49d, adhesion molecules involved in T‐cell recruitment towards inflamed tissues. This was consistent with a significantly increased number of CD27+ CD25high CD4+ T cells in the cerebrospinal fluid (CSF), as compared with peripheral blood, in RR‐MS patients. Taken together, these data show aberrant FOXP3 expression at the single‐cell level correlated with Treg dysfunction in RR‐MS patients. Our results also suggest that Tregs accumulate in the CSF of RR‐MS patients, in an attempt to down‐regulate local inflammation in the central nervous system.

Secondary progressive in contrast to relapsing-remitting multiple sclerosis patients show a normal CD4+CD25+ regulatory T-cell function and FOXP3 expression

Accumulating evidence indicates an immunosuppressive role for CD4+CD25+ regulatory T cells (Tregs) in autoimmune diseases. Although an impaired Treg function in patients with relapsing‐remitting multiple sclerosis (RR‐MS) has been reported recently, no information is available so far about Treg function in the progressive stage of the disease. In the present study, the phenotypic and functional characteristics of CD4+CD25+ T cells isolated from the peripheral blood of patients with RR‐MS and secondary progressive multiple sclerosis (SP‐MS) were investigated. No significant quantitative or phenotypic abnormalities in CD4+CD25+ T cells from RR‐ and SP‐MS patients were detected. However, whereas a reduced suppressor function of CD4+CD25+ T cells toward proliferation and interferon‐γ production of CD4+CD25– responder T cells was found in RR‐MS patients, SP‐MS patients showed a normal Treg function. The suppressive capacity of MS‐derived CD4+CD25+ T cells was correlated with disease duration but not with age, indicating that Treg function is more affected in the early phase of the disease process. Consistently with the suppressive capacity, CD4+CD25+ T cells from SP‐MS patients showed normal levels of FOXP3 mRNA in contrast to RR‐MS patients that had a reduced FOXP3 expression. These data are the first to demonstrate differences in function and FOXP3 expression of CD4+CD25+ T cells from patients with RR‐ and SP‐MS.

T-cell reactivity to multiple myelin antigens in multiple sclerosis patients and healthy controls

Myelin proteins, including myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) are candidate autoantigens in MS. It is not clear whether MS patients show a predominant reactivity to one or several myelin antigens. We evaluated the IFN‐γ production induced by MBP and MOG and selected MBP‐, MOG‐ and PLP‐peptides in MS patients and healthy controls using the IFN‐γ ELISPOT assay. Most MS patients and healthy controls showed a heterogeneous anti‐myelin T‐cell reactivity. Interestingly in MS patients a positive correlation was found between the anti‐MOG and anti‐MBP T‐cell responses. No myelin peptide was preferentially recognized among the peptides tested (MBP 84–102, 143–168, MOG 1–22, 34–56, 64–86, 74–96, PLP 41–58, 184–199, 190–209). In addition the frequency of IL2R+ MBP reactive T‐cells was significantly increased in blood of MS patients as compared with healthy subjects, indicating that MBP reactive T‐cells exist in an in vivo activated state in MS patients. Most of the anti‐MBP T‐cells were of the Th1‐type because reactivity was observed in IFN‐γ but not in IL‐4 ELISPOT‐assays. Using Th1 (IL‐12) and Th2 (IL‐4) promoting conditions we observed that the cytokine secretion pattern of anti‐MBP T‐cells still is susceptible to alteration. Our data further indicate that precursor frequency analysis of myelin reactive T‐cells by proliferation‐based assays may underestimate the true frequency of myelin specific T‐cells significantly. J. Neurosci. Res. 63:290–302, 2001.

Risks of multiple sclerosis in relatives of patients in Flanders, Belgium.

OBJECTIVES: To calculate age adjusted risks for multiple sclerosis in relatives of Flemish patients with multiple sclerosis. METHODS: Lifetime risks were calculated using the maximum likelihood approach. RESULTS: Vital information was obtained on 674 probands with multiple sclerosis in Flanders and a total of their 26225 first, second, and third degree relatives. Full medical information to allow documentation of multiple sclerosis status was available for 21351 (81.4%) relatives. The age adjusted risk for parents was 1.61 (SEM 0.35)%, for siblings 2.10 (SE 0.36)%, and for children 1.71 (SEM 0.70)%. For aunts and uncles, the risk was 0.66 (SEM 0.13)%. CONCLUSIONS: The risk for first degree relatives of patients with multiple sclerosis in Flanders is increased 10-fold to 12-fold; for second degree relatives, it is increased threefold. This information can be used for risk counselling in families and provides additional support for the role of more than one locus contributing to the susceptibility of multiple sclerosis.

Longitudinal study of antimyelin T-cell reactivity in relapsing-remitting multiple sclerosis: association with clinical and MRI activity.

In multiple sclerosis (MS), T-cells are considered to be critical in coordinating an immunopathological cascade that results in myelin damage. We investigated whether clinical disease activity or brain inflammatory activity as measured by magnetic resonance imaging (MRI) was associated with changes in autoreactive T-cell reactivities in MS patients. To this end, a longitudinal study was performed in which T-cell-related immune parameters and clinical parameters (including MRI) were monitored in seven relapsing-remitting (RR) MS patients and two healthy controls with bimonthly intervals over a period of 18 months. The serial evaluation of antimyelin (MBP, PLP, MOG) T-cell responses revealed highly dynamic shifts and fluctuations from one pattern to another in a patient-dependent manner. In some of the patients, changes in T-cell-related immune variables were found to concur with MRI activity and generally preceded clinical relapses. These alterations include: increased number of myelin-reactive IFN-gamma secreting T-cells, detection of clonally expanded myelin-reactive T-cells, elevated proinflammatory and decreased antiinflammatory cytokine production, upregulation of ICAM-1 membrane expression and highly increased serum levels of soluble VCAM-1. However, not all exacerbations and MRI changes were associated with changes in antimyelin reactivity. Some of the observed immune alterations were also detected in the healthy controls, indicating that additional regulatory mechanisms-which may be defective in MS-play a role in the downregulation of potentially pathological T-cell responses. In conclusion, this study provides further support for an important role of myelin-reactive T-cells in the pathogenesis of MS. In addition, the observed dynamic changes in the antimyelin T-cell reactivity pattern may be a major obstacle for the development of antigen-specific immunotherapies.

Vaccination with autoreactive T cell clones in multiple sclerosis: overview of immunological and clinical data.

Although the etiology and pathogenesis of Multiple Sclerosis (MS) remain elusive, accumulating evidence indicates that MS is a chronic inflammatory disease with an autoimmune component, mediated by autore‐active T lymphocytes specific for myelin antigens. The triggering T cell autoantigen has not been identified yet, but recent immunological studies in MS and parallel experiments in experimental allergic encephalomyelitis (EAE), the animal model of MS, have indicated that myelin basic protein (MBP) can be considered as one of the major candidate autoantigens in the pathogenesis of the disease. Based on these observations, several therapeutic strategies have been developed aimed at the specific elimination or inactivation of MBP reactive T cells in MS. One of these approaches involves the immunization of MS patients with autologous attenuated autoreactive T cells to induce an immune response specifically targeted at these autoreactive T cells. This method, termed T cell vaccination, has been shown to prevent and treat EAE. We have recently conducted a pilot trial of T cell vaccination in a limited group of MS patients to evaluate the immunological responses to the injected cells. The data obtained indicate that this type of vaccination induces an effective anti‐clonotypic T cell response leading to a specific depletion of circulating MBP reactive T cells. Preliminary data on the clinical effects are promising, encouraging further clinical trials.

Myelin reactive T cells after T cell vaccination in multiple sclerosis: cytokine profile and depletion by additional immunizations

Pathogenic autoreactive T cells can be targeted by T cell vaccination (TCV), a procedure in which patients are immunized with autologous attenuated pathogenic T cells. We reported previously that TCV with myelin basic protein (MBP) reactive T cell clones in multiple sclerosis (MS) patients induced T cell immune responses, resulting in a clonal depletion of MBP reactive T cells in all patients. Five years after TCV, MBP reactive T cells were observed in five out of nine MS patients. These clones had a different clonal origin from those isolated before vaccination. We have studied the cytokine profile, cytotoxicity and epitope specificity of these reappearing clones. Our data indicate that the clones express similar effector functions as those isolated before TCV, suggesting that they also could play a pathogenic role in the disease. We demonstrated that these clones can be depleted by an additional sequence of immunizations. These findings may be relevant to other T cell targeted immunotherapies for MS and other autoimmune diseases.

MYELIN REACTIVE T CELLS IN THE AUTOIMMUNE PATHOGENESIS OF MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) leading to demyelination. Although it is widely accepted that demyelination in MS results from an active inflammatory process, the cause of the inflammation is still not completely resolved. Findings in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and observations in human MS have led to the hypothesis that MS is an autoimmune disease mediated by autoreactive T cells with specificity for myelin antigens. The identity of the brain antigen(s) which is (are) the primary target(s) of the autoimmune process is not known, but current evidence indicates that myelin basic protein (MBP) is a likely candidate. In this paper we will overview some of the experimental evidence suggesting that MBP reactive T cells hold a central position in the pathogenesis of MS, and discuss some of the currently tested therapeutic strategies in MS which are directed towards the pathogenic MBP reactive T cells. Although there appears to be no direct correlation between anti-MBP T cell responses and clinical disease activity, some recent observations suggest that monitoring of anti-MBP T cell responses could be helpful to study immunological efficacy of experimental immunotherapies in MS.

Preferential peptide specificity and HLA restriction of myelin basic protein-specific T cell clones derived from MS patients

A panel of 17 myelin basic protein (MBP)-specific T lymphocyte clones were generated from four multiple sclerosis (MS) patients. All T cell clones expressed CD4 phenotype and 14 clones exhibited substantial cytotoxic activity on MBP-coated target cells. T cell recognition sites of the clones on human MBP were identified by using MBP fragments and synthetic peptides. Despite the fact that at least three epitopes were defined, these T cell clones displayed a striking bias to the C-terminal peptide 149-171 independent of differences in HLA-DR and DQ expression. In addition, the T cell responses of the clones appeared to be restricted by HLA-DR molecules irrespective of peptide specificities. The present study suggests an immunodominant property of the C-terminal peptide for HLA-DR-restricted T cell responses to MBP. However, its association with encephalitogenicity in humans and its potential pathologic importance in MS await further clarification.

Boosting endogenous neuroprotection in multiple sclerosis: the ASsociation of Inosine and Interferon \beta in relapsing- remitting Multiple Sclerosis (ASIIMS) trial

Anti-inflammatory drugs are effective on relapses, but neuroprotective agents to prevent disability are still unavailable. Uric acid has neuroprotective effects in experimental models including encephalomyelitis and appears to be involved in multiple sclerosis. Oral administration of inosine, a precursor of uric acid, increases serum uric acid levels and is well tolerated. Our objective was to test the possibility that a combination therapy associating an anti-inflammatory drug (interferon β) and an endogenous neuroprotective molecule (uric acid) would be more effective than interferon β alone on the accumulation of disability. Patients with relapsing—remitting multiple sclerosis on interferon β for at least 6 months were randomized to interferon β + inosine or interferon β + placebo for 2 years. The dose of inosine was adjusted to maintain serum uric acid levels in the range of asymptomatic hyperuricaemia (≤10 mg/dl). The primary end points were percentage of patients with progression of disability and time to sustained progression (Kaplan—Meier analysis). The combination of interferon β and inosine was safe and well tolerated but did not provide any additional benefit on accumulation of disability compared with interferon β alone. We conclude that endogenous neuroprotective mechanisms recently identified in multiple sclerosis are complex and uric acid does not reflect the entire story.