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

Abstract

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-γ 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.

Introduction

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS) of presumed autoimmune origin. There is evidence that autoimmune T-cells recognize components of the myelin sheath and trigger a cascade of events leading to demyelination (Hellings et al., 2002). The potential pathogenic role of myelin-reactive T-cells in MS is largely based on studies in experimental autoimmune encephalomyelitis, the animal model of MS (Bernard et al., 1976). However, several important questions related to the role of autoreactive T-cells in the disease process of MS remain unanswered. For instance, it is not known which components within the CNS myelin are the major target of the pathological autoimmune response. Some of the candidate autoantigens include myelin basic protein (MBP), proteolipid protein (PLP) and myelin oligodendrocyte protein (MOG) (Hellings et al., 2002). Moreover, it remains unclear whether antimyelin T-cell responses are stable or change over time as a result of determinant spreading or shifting (Tuohy et al., 1997). In addition, it is not apparent if meaningful functional differences exist between myelin-reactive T-cells of MS patients and healthy controls. MBP-reactive T-cells have been shown to be in vivo activated in the blood of patients with MS Zhang et al., 1994, Vandevyver et al., 1995, Hellings et al., 2001. However, it remains unknown whether these activated T-cells are actually involved in inducing clinical exacerbation. Other T-cell-related events including production of inflammatory cytokines and expression of adhesion molecules have also been indicated to contribute in some way to the disease pathogenesis (Hartung et al., 1995a). One strategy to correlate T-cell-related immune events with clinical disease activity and, thus, to implicate their direct role in the disease process, is to look for possible correlations between this immune event and the disease activity in a longitudinal study.

New treatments including interferon-beta and copolymer-1 were recently approved for MS The IFNB Multiple Sclerosis Study Group, 1993, Johnson et al., 1995. There is, however, still a need for more effective therapies. Clinical studies in MS have been hampered by the lack of a paraclinical or laboratory marker of disease activity. Such surrogate markers of disease activity would be extremely helpful to study the efficacy of new treatments for MS (Sorensen., 1999). Although brain lesions as identified by magnetic resonance imaging (MRI) are now used as standard outcome measures in clinical studies of MS, it is known that brain MRI activity does not always correlate well with clinical disease progression (Miller et al., 1996). In addition, the correlation between disease activity and immune-related parameters may provide further clues about the autoimmune pathogenesis of the disease.

Previous reports indicated that some immune parameters may potentially be related to disease activity. A number of cytokines were found to be increased in different clinical stages of relapsing–remitting MS (RR-MS). Increased levels of the proinflammatory cytokines IFN-γ and TNF-α were correlated with clinical exacerbation, whereas elevated levels of the antiinflammatory cytokines IL-10 and TGF-β were found to be related to remission (Navikas and Link., 1996). The expression of various adhesion molecules and the levels of circulating soluble adhesion molecules are indicators for cell trafficking and blood–brain barrier abnormalities and may correlate with clinical and MRI activity Dore-Duffy et al., 1995, Kraus et al., 1998. In addition, some other biological markers were proposed to be of potential use for predicting disease activity in RR-MS, including markers of cellular activation, matrix-metalloproteinases and T lymphocyte calcium fluxes Oger et al., 1988, Martino et al., 1996, Kieseier et al., 1998. Although myelin-reactive T-cells are considered to play an important role in the MS pathogenesis, only few studies addressed the question whether antimyelin T-cell responses may correlate with disease activity. While one study found a correlation between antimyelin T-cells responses and disease activity, another group did not find a correlation with clinical variables Soderstrom et al., 1994, Chou et al., 1994.

Studies designed to correlate disease activity with a laboratory parameter have often led to conflicting results. Several explanations could account for these discrepancies. First, it may be possible that different pathological changes occur in the clinical subtypes of MS: relapsing–remitting (RR), primary and secondary progressive disease. In addition, many of these studies analyzed a single biological marker only. Several studies were based on single measurements in cross-sectional studies but not on longitudinal studies. Since immune markers may significantly fluctuate over time in individual patients, single measurements would lead to a one-dimensional view, limiting the success of finding a reliable disease marker.

We performed a longitudinal analysis of T-cell-related parameters to provide further information about the role of T-cells in the MS pathogenesis. Several immunologic and clinical parameters were monitored in seven RR-MS patients and two healthy controls during a study period of 18 months. The patients were subjected to a clinical examination (EDSS, number of relapses) every 2 months. MRI scans were taken every 4 months to determine the number of T1- and T2-weighted and gadolinium enhancing lesions. Serial analyses were performed to follow-up the myelin-specificity, phenotype and cytokine profile of T-cell responses in the blood of the patients and control subjects. T-cell reactivity to several myelin antigens was determined by IFN-γ ELISPOT assays to test whether the antimyelin reactivity is stable over time in a given patient or may shift to other antigens due to determinant spreading. The cytokine production of in vitro stimulated PBMC further provided information on the Th-subtype of the immune responses. Every 6 months, MBP- and PLP-reactive T-cells were isolated and characterized for their TCR V gene expression to determine if clonally expanded or persisting myelin-reactive T-cell clones are present in the blood of MS patients. Serum levels of soluble adhesion molecules were measured in ELISA assays. The immunological, clinical and MRI parameters were correlated to provide further information about the role of antimyelin T-cell responses in MS, and to identify potential paraclinical disease markers that can be used for diagnostic or prognostic purposes.