Veerle Somers

Premature immunosenescence in rheumatoid arthritis and multiple sclerosis patients.

Abstract: Patients with T‐cell‐mediated autoimmune diseases show immune system abnormalities that resemble the typical characteristics of autoimmune dysfunction described in the elderly. In addition, the incidence of autoimmune disease increases with advancing age. To evaluate whether patients with rheumatoid arthritis (RA) and multiple sclerosis (MS) have premature immuno‐senescence, we measured two indicators of aging: the number of T‐cell‐receptor excision circles (TRECs) and the percentage of CD4+CD28null T cells. We studied them in the peripheral blood mononuclear cells (PBMCs) of 60 RA patients, 32 MS patients, and 40 healthy controls (HCs). We found that TREC numbers were lower in RA and MS patients than in age‐matched HCs, indicating premature thymic involution. Moreover, a subset of these patients contained age‐inappropriate high frequencies of CD4+CD28null T cells. This study provides evidence of premature immune system senescence in both RA and MS patients. Premature aging could be a risk factor for developing autoimmune disorders in genetically predisposed individuals in a susceptible environment.

Targets of the humoral autoimmune response in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory disorder of the central nervous system (CNS) with heterogeneous clinical, genetic and pathophysiological characteristics. The establishment of reliable biomarkers for diagnosis, prognosis and treatment of MS has therefore proven to be very difficult. During the last decades, mounting evidence has been collected for the involvement of B cells and antibodies in MS pathogenesis. A wide variety of autoantibodies has been described in MS and these autoantibodies could be useful biomarkers for MS. Since demyelination is a key component of MS pathogenesis, myelin antigens were first investigated as primary targets of autoantibodies in MS. More recently, it became evident that the humoral autoimmune response is not restricted to myelin but is much more widespread throughout the brain. Autoantibodies are formed against different CNS cell types, including neurons, oligodendrocytes and astrocytes, and even immune cells, indicating the complex heterogeneity of the disease. In this review, we give an extensive overview of the known autoantibody targets in MS, not according to the traditional subdivision of myelin and non-myelin components but according to each of the affected cell types, including the most recently described target antigens.

The auto-antigen repertoire in myasthenia gravis

Myasthenia Gravis (MG) is an antibody-mediated autoimmune disorder affecting the postsynaptic membrane of the neuromuscular junction (NMJ). MG is characterized by an impaired signal transmission between the motor neuron and the skeletal muscle cell, caused by auto-antibodies directed against NMJ proteins. The auto-antibodies target the nicotinic acetylcholine receptor (nAChR) in about 90% of MG patients. In approximately 5% of MG patients, the muscle specific kinase (MuSK) is the auto-antigen. In the remaining 5% of MG patients, however, antibodies against the nAChR or MuSK are not detectable (idiopathic MG, iMG). Although only the anti-nAChR and anti-MuSK auto-antibodies have been demonstrated to be pathogenic, several other antibodies recognizing self-antigens can also be found in MG patients. Various auto-antibodies associated with thymic abnormalities have been reported, as well as many non-MG-specific auto-antibodies. However, their contribution to the cause, pathology and severity of the disease is still poorly understood. Here, we comprehensively review the reported auto-antibodies in MG patients and discuss their role in the pathology of this autoimmune disease.

Autoantibody Profiling in Multiple Sclerosis Reveals Novel Antigenic Candidates

An important contribution of B cells and autoantibodies has been demonstrated in the pathogenesis of multiple sclerosis (MS), leading to interest in the use of such autoantibodies as diagnostic or prognostic biomarkers. The objective of this study was to identify novel Ab biomarkers for MS using “serological Ag selection”. Using a phage display library derived from MS brain plaques, we applied serological Ag selection to identify antigenic targets specifically interacting with Abs present in the cerebrospinal fluid (CSF) of 10 relapsing-remitting MS patients. These antigenic targets were further evaluated on a large panel of CSF from 63 other MS patients, 30 patients with other inflammatory disorders, and 64 patients with noninflammatory neurological disorders. A panel of eight antigenic targets was identified that showed a 86% specificity and 45% sensitivity in discriminating MS patients and controls. Four of the antigenic targets showed exclusive reactivity (100% specificity; 23% sensitivity) in the MS group as compared with the control group. Detailed bio-informatic analyses revealed a novel Ag, SPAG16. Among the novel phage peptides identified, novel epitopes were generated from untranslated sequences and out-of-frame sequences. Of 10 relapsing-remitting patients used for serological Ag selection, Ab reactivity toward one of the eight antigenic targets was also demonstrated in serum of 38% CSF-positive patients. Autoantibody profiles against epitopes derived from MS brain tissue could serve as diagnostic markers or form the basis for the identification of a subgroup of MS patients.

Peripheral blood but not synovial fluid natural killer T cells are biased towards a Th1-like phenotype in rheumatoid arthritis

Natural killer T (NKT) cells have been implicated in the regulatory immune mechanisms that control autoimmunity. However, their precise role in the pathogenesis of rheumatoid arthritis (RA) remains unclear. The frequency, cytokine profile and heterogeneity of NKT cells were studied in peripheral blood mononuclear cells (PBMCs) from 23 RA patients and 22 healthy control individuals, including paired PBMC–synovial fluid samples from seven and paired PBMC–synovial tissue samples from four RA patients. Flow cytometry revealed a decreased frequency of NKT cells in PBMCs from RA patients. NKT cells were present in paired synovial fluid and synovial tissue samples. Based on the reactivity of PBMC-derived NKT cells toward α-galactosylceramide, RA patients could be divided into responders (53.8%) and nonresponders (46.2%). However, NKT cells isolated from synovial fluid from both responders and nonresponders expanded upon stimulation with α-galactosylceramide. Analysis of the cytokine profile of CD4+ and CD4- PBMC derived NKT cell lines from RA patients revealed a significantly reduced number of IL-4 producing cells. In contrast, synovial fluid derived NKT cell lines exhibited a Th0-like phenotype, which was comparable to that in healthy control individuals. This suggests that synovial fluid NKT cells are functional, even in patients with nonresponding NKT cells in their blood. We conclude that, because the number of Vα24+Vβ11+CD3+ NKT cells is decreased and the cytokine profile of blood-derived NKT cells is biased toward a Th1-like phenotype in RA patients, NKT cells might be functionally related to resistance or progression of RA. Providing a local boost to the regulatory potential of NKT cells might represent a useful candidate therapy for RA.

B cell characterization and reactivity analysis in multiple sclerosis

Abstract B cells are one of the key players in the pathogenesis of multiple sclerosis (MS). The peripheral B cell distributions are similar in healthy persons and MS patients. In healthy controls, B cells are rarely present in the cerebrospinal fluid (CSF) while in MS patients, a clonally expanded B cell population is detected. This consists of memory B cells, centroblasts and antibody-secreting plasma blasts and plasma cells that are responsible for intrathecal immunoglobulin G production and oligoclonal band formation in more than 90% of MS patients. Unfortunately, the targets of the autoreactive B cells and antibodies remain largely unknown. Various candidate antigens have been identified but often their involvement in the disease process is still unclear. Most studies characterizing these target antigens examined autoantibodies by analyzing sera or CSF of MS patients. An alternative approach is focusing on the clonally expanded B cells. In this way B cells directed against myelin, astroglia and axons have been denoted in MS patients. B cell immortalization, that is based on the antibody-producing potential of Epstein–Barr virus (EBV) transformed B cells, can be used to expand B cells from MS patients for the production of antibodies, that ultimately can be analysed for target identification.

Identification of a genetic variant for joint damage progression in autoantibody-positive rheumatoid arthritis

Background Joint destruction is a hallmark of autoantibody-positive rheumatoid arthritis (RA), though the severity is highly variable between patients. The processes underlying these interindividual differences are incompletely understood. Methods We performed a genome-wide association study on the radiological progression rate in 384 autoantibody-positive patients with RA. In stage-II 1557 X-rays of 301 Dutch autoantibody-positive patients with RA were studied and in stage-III 861 X-rays of 742 North American autoantibody-positive patients with RA. Sperm-Associated Antigen 16 (SPAG16) expression in RA synovium and fibroblast-like synoviocytes (FLS) was examined using Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) and immunohistochemistry. FLS secrete metalloproteinases that degrade cartilage and bone. SPAG16 genotypes were related to matrix metalloproteinase (MMP)-3 and MMP-1 expression by FLS in vitro and MMP-3 production ex vivo. Results A cluster of single nucleotide polymorphisms (SNPs) at 2q34, located at SPAG16, associated with the radiological progression rate; rs7607479 reached genome-wide significance. A protective role of rs7607479 was replicated in European and North American patients with RA. Per minor allele, patients had a 0.78-fold (95% CI 0.67 to 0.91) progression rate over 7 years. mRNA and protein expression of SPAG16 in RA synovium and FLS was verified. FLS carrying the minor allele secreted less MMP-3 (p=1.60×10−2). Furthermore, patients with RA carrying the minor allele had lower serum levels of MMP-3 (p=4.28×10−2). In a multivariate analysis on rs7607479 and MMP-3, only MMP-3 associated with progression (p=2.77×10−4), suggesting that the association between SPAG16-rs7607479 and joint damage is mediated via an effect on MMP-3 secretion. Conclusions Genetic and functional analyses indicate that SPAG16 influences MMP-3 regulation and protects against joint destruction in autoantibody-positive RA. These findings could enhance risk stratification in autoantibody-positive RA.

Selective Identification of Macrophages and Cancer Cells Based on Thermal Transport through Surface-Imprinted Polymer Layers

In this article, we describe a novel straightforward method for the specific identification of viable cells (macrophages and cancer cell lines MCF-7 and Jurkat) in a buffer solution. The detection of the various cell types is based on changes of the heat transfer resistance at the solid-liquid interface of a thermal sensor device induced by binding of the cells to a surface-imprinted polymer layer covering an aluminum chip. We observed that the binding of cells to the polymer layer results in a measurable increase of heat transfer resistance, meaning that the cells act as a thermally insulating layer. The detection limit was found to be on the order of 10(4) cells/mL, and mutual cross-selectivity effects between the cells and different types of imprints were carefully characterized. Finally, a rinsing method was applied, allowing for the specific detection of cancer cells with their respective imprints while the cross-selectivity toward peripheral blood mononuclear cells was negligible. The concept of the sensor platform is fast and low-cost while allowing also for repetitive measurements.

Compositional Changes of B and T Cell Subtypes during Fingolimod Treatment in Multiple Sclerosis Patients: A 12-Month Follow-Up Study

Background and objective The long term effects of fingolimod, an oral treatment for relapsing-remitting (RR) multiple sclerosis (MS), on blood circulating B and T cell subtypes in MS patients are not completely understood. This study describes for the first time the longitudinal effects of fingolimod treatment on B and T cell subtypes. Furthermore, expression of surface molecules involved in antigen presentation and costimulation during fingolimod treatment are assessed in MS patients in a 12 month follow-up study. Methods Using flow cytometry, B and T cell subtypes, and their expression of antigen presentation, costimulation and migration markers were measured during a 12 month follow-up in the peripheral blood of MS patients. Data of fingolimod-treated MS patients (n = 49) were compared to those from treatment-naive (n = 47) and interferon-treated (n = 27) MS patients. Results In the B cell population, we observed a decrease in the proportion of non class-switched and class-switched memory B cells (p<0.001), both implicated in MS pathogenesis, while the proportion of naive B cells was increased during fingolimod treatment in the peripheral blood (PB) of MS patients (p<0.05). The remaining T cell population, in contrast, showed elevated proportions of memory conventional and regulatory T cells (p<0.01) and declined proportions of naive conventional and regulatory cells (p<0.05). These naive T cell subtypes are main drivers of MS pathogenesis. B cell expression of CD80 and CD86 and programmed death (PD) -1 expression on circulating follicular helper T cells was increased during fingolimod follow-up (p<0.05) pointing to a potentially compensatory mechanism of the remaining circulating lymphocyte subtypes that could provide additional help during normal immune responses. Conclusions MS patients treated with fingolimod showed a change in PB lymphocyte subtype proportions and expression of functional molecules on T and B cells, suggesting an association with the therapeutic efficacy of fingolimod.

Multiplexing approaches for autoantibody profiling in multiple sclerosis

The preliminary positive effects of B cell depletion therapy in multiple sclerosis (MS) have renewed interest in a potential role of B cells and autoantibodies in the MS disease process. Regardless of a possible pathogenic role of the humoral immune response in MS, the analysis of autoantibodies as disease markers is valuable. Despite intense research, there is no known MS-associated antibody specificity that can individually discriminate between MS patients and controls. Due to the overlap in autoantibody profiles in autoimmune diseases, and due to the complexity of MS, multiplex autoantibody profiling approaches are needed to generate a panel of MS-associated autoantibodies with high combined sensitivity and specificity for MS. In recent years, several multiplexing approaches have been applied in MS autoantibody profiling with promising results regarding the generation of a so-called MS-specific autoantibody fingerprint. We also recently applied a high-throughput autoantibody profiling technique for MS cerebrospinal fluid resulting in the identification of a novel panel of 8 antigenic targets with 45% sensitivity and 86% specificity for the disease. Identification of MS-specific autoantibody specificities is important for the development of diagnostic and prognostic markers for MS. Moreover, it can provide more knowledge regarding underlying MS disease processes and novel therapeutic targets.