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Two validated assays, a bridging ELISA and a luciferase-based bioassay, were compared for detection of anti-drug antibodies (ADA) against interferon-beta (IFN-β) in patients with multiple sclerosis. Serum samples were tested from patients enrolled in a prospective study of 18 months. In contrast to the ELISA, when IFN-β-specific rabbit polyclonal and human monoclonal antibodies were tested, the bioassay was the more sensitive to detect IFN-β ADA in patients' sera. For clinical samples, selection of method of ELISA should be evaluated prior to the use of a multi-tiered approach. A titer threshold value is reported that may be used as a predictor for persistently positive neutralizing ADA.

Beta-interferons are still among the most commonly used drugs to treat Multiple Sclerosis (MS). The use of beta-interferons is limited by the development of anti-drug antibodies (ADA), which may abrogate the treatment effect of the drug. Although the antibody response has been well studied, little is known about the T cell response to interferon-beta (IFN-β). We investigated T cell responses in four treatment naive MS patients and twenty-three patients treated with IFN-β who had or had not developed ADA to IFN-β. T cell responses were determined by split-well and primary proliferation assays against different IFN-β protein preparations and a set of overlapping peptides covering the full sequence of IFN-β. T cell responses to IFN-β were observed in all donors. ADA positive patients showed higher T cell responses to IFN-β protein than ADA negative patients and untreated controls. We identified two immunodominant regions; T cell responses to IFN-β1-40 were observed in all patients independent of ADA status, while T cell responses to IFN-β125-159 were stronger in ADA positive than ADA negative patients. IFN-β specific T cell responses were HLA class II restricted and in ADA positive patients skewed towards a Th2 phenotype. In IFN-β treated patients we observed a correlation between IFN-β specific T cell responses, serum ADA titer and loss of biological activity of IFN-β treatment. Our studies demonstrate the occurrence of an antigen specific HLA class II restricted Th2 T cell response associated with the development of ADA in IFN-β treated patients.

Background: Inward rectifier potassium channel 4.1 (KIR4.1) specific serum antibodieswere reported in a sub-population ofmultiple sclerosis (MS) patients. KIR4.1 is a glycosylated channel protein expressed in oligodendrocytes and a subset of astrocytes. Objective: To determine the posttranslational modifications of KIR4.1 occurring in human brain and their impact on autoantibody binding. Methods: KIR4.1 protein was isolated from human subcortical white and cortical gray matter and analyzed by immunoblot, immunoprecipitation and N-deglycosylation assays. We purified recombinantly expressed KIR4.1 protein from HEK293 and Oli-neu cells by affinity and gel filtration chromatography to study its molecular heterogeneity and N-linked glycosylation. We analyzed the expression pattern of differently glycosylated KIR4.1 in human subcortical brain sections and transfected cells by immunochemistry using in-house generated monoclonal antibodies (mAb) against the extracellular domain, monoand polyclonal antibodies against the C-terminal domain, and KIR4.1-specific IgGs purified from MS sera (KIR4.1-IgG). Results: KIR4.1 contains a functional N-glycosylation site in its first extracellular domain. In a cell dependent fashion KIR4.1 undergoes simple or complex N-glycosylation. In astrocytes most of KIR4.1 is highly glycosylated (51–60 kD) while oligodendrocytes express predominantly lower glycosylated KIR4.1 (40–42 kD). MAb generated against the extracellular domain and KIR4.1-IgG from MS sera specifically precipitate the lower glycosylated KIR4.1 but not higher glycosylated KIR4.1. This mAb and KIR4.1-IgG predominantly bind to oligodendrocytes and to a much lesser extent to astrocytes. In KIR4.1 transfected HEK293 cells most protein is highly glycosylated while only a minor fraction in the endoplasmic reticulum is weakly glycosylated corresponding to the protein expressed in oligodendrocytes. Separation of different KIR4.1 glycosylation forms demonstrates thatMS specific antibody reactivity is observed toweakly but not highly glycosylated KIR4.1. Conclusion: Our findings demonstrate that KIR4.1 is differentially glycosylated in a cell specific manner. KIR4.1-IgG from MS sera is directed against the weakly glycosylated KIR4.1 expressed predominantly in oligodendrocytes, while higher glycosylation of the extracellular domain interferes with binding to KIR4.1. These findings demonstrate for the first time that posttranslational modification of an autoantigen can influence autoantibody binding and focus the antibody response to a particular subset of cells. These findings have important implications for the understanding of autoantibody responses but also for the development of assays to detect autoantibodies in autoimmune diseases.