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B cells and endogenous retroviruses in multiple sclerosis

Zimmermann, Maria. B cells and endogenous retroviruses in multiple sclerosis. 2016, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_11846

Abstract

Part I:
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Although neither the cause nor the pathomechanism of MS is fully understood, it is believed to be caused by autoimmune destruction of the myelin. Various findings in patients with MS suggest the involvement of B cells in the pathogenesis. The most prominent finding is the presence of oligoclonal bands in patients with MS and the success of B cell depleting therapies. Despite tremendous efforts, the target antigens of B cells in MS have not yet been identified.
The goal of our first study (Results I of Part I of the presented work), was to develop a sensitive method for identifying antigen-specific B cells in humans, with the future goal of being able to identify autoantigen-specific B cells in MS patients. However, in many patients we do not know the specific antigens involved, and we expect the frequency of autoreactive B cells to be low. Thus, in order to develop a method for isolating such B cells we needed a model in which the target antigen is known and B cells specific for that antigen are present in normal human donors. Influenza hemagglutinin (HA) was chosen as an appropriate model antigen since healthy individuals are commonly immunized against influenza and the frequency of influenza-specific B cells is relatively high following immunization. Our method is based on culturing human B cells with fluorescently labeled hemagglutinin-expressing cells, followed by a FACS-sort to isolate putatively hemagglutinin-specific B cells from other B cells. With this method we were able to enrich hemagglutinin-specific B cells approximately 150-fold, as shown by ELISpot. We EBV-transformed FACS-sorted B cells to enable the production of monoclonal antibodies needed for the analysis of the B cell specificity. The transformations with EBV were performed with B cells from four different donors by adding 30-60 sorted B cells per well, yielding transformed B cell colonies in 48-75% of wells. Antibodies in the supernatants from those cultures were examined for their binding capacity to HA. Between 15% and 26% of GFP-capturing B cell cultures, and between zero and 3% of non-capturing B cell cultures produced antibodies that showed binding to HA by ELISA. By a cell-based flow cytometric assay 2.7-26% of GFP-capturing B cell cultures and none of the non-capturing B cell cultures produced antibodies that showed binding to HA. In addition, FACS-sorted B cells were subjected to an in vitro expansion protocol that also allowed the recombinant expression of immunoglobulins. Seventy-two combinations of heavy and light chains were expressed recombinantly and the cloned antibodies were tested by IgG ELISA for binding to HA, tetanus toxoid, BSA and anti-IgG. Of those 72 combinations, 42 showed binding to HA and 5 showed polyspecific binding to BSA, tetanus or both, in addition to HA. Because this method isolates intact cells, rather than soluble antibodies, it can be used for the identification of B cells whose B cell receptor (BCR) is specific for unknown antigens, e.g. antigens expressed on the myelin sheath but of unknown identity.
In a connected project that also makes use of the phenomenon of membrane capture by B cells (Results II of Part I of the presented work), we tested a novel hypothesis of B cell autoimmunity in an in vitro system. We examined whether B cells induce autoimmunity indirectly by activating autoreactive T cells via the following mechanism: a B cell binds and captures its cognate antigen from an antigen-expressing cell, and simultaneously captures other membrane components including self-antigens. The possibility arises that this B cell, in parallel with processing its cognate antigen, processes and presents this "bystander antigen" to T cells of the appropriate specificity, which in an inflammatory context would induce a self-destructive T cell response. We tested this hypothesis using adherent cells that express the CNS-restricted membrane protein myelin oligodendrocyte glycoprotein (MOG) as a model self-antigen, influenza hemagglutinin (HA) as a model viral antigen, and transgenic mouse B and T cells specific for each antigen. This antigen pair was chosen because both are integral membrane proteins, with a plausible role in autoimmunity. MOG is implicated in autoimmune neurological disorders and animal models thereof, notably in acute demyelinating encephalomyelitis, and influenza is among the most important known viral triggers of this autoimmune condition. In our study we observed the following two mechanisms: (i) cognate antigen is rapidly captured from membranes and induces strong activation of the capturing B cell and (ii) smaller quantities of other co-expressed, "bystander" antigens are co-captured at the same time and can be presented to T cells as well. This non-cognate antigen co-capture was observed in two different paradigms. HA-specific B cells co-capture MOG and present it to autoreactive MOG-specific T cells; and MOG-specific B cells co-capture a fusion protein of HA and ovalbumin and present it to ovalbumin-specific T cells. The fusion protein was generated because HA-specific T cells were not available. This phenomenon thus has two kinds of implications for autoimmunity: viral antigen-specific B cells can activate self-reactive T cells, and conversely, self-antigen-specific B cells can receive T cell help from virus-specific T cells, leading to the production of autoantibodies. These findings offer a possible explanation for the link between autoimmunity and viral infections.
Part II:
We reported the results of immunological monitoring of patients enrolled in a randomized, placebo-controlled, single-blind, phase 2a study testing two doses of GNbAC1 in patients with multiple sclerosis (MS). GNbAC1 is a recombinant, humanised, IgG4 monoclonal antibody directed against the envelope protein (Env) of the multiple sclerosis associated retrovirus (MSRV), a human endogenous retrovirus of the HERV-W family. The hypothesis that binding of MSRV-Env protein to Toll-like receptor 4 (TLR4) activates monocytes predicts that blocking this binding with GNbAC1 should decrease monocyte activation in patients with MS.
Ten MS Patients with a mean age of 53 years and a mean expanded disability status scale (EDSS) score of 4.8 were randomized into two groups (2 or 6 milligrams of GNbAC1 per kilogram of body weight given at 4 week intervals for 12 months). In addition to safety and clinical monitoring, three immunological parameters were followed: (i) T cell response to myelin and viral antigens by ELIspot using peripheral blood mononuclear cells (PBMC); (ii) Monocyte TLR4 signaling measured by flow cytometry of p38 phosphorylation; and (iii) general immune status as reflected in proportions of lymphocytes with given phenotypes.
In the immunological substudy (Results I of Part II of the presented work) ten patients and yoked healthy volunteers were followed. T cell response, as measured by interferon-gamma (IFN-γ) ELISpot, to viral antigens was robust throughout the study for both healthy subjects (range 22 to 322 spots per 200,000 PBMC) and patients (range 30 to 534 spots per 200,000 PBMC). Response to the myelin antigen Myelin Basic Protein (MBP) was not different from negative control in any subject at any time point. TLR4 signaling was analyzed before treatment start and at several timepoints after treatment by measuring phosphorylated p38 using intracellular immunofluorescence flow cytometry of CD33-positive cells, with and without lipopolysaccharide (LPS) stimulation. Without stimulation, fewer than 10% of monocytes showed p38 phosphorylation in patients and in controls at all time points, other than one time-point for one patient, in whom elevated C-reactive protein was also detected, suggesting an inflammatory infection. LPS stimulation (1 nanogram per milliliter in whole blood) resulted in almost 100% monocyte p38 phosphorylation in both patients and controls. There was a non-significant trend towards a reduced monocyte activation during treatment compared to before treatment. Immune cell subsets (CD4+ and CD8+ T cells, monocytes, and B cells) were stable over the treatment.
These results suggest that GNbAC1 administration is not associated with any deficit in T cell response to viral antigens. TLR4 activation might be reduced by the treatment. However, this result did not reach statistical significance probably due to the small number of investigated patients.
Advisors:Rolink, Antonius G. and Derfuss, Tobias Johannes and Lindberg Gasser, Raija L.P.
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Further Research Groups at DBM > Developmental and Molecular Immunology (Rolink)
Item Type:Thesis
Thesis no:11846
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (89 Seiten)
Language:English
Identification Number:
Last Modified:25 Oct 2016 10:59
Deposited On:25 Oct 2016 10:58

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