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High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum
Published on: November 23, 2013
Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured
A Møller-Larsen1, T Brudek, T Petersen
1Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
Abstract:
Damage of target cells by cytotoxicity, either mediated by specific lymphocytes or via antibody-dependent reactions, may play a decisive role in causing the central nervous system (CNS) lesions seen in multiple sclerosis (MS). Relevant epitopes, antibodies towards these epitopes and a reliable assay are all mandatory parts in detection and evaluation of the pertinence of such cytotoxicity reactions. We have adapted a flow cytometry assay detecting CD107a expression on the surface of cytotoxic effector cells to be applicable for analyses of the effect on target cells from MS patients expressing increased amounts of human endogenous retrovirus antigens. MS patients also have increased antibody levels to these antigens. The target cells are spontaneously growing peripheral blood mononuclear cells (PBMCs) of B cell lineage, expressing human endogenous retrovirus HERV epitopes on their surface. Polyclonal antibodies against defined peptides in the Env- and Gag-regions of the HERVs were raised in rabbits and used in antibody-dependent cell-mediated cytotoxicity (ADCC) -assays. Rituximab® (Roche), a chimeric monoclonal antibody against CD20 expressed primarily on B cells, was used as control antibody. Without antibodies this system is suitable for analyses of natural killer cell activity. In optimization of the assay we have used effector lymphocytes from healthy donors. The most effective effector cells are CD56(+) cells. CD8(+) T cells also express CD107a in ADCC. Using the adapted assay, we demonstrate significant ADCC activity to target cells expressing HERV epitopes, and additionally a low level of NK activity.
Insights
Cytotoxicity against human endogenous retrovirus (HERV) antigens on B cells may contribute to multiple sclerosis (MS) central nervous system lesions. This study developed a flow cytometry assay to detect antibody-dependent cell-mediated cytotoxicity (ADCC) against HERV-expressing cells in MS patients.
Area of Science:
- Immunology
- Neuroscience
- Virology
Background:
- Central nervous system (CNS) lesions in multiple sclerosis (MS) may result from cell damage via cytotoxicity, including antibody-dependent reactions.
- Human endogenous retrovirus (HERV) antigens are upregulated on target cells in MS patients, who also exhibit increased antibody levels against these antigens.
- Evaluating cytotoxicity requires specific epitopes, antibodies, and a reliable assay to assess their relevance.
Purpose of the Study:
- To adapt a flow cytometry assay for detecting CD107a expression on cytotoxic effector cells.
- To analyze the impact of this assay on target cells from MS patients expressing HERV antigens.
- To investigate antibody-dependent cell-mediated cytotoxicity (ADCC) against HERV-expressing B cells in MS.
Main Methods:
- Adapted a flow cytometry assay to detect CD107a surface expression on cytotoxic effector cells.
- Utilized peripheral blood mononuclear cells (PBMCs) from MS patients as target cells, expressing HERV epitopes.
- Employed polyclonal antibodies against HERV Env and Gag regions, and Rituximab as a control, in ADCC assays with effector lymphocytes from healthy donors.
Main Results:
- Demonstrated significant ADCC activity against target cells expressing HERV epitopes.
- Identified CD56(+) cells as the most effective effector cells, with CD8(+) T cells also contributing to ADCC.
- Observed a low level of natural killer (NK) cell activity in the adapted assay system.
Conclusions:
- The adapted flow cytometry assay is suitable for analyzing cytotoxicity reactions involving HERV antigens in MS.
- ADCC against HERV-expressing cells is a significant finding in the context of MS pathogenesis.
- The assay also allows for the assessment of NK cell activity, providing a comprehensive tool for immunological studies in MS.
