title: Flow cytometric assay detecting cytotoxicity against human endogenous retrovirus antigens expressed on cultured multiple sclerosis cells.
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.
authors: Department of Biomedicine, Aarhus University, Aarhus C, Denmark.
authors: A
authors: A
authors: Møller-Larsen
authors: T
authors: T
authors: Brudek
authors: T
authors: T
authors: Petersen
authors: E L
authors: EL
authors: Petersen
authors: M
authors: M
authors: Aagaard
authors: D T
authors: DT
authors: Hansen
authors: T
authors: T
authors: Christensen
mesh_terms: D000328:Adult; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology / Q000378:metabolism; D000920:Antibody-Dependent Cell Cytotoxicity; D000941:Antigens / Q000276:immunology*; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D020077:Endogenous Retroviruses / Q000276:immunology*; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007694:Killer Cells, Natural / Q000276:immunology; D008297:Male; D008875:Middle Aged; D009103:Multiple Sclerosis / Q000276:immunology* / Q000378:metabolism; D000069283:Rituximab; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism; D055815:Young Adult
chemical_list: D000906:Antibodies; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D000941:Antigens; D000069283:Rituximab
keywords: ADCC; CD107a; HERV; NK cells; flow cytometry
references: Clin Exp Immunol. 2002 Nov;130(2):172-82
references: 12390303
references: Retrovirology. 2009 Nov 16;6:104
references: 19917105
references: Exp Hematol. 2006 Jun;34(6):753-9
references: 16728280
references: J Virol. 2006 Apr;80(7):3634-43
references: 16537631
references: Trends Neurosci. 2002 Jun;25(6):313-9
references: 12086750
references: Immunol Rev. 2005 Apr;204:208-31
references: 15790361
references: Acta Neurol Scand Suppl. 1997;169:49-58
references: 9174640
references: J Immunol Methods. 2005 Jan;296(1-2):63-76
references: 15680151
references: Methods Mol Biol. 2010;612:335-52
references: 20033652
references: Mult Scler. 2012 Dec;18(12):1721-36
references: 22457345
references: Leukemia. 2005 May;19(5):707-9
references: 15744339
references: Mult Scler. 1998 Jun;4(3):111-7
references: 9762657
references: J Immunol. 2009 Jan 15;182(2):1202-10
references: 19124764
references: Immunology. 2009 Apr;126(4):458-65
references: 19278419
references: Biol Res Nurs. 2003 Oct;5(2):142-52
references: 14531218
references: Virology. 2001 Jan 20;279(2):558-69
references: 11162811
references: Curr Opin Immunol. 2009 Dec;21(6):634-40
references: 19892538
references: Immunobiology. 2008;213(8):629-40
references: 18765168
references: J Neuroimmunol. 2007 Feb;183(1-2):175-88
references: 17113160
references: Brain. 2005 Jul;128(Pt 7):1667-76
references: 15800022
references: Autoimmun Rev. 2011 Apr;10(6):364-7
references: 21195807
references: J Neuroimmunol. 2008 Feb;194(1-2):147-52
references: 18155780
references: J Immunol. 2011 Jul 1;187(1):570-9
references: 21622858
references: Arch Neurol. 2005 Oct;62(10):1620-3
references: 16216948
references: Mult Scler. 1995 Jun;1(2):78-81
references: 9345456
references: Clin Immunol. 1999 Dec;93(3):283-93
references: 10600340
references: Ann Neurol. 2009 Aug;66(2):132-41
references: 19743458
references: J Immunol Methods. 2003 Oct 1;281(1-2):65-78
references: 14580882
references: Rev Med Virol. 2005 May-Jun;15(3):179-211
references: 15782388
references: Methods Mol Med. 2007;131:221-30
references: 17656786
references: J Neuroimmunol. 2010 Apr 15;221(1-2):7-14
references: 19931190
references: Am J Pathol. 2009 Sep;175(3):1160-6
references: 19700745
references: J Immunol Methods. 2004 Nov;294(1-2):15-22
references: 15604012
references: AIDS Res Hum Retroviruses. 2006 Dec;22(12):1253-9
references: 17209768
references: Neurology. 2010 Jan 5;74 Suppl 1:S2-8
references: 20038759
references: Breast Cancer Res Treat. 2005 Jul;92(1):85-93
references: 15980996
references: Proc Natl Acad Sci U S A. 2004 Feb 10;101(6):1731-6
references: 14757826
references: J Virol. 2012 Apr;86(7):3713-22
references: 22278236
references: Lancet Neurol. 2008 Sep;7(9):852-8
references: 18703007
references: Neurol Sci. 2001 Apr;22(2):171-3
references: 11603622