Abstract
Among the different techniques available for determining physiological cell death or apoptosis in immune cells, fluorescence-activated cell sorting-based approaches prove to be one of the most efficient and quantitative assays in capturing cells that are actively undergoing apoptosis elicited by either extrinsic or intrinsic forms of cell death. The key advantage of the technique is to allow the user to determine apoptotic responses of multiple immune cell types or subsets of the same lineage of immune cells without the prior requirement for cell separation. Here, we describe a “multiparameter” flow method to rapidly determine apoptosis-sensitivity induced by the Fas receptor pathway within different CD4 T cell subsets in a mixed pool of analyzed ex vivo peripheral blood mononuclear cells.
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References
Siegel RM, Chan FK, Chun HJ, Lenardo MJ (2000) The multifaceted role of Fas signaling in immune cell homeostasis and autoimmunity. Nat Immunol 1:469–474
Fisher GH, Rosenberg FJ, Straus SE, Dale JK, Middleton LA, Lin AY, Strober W, Lenardo MJ, Puck JM (1995) Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome. Cell 81:935–946
Straus SE, Sneller M, Lenardo MJ, Puck JM, Strober W (1999) An inherited disorder of lymphocyte apoptosis: the autoimmune lymphoproliferative syndrome. Ann Intern Med 130:591–601
Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S (1992) Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature 356:314–317
Ramaswamy M, Cruz AC, Cleland SY, Deng M, Price S, Rao VK, Siegel RM (2011) Specific elimination of effector memory CD4+ T cells due to enhanced Fas signaling complex formation and association with lipid raft microdomains. Cell Death Differ 18:712–720
Hughes PD, Belz GT, Fortner KA, Budd RC, Strasser A, Bouillet P (2008) Apoptosis regulators Fas and Bim cooperate in shutdown of chronic immune responses and prevention of autoimmunity. Immunity 28:197–205
Oliveira JB, Bleesing JJ, Dianzani U, Fleisher TA, Jaffe ES, Lenardo MJ, Rieux-Laucat F, Siegel RM, Su HC, Teachey DT, Rao VK (2010) Revised diagnostic criteria and classification for the autoimmune lymphoproliferative syndrome (ALPS): report from the 2009 NIH International Workshop. Blood 116:e35–e40
Turbyville JC, Rao VK (2010) The autoimmune lymphoproliferative syndrome: a rare disorder providing clues about normal tolerance. Autoimmun Rev 9:488–493
Kovacs B, Vassilopoulos D, Vogelgesang SA, Tsokos GC (1996) Defective CD3-mediated cell death in activated T cells from patients with systemic lupus erythematosus: role of decreased intracellular TNF-alpha. Clin Immunol Immunopathol 81:293–302
Snow AL, Marsh RA, Krummey SM, Roehrs P, Young LR, Zhang K, van Hoff J, Dhar D, Nichols KE, Filipovich AH, Su HC, Bleesing JJ, Lenardo MJ (2009) Restimulation-induced apoptosis of T cells is impaired in patients with X-linked lymphoproliferative disease caused by SAP deficiency. J Clin Invest 119: 2976–2989
Acknowledgements
This work was supported by the Intramural Research Program of National Institute of Allergy and Infectious Diseases and National Institute of Arthritis and Musculoskeletal and Skin Diseases.
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Lo, B., Ramaswamy, M. (2013). Determination of Apoptosis Sensitivity in Specific T Cell Subsets from Human Peripheral Blood by Utilizing a Multiparameter Fluorescence-Activated Cell Sorting-Based Technique. In: Snow, A., Lenardo, M. (eds) Immune Homeostasis. Methods in Molecular Biology, vol 979. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-290-2_4
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DOI: https://doi.org/10.1007/978-1-62703-290-2_4
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Publisher Name: Humana Press, Totowa, NJ
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