A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes

Journal of Immunological Methods
Andrea WerenAndreas Ambach

Abstract

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by...Continue Reading

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Citations

Jun 4, 2010·Expert Review of Vaccines·Liubov ZaritskayaAnatoli M Malyguine
Nov 30, 2010·Journal of Immunological Methods·Theodore S JohnsonJack J Bleesing
Feb 10, 2006·Journal of Photochemistry and Photobiology. B, Biology·Andreas AmbachHarald Gollnick
May 4, 2013·Seizure : the Journal of the British Epilepsy Association·Gang LiHajo M Hamer
Feb 4, 2014·Journal of Immunological Methods·Janet Kerstin PeperStefan Stevanović
Jul 15, 2016·Journal of Immunology Research·Snehal ShabrishManisha Madkaikar
Sep 27, 2018·The Journal of Infectious Diseases·Elisabeth A van ErpPuck B van Kasteren

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