Release of preformed Fas ligand in soluble form is the major factor for activation‐induced death of Jurkat T cells

Abstract

SUMMARYInteraction of Fas/APO-1 (CD95) and its ligand (FasL) plays an important role in the activation-induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T-cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non-activated Jurkat cells, and also of L1210Fas, but not that of Fasnegative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non-activated counterparts. Time-course analysis revealed that PHA-stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti-Fas monoclonal antibody, suggesting that PHA-stimulated Jurkat cells exert Fas-based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non-activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti-FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of preformed FasL in soluble form upon stimulation.

Enhanced expression of IFN-γ mRNA in CD4+ or CD8+ tumour-infiltrating lymphocytes compared to peripheral lymphocytes in patients with renal cell cancer

“…It may be concluded from the concomitantly high levels of IL-10 mRNA and IFN-γ mRNA in CD8 + TIL that Th1 as well as Th2 lymphocytes are activated, which is in accordance with a recently published study showing also high levels of IL-10 and IFN-γ mRNA in TIL from non-small cell lung cancer and ovarian cancer biopsies (Asselin-Paturel et al, 1998;Pisa et al, 1992). Since it has recently been shown that in vitro activated T cells express high levels of FasL mRNA and protein (Alderson et al, 1995;Brunner et al, 1995;Dhein et al, 1995;Martinez-Lorenzo et al, 1996), the elevated FasL mRNA levels in RCC TIL also indicate an immunological activation of these lymphocytes.…”

Induction of apoptosis and modulation of activation and effector function in T cells by immunosuppressive drugs

“…All other ISD tested did not modulate CD95-sensitivity in Jurkat cells or in peripheral T cells. AICD in Jurkat T cells [45] and peripheral human T cells [46] is mediated mainly by an increased production and release of CD95L. Therefore we hypothesized that ISD might induce an accelerated AICD by increased CD95L production.…”

Vesicles Released by Activated T Cells Induce Both Fas-Mediated RIP-Dependent Apoptotic and Fas-Independent Nonapoptotic Cell Deaths

“…Cytotoxic activity was detectable within 15 min after stim-ulation and could be attenuated by anti-Fas neutralizing Ab, providing evidence for functional, preformed FasL secreted upon PHA activation (26)(27)(28)(29)32). Following CSup ultracentrifugation, the cytotoxic activity was retained in the pellet, and FasL could be visualized by electron microscopy in structures of 50-200 nm in diameter either in intracellular cellular stores or in the supernatant of the activated cells by Western blot (25,30,32).…”

“…The presence of FasL-expressing soluble granules has been observed in several culture supernatants (CSups) derived from PHA-prestimulated Jurkat cells, PBMC, or melanoma cells (25)(26)(27)(28)(29)(30)(31)(32)(33). Cytotoxic activity was detectable within 15 min after stim-ulation and could be attenuated by anti-Fas neutralizing Ab, providing evidence for functional, preformed FasL secreted upon PHA activation (26)(27)(28)(29)32).…”

“…A functional, soluble form of microvesicle-bound FasL could also be obtained after CD3 engagement (27,31,34) or upon stimulation of cells by PHA or PMA plus ionomycin (32).…”