Fas ligand-positive membranous vesicles isolated from sera of patients with oral cancer induce apoptosis of activated T lymphocytes.

Abstract

In patients with oral squamous cell carcinoma, a high proportion of T cells in the tumor undergo apoptosis, which correlates with Fas ligand (FasL) expression on tumor cells. The present study was done to identify mechanisms responsible for apoptosis of T cells seen in the peripheral circulation of these patients. Sera of 27 patients, normal donor sera, and supernatants of cultured normal or tumor cells were fractionated by size exclusion chromatography and ultracentrifugation to isolate microvesicles. The presence of microvesicle-associated FasL was studied by Western blots, blocking with anti-Fas reagents, and immunoelectron microscopy. Biological activities of microvesicles were tested including the ability to induce apoptosis of Jurkat and T-cell blasts. Semiquantitative analysis of FasL in microvesicles was correlated with caspase-3 activity, DNA fragmentation, cytochrome c release, loss of mitochondrial membrane potential, and TCR-zeta chain expression in lymphocytes. FasL-positive (FasL+) microvesicles were detected in sera of 21 of 27 patients. Microvesicles contained 42 kDa FasL. These microvesicles induced caspase-3 cleavage, cytochrome c release, loss of mitochondrial membrane potential, and reduced TCR-zeta chain expression in target lymphocytes. Biological activity of the FasL+ microvesicles was partially blocked by ZB4 anti-Fas monoclonal antibody. Microvesicle-associated FasL levels correlated with the patients' tumor burden and nodal involvement. Sera of patients with active oral squamous cell carcinoma contain FasL+ microvesicles, which induce the receptor and mitochondrial apoptotic pathways in Jurkat and activated T cells.