Our previously reported data indicated that T cells found in the peripheral circulation and at tumor sites of patients with cancer had high rates of spontaneous apoptosis, which was, in part, mediated by the Fas/FasL pathway (Reichert et al., Clin. Cancer Res., 8:3137, 2002). Although T-cell apoptosis in the tumor correlated with FasL expression on tumor cells, no adequate explanation was provided for apoptosis of circulating peripheral blood T cells. This study attempts to identify a mechanism that could explain the observed increased rates of apoptosis in circulating T cells of patients with cancer. Sera of 27 patients with oral squamous cell carcinoma (OSCC), normal sera and supernatants of normal and tumor cells were fractionated by size exclusion chromatography and ultracentrifugation. The microvesicle (MV)-containing fractions were isolated. MV-associated FasL (42 kDa) and MHC class I antigens were quantified by Western blots. Biologic activities of MV were studied by 24 h co-incubation of Fas-sensitive and CH11 antibody (Ab)-resistant (Fas-resistant) Jurkat T cells, with each MV fraction normalized/μg protein; apoptosis was measured in JAM assays, and caspase 8 and 3 cleavage was detected in Western blots. Quantitative flow cytometry-based assays for TCR-ζ chain expression and mitochondrial instability in Jurkat cells co-incubated with MV were also performed. FasL+ MV were detected in sera of 21/ 27 patients but not in control sera. Both FasL+ and FasL- MV fractions were found to be biologically active. They induced apoptosis in both Fas-sensitive and CH11 Ab-resistant Jurkat cells in DNA fragmentation (JAM) assays. In selected fractions, a 32 kDa prodomain of caspase 3 was cleaved in the presence of Fas neutralizing mAb. MHC class I antigen-containing MV were also found to be involved in the induction of apoptosis in Jurkat T cells. MV fractions also caused a significant reduction in TCR-ζ chain expression in T cells. The data are consistent with hypothesis that both Fas/FasL-dependent and –independent pathways are involved in apoptosis mediated by MV. Importantly, the level of MV-associated FasL was significantly correlated to tumor burden, nodal involvement and disease stage. Our results show that sera of patients with cancer contain MV which can induce apoptosis of activated T cells. Both the receptor and mitochondrial apoptotic pathways in T cells are induced by MV. Biologic importance of MV is underlined by the fact that sera of patients with advanced disease contained MV with significantly higher levels of FasL than sera of patients with less advanced disease.
[Proc Amer Assoc Cancer Res, Volume 45, 2004]