CD4+T Cells in CIKs (CD4+ CIKs) Reversed Resistance to Fas-Mediated Apoptosis Through CD40/CD40L Ligation Rather Than IFN-γ Stimulation

摘要

Background: Cytokine-induced killer cells (CIKs) are nonspecific antitumor effectors with superior advantages.nCD4u0002 CIKs can induce Fas-dependent apoptosis in sensitive Raji cells. Here, a Fas-dependentnapoptosis was detected in resistant breast cancer MDA-MB-231 cells, and underlying mechanisms werendiscriminated. Methods: Amplification of CIKs and purification of CD4u0002 CIKs were performed in 15 patientsnwith malignant solid tumors. The expression of CD40L and soluble cytokines in CD4u0002 CIKs werenanalyzed. The apoptotic rates of tumor cells and the expression of Fas on membranes were detected usingnflow cytometry assay. The specific blocking antibodies against FasL, CD40L, and interferon-u0002 (IFN-u0002)nwere added to abolish their effects. The changes of 4 apoptosis-related genes (Bcl-2, Bax, Fas-associatingnprotein with death domain [FADD], and FLICE inhibitory protein [c-FLIP]) in MDA-MB-231 cellsncocultured with CD4u0002 CIKs were measured by real-time quantitative reverse-transcriptase polymerasenchain reaction after 6 hours and 24 hours with or without blocking antibodies. Results: Upregulated expressionnof membrane-attached CD40L and dramatically increased secretion of soluble CD40L and IFN-nu0002 were identified in CD4u0002 CIK. The susceptibility to Fas-mediated apoptosis of insensitive MDA-MB-231ncells was elevated after being pretreated with supernatants from CD4u0002 CIK. After coculture with CD4u0002nCIK, apoptosis in MDA-MB-231 cells paralleled with enhanced expression of Fas was blocked fully byneither anti-FasL or anti-CD40L, but only partly by anti-IFN-u0002 antibodies. The anti-CD40L monoclonalnantibody (McAb) rather than anti-IFN-u0002 McAb induced significant increase of c-FLIP, which negativelyncorrelated with the apoptosis observed in MDA-MB-231 cells. Conclusions: Apoptosis in MDA-MB-231ncells induced by CD4u0002 CIK is Fas-dependent. The reversion of Fas resistance is mediated throughnCD40/CD40L ligation rather than IFN-u0002 stimulation by inhibiting synthesis of c-FLIP.