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In a previous study, we constructed a MHSP65-TCL anti-lung cancer vaccine with Lewis lung carcinoma TCL plus MHSP65, and illustrated its anti-lung cancer effect through specific and nonspecific anti-tumor immunity. However, TCL contains some immunoinhibit components such as FasL. If this component can be eliminated from TCL, the anti-tumor immunity of MHSP65-TCL constructed with TCL should be improved. In the present study, we knocked down FasL from Lewis lung carcinoma cells and prepared MHSP65-(FasL-/TCL) with this cell line's TCL. After further investigation, MHSP65-(FasL-/TCL) exhibited a better ability to reduce splenocytes apoptosis, promote its activation and secretion of secretingTNF-β, IL-2 compared with MHSP65-(FasL +/TCL). Accordingly, specific and nonspecific antitumor immunity induced by MHSP65-(FasL-/TCL) is stronger than that of MHSP65-(FasL +/TCL). In vivo, MHSP65-(FasL-/TCL) immunization can prolong survival of Lewis lung carcinoma bearing mice. Thus, we report that the anti-lung cancer effect of MHSP65-TCL can be improved by removal of FasL from the TCL. It provides a new route to construct MHSP65-TCL and other antitumor vaccines based on TCL.Constructed a MHSP65-(FasL-/TCL) anti-lung cancer vaccine with the TCL removed FasL (FasL-/TCL).MHSP65-(FasL-/TCL) can induce stronger nonspecific and specific anti-lung cancer immunity than MHSP65-(FasL+/TCL).MHSP65-(FasL-/TCL) immunization can prolong survival of tumor bearing mice compared with that of MHSP65-(FasL+/TCL).Theoretically, the method eliminated FasL from TCL can be applied in all the immunotherapy based on TCL.