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Background: Currently, many studies suggest that cancer stem cells (CSCs) are responsible for tumor initiation, tumorigenesis, metastasis and recurrence. CSCs have been identified from various human and murine tumors. The identification of CSCs allows us to develop strategies to target the CSCs. Methods and Results: In this study, we used ALDEFLUOR as a single marker to isolate the CSCs from the human lung cancer cell line H460. We then characterized the CSCs by testing their sphere formation ability and tumorigenicity. Furthermore, we used CSC lysate-pulsed dendritic cells to stimulate CD8+T cells as a treatment strategy. Our study demonstrated that ALDEFLUOR could be used as a single marker to identify CSCs from the human lung cancer cell line H460. The ALDHhigh cells could form more spheres and were more tumorigenic than the ALDHlow cells. Further study demonstrated that ALDHhigh-CD8+T cells conferred more significant antitumor effects, resulting in the inhibition of tumor growth and prolonged survival. And the ALDHhigh-CD8+T cells-mediated anti-tumor immunity might be due to the directly targeting against ALDHhigh cancer stem cells (CSCs). Conclusions: This study shows that ALDHhigh-CD8+T cells mediate anti-tumor immunity by selectively targeting cancer stem cells, which result in inhibiting tumor growth and prolonging the survival of tumor-bearing mice, which provides a new strategy using ALDHhigh-CD8+T cells to treat tumors. [ABSTRACT FROM AUTHOR]