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The immune checkpoint blockade (ICB) immunotherapy has prolonged overall survival for cancer patients but the response rates are low. The resistance to ICB is likely due to compensatory upregulation of additional immune inhibitory molecules. In this study, we first systematically examined Tim-3 expression in immune cells in mouse tumors and found that Tim-3 was specifically up-regulated in a large number of Treg, conventional CD4 + , CD8 + T cells, dendritic cell 1 (DC1), and macrophage 1 (M1) in the tumor microenvironment (TME). Interestingly, Tim-3 + T cells in the TME were phenotypically effector but not "exhausted" T cells because Tim-3 + PD-1 + CD8 + T cells had a higher number of mitochondria, greater levels of glycolysis, and higher tumor-specific cytolytic activities compared to Tim-3 - PD-1 - CD8 + T cells. The combination treatment with Tim-3 and PD-1 mAbs resulted in a synergistic antitumor activity but also increased the expression of Lag-3 and GITR in TIL, demonstrating cross-regulation between multiple checkpoint molecules. Furthermore, we found that the antitumor efficacy with triple combination of Tim-3, PD-1, and Lag3 mAbs was much greater than any two antibodies. Mechanistically, we demonstrated that simultaneous targeting of Tim-3, PD-1, and Lag-3 cooperatively increased the levels of granzyme B and tumor-specific cytolytic activities of CD8 + TIL. Our data indicate that multiple checkpoint molecules are coordinately upregulated to inhibit the function of hyperactivated T cells in the TME and requirement for the simultaneous blockade of PD-1, Tim-3 and Lag3 for cancer treatment., (© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.)