Abstract: Objective: To purify the anti-T cell immunoglobulin mucin (TIM)-3 monoclonal antibody 4E8 and examine its biological function in vitro. Methods: The mouse monoclonal antibody against human TIM-3, clone 4E8, was obtained by standard protocol for monoclonal antibody purification. The cell lines expressing human TIM-3 molecule were obtained by cell transfection technique. We examined the ability of 4E8 binding to human TIM-3 by flow cytometry. The ability of 4E8 blocking the binding of fusion protein TIM-3 Ig-huFc with phosphatidylserine(PtdSer), the apoptotic cell surface TIM-3 ligand, was also analyzed by flow cytometry. Mixed lymphocyte reaction (MLR) and ELISA assays were used to determine the effect of TIM-3 monoclonal antibody (4E8) on IFN-γ secretion in CD4⁺ T cells. Results: 4E8 specifically bound to human TIM-3 but could not block the binding of TIM-3 to Ptdser. Compared with the negative control (IFN-γ secretion: 958.3±153.2), 4E8 enhanced the ability of CD4⁺ T cells to secrete IFN-γ in MLR (4E8 of 10 μg/mL group: IFN-γ secretion 2 563±150.3 and 4E8 of 3.33 μg/mL group: IFN-γ secretion 1 981±211.5) with statistically significant difference (P＜0.05). In addition, the combined application of 4E8 with the anti-programmed death-1 (PD-1) monoclonal antibody nivolumab showed synergistic effects for increasing IFN-γ secretion in MLR assay (4E8 of 10 μg/mL group: IFN-γ secretion 3 049±80.5 and 4E8 of 0.33 μg/mL group: IFN-γ secretion 1 957±321.3) as compared with 4E8 alone (10 μg /mL group: IFN-γ secretion 2 563±150.3 and 0.33 μg/mL group: IFN-γ secretion 844±76.2) with statistically significant difference (P＜0.05). Conclusion: We successfully obtained a 4E8 clone of monoclonal antibody to human TIM-3 which may enhance the capacity of IFN-γ secretion from CD4⁺ T cells. The effect of enhancing IFN-γ secretion of CD4⁺T cells by TIM-3 monoclonal antibody was independent from blocking the binding of TIM-3 with Ptdser.