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The canonical Wnt-β-catenin signaling pathway arrests the differentiation of T cells and plays an important role in phenotypic maintenance of naive T cells and stem cell-like memory T cells in human peripheral blood, but its effect on tumor-infiltrating lymphocytes (TILs) from non–small cell lung cancer is little known. In this study, we showed that glycogen synthase kinase-3β inhibitor TWS119 has different effects on CD4+ and CD8+ T cells in TILs. TWS119 preserved the expansion of naive T cell and CD8+ stem cell-like memory T cells, and induced CD8+ effector T-cell proliferation in TILs. To further determine whether TWS119 impaired the effector function of TILs, TILs were stimulated with polyclonal stimulation, IL-2 and IFN-γ production were detected. Our data showed that TWS119 does not affect the production of IFN-γ in TILs compared with the control group; whereas TWS119 inhibited IFN-γ secretion of T cells from healthy donor. IL-2 production in CD4+ central memory T cells and CD4+ effector memory T cells from TILs was significantly increased with the TWS119 treatment; TWS119 also promoted the secretion of IL-2 in all cell subsets of CD8+ TILs. These findings reveal that TWS119 has a distinct effect on the proliferation and cytokine production of TILs, and provide new insights into the clinical application of TILs with TWS119 treatment for the adoptive immunotherapy.