Tim-3 upregulation after PD-1 blockade is PI3K/Akt pathway dependent. TIL from HNSCC patient were treated with anti-CD3/CD28 beads in combination with DMSO, broad PI3K inhibitor (LY294002), PI3K p110α/mTOR inhibitor (CAY10626), Akt inhibitor, PI3K p110δ inhibitor (GS-1101) and PI3K p110γ inhibitor (AS041164) with or without anti-PD-1 mAbs (Nivolumab) for 48 h. Cells are collected and Tim-3, p-S6 expressions were assessed by flow cytometry. (A) Summary data showing Tim-3 and p-S6 MFI, (n = 4), significance were calculated with ordinary one-way ANOVA, *p < 0.05, **p < 0.001. (B) Summary data showing Tim-3 MFI fold change in the presence of selective PI3K inhibitors with anti-PD-1 or IgG4. (n = 4) Significance was calculated with unpaired t test, *p < 0.05.