摘要Objective:Conventional tumor-infiltrating lymphocyte(TIL)therapy for solid tumors relies on high-dose interleukin-2(IL-2)during expansion and post-infusion,and promotes T-cell exhaustion and toxicity.Herein,we developed a feeder-free,low-dose IL-2 TIL expansion protocol and evaluated whether hydroxychloroquine(HCQ)or programmed cell death protein 1(PD-1)blockade might enhance therapeutic efficacy and decrease IL-2 dependence.Methods:TILs from multiple solid tumors were expanded ex vivo with decreased-dose IL-2,IL-7,and IL-15 plus CD3/CD28 co-stimulation,without feeder cells.TIL products were assessed via quality control,T-cell phenotypes,and exhaustion markers.Cytotoxic activity was measured in vitro through interferon-gamma(IFN-γ)release and real-time cell analysis(RTCA).HCQ-induced changes in major histocompatibility complex class Ⅰ(MHC-I)and programmed death-ligand 1(PD-L1)expression were assessed in tumor cell lines,and RTCA-based cytotoxicity was evaluated using T-cell receptor-engineered T cells(TCR-T cells).The in vivo efficacy of HCQ and PD-1 blockade separately combined with TIL therapy was examined in a colorectal cancer patient-derived xenograft(PDX)model.Results:The protocol consistently produced viable TILs of favorable quality across tumor types,with variable CD8+and memory T-cell profiles.Expanded TILs showed effector-to-target(E:T)ratio-dependent tumor cell killing in RTCA and secreted IFN-γ across multiple tumor types.HCQ significantly upregulated MHC-I expression in vitro(P<0.05)without affecting PD-L1 expression or impairing TIL proliferation,and enhanced early TCR-T-mediated killing.In the PDX model,TIL plus HCQ,compared with TIL,showed less tumor growth and greater MHC-I expression,although these differences were not significant,given the small sample size.TIL plus low-dose PD-1 blockade significantly reduced tumor volume versus the control group(P=0.002)and maintained higher body weights than the TIL-only and control groups.Conclusions:The feasibility of a feeder-free,low-dose IL-2 TIL expansion system was demonstrated.PD-1 blockade significantly enhanced antitumor activity and treatment tolerability,thus supporting its promise as an alternative to high-dose IL-2.HCQ demonstrated potential immunomodulatory effects,although its in vivo benefit was minimal.This strategy warrants further clinical evaluation in solid tumors.