TY  - JOUR
AU  - Zhang, Ying
AU  - Du, Sicheng
AU  - Liu, Rongrui
AU  - Zhao, Chuanhua
AU  - Li, Juan
AU  - Ye, Sisi
AU  - Zhang, Man
AU  - Ma, Xingming
AU  - He, Zhou
AU  - Zhuang, Wenjia
AU  - Jin, Huajun
AU  - Xu, Jianming
PY  - 2026
TI  - Expansion of IL-2-independent tumor-infiltrating lymphocytes through a feeder-free process: a preclinical study for solid tumors
JO  - Cancer Biology & Medicine
SN  - 2095-3941
SP  - 910
EP  - 924
VL  - 23
IS  - 6
AB  - ObjectiveConventional 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.MethodsTILs 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.ResultsThe 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.ConclusionsThe 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.
UR  - https://doi.org/10.20892/j.issn.2095-3941.2025.0441
DO  - 10.20892/j.issn.2095-3941.2025.0441