@article{Zhang2026, 
author = {Ying Zhang and Sicheng Du and Rongrui Liu and Chuanhua Zhao and Juan Li and Sisi Ye and Man Zhang and Xingming Ma and Zhou He and Wenjia Zhuang and Huajun Jin and Jianming Xu},
title = {Expansion of IL-2-independent tumor-infiltrating lymphocytes through a feeder-free process: a preclinical study for solid tumors},
year = {2026},
journal = {Cancer Biology & Medicine},
volume = {23},
number = {6},
pages = {910-924},
keywords = {Tumor-infiltrating lymphocytes, hydroxychloroquine, solid tumors, PD-1 blockade, MHC class Ⅰ},
url = {https://www.sciopen.com/article/10.20892/j.issn.2095-3941.2025.0441},
doi = {10.20892/j.issn.2095-3941.2025.0441},
abstract = {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.}
}