通过肿瘤类器官-免疫细胞共培养分析自体 TIL 与异体 γδ T 细胞的肿瘤反应性

The efficacy of conventional T cell-based immunotherapies is often limited by tumor immune evasion. T cells can bypass these limitations through MHC-independent tumor recognition, but their function within the tumor microenvironment (TME) remains poorly characterized due to a lack of relevant preclinical models. This study aims to establish a patient-derived tumor organoid-immune cell coculture system to evaluate T-cell reactivity and cytotoxicity in a human-relevant TME (n = 10 PDTO lines; n = 3 independent experiments).

We developed an innovative coculture system believed to be the first to integrate patient-derived tumor organoids (PDTOs) and autologous tumor-infiltrating lymphocytes (TILs) or healthy donor-derived allogeneic V 9V 2 T cell. T-cell activation was quantified by comparing CD137 expression on T cells versus CD4+ and CD8+ T cells via flow cytometry after coculture. The cytotoxicity of V 9V 2 T cells was evaluated at various effector-to-target (E:T) ratios using a live-cell imaging assay to track BCO infiltration and apoptosis over 24 hours. Secreted effector molecules (IFN- , granzyme B, perforin) were measured from coculture supernatants using a cytometric bead array.

Baseline analysis revealed that T cells represent the most reactive subset within expanded TILs, with 12.3% expressing CD137 compared to 3.49% of CD8+ T cells (p = 0.0118). Notably, autologous BCO TIL coculture preferentially enhanced T-cell activation, reaching 24.85% CD137+ a frequency significantly higher than that of CD8 (9.15%, p = 0.0062) and CD4 (9.99%) subsets. The net increase in CD137 positivity for T cells was more than double that of CD8 T cells (12.55% vs. 5.66%, p = 0.0467). Allogeneic V 9V 2 T cells demonstrated significant, dose-dependent cytotoxicity against BCOs (p < 0.0001). Maximal cell death (RFU fold-change: 5.2 0.5) was achieved at an effector-to-target (E:T) ratio of 10:1 (p < 0.0001). This cytotoxic efficacy was highly correlated with the secretion of lytic effectors, including IFN- ( > 1000 pg/mL; p< 0.001), granzyme B ( > 2000 pg/mL), and perforin ( > 350 pg/mL).

Our findings demonstrate that 3D autologous coculture leads to the preferential stimulation of T cells, establishing them as a primary reactive subset capable of bypassing MHC-restriction bottlenecks. The potent dismantling of tumor architecture by allogeneic V 9V 2 T cells, supported by high-resolution kinetic and cytokine data, substantiates their development as off-the-shelf therapies. Collectively, this platform provides a standardized, high-fidelity engine for the rapid preclinical assessment of next-generation cellular immunotherapies.