Analysis of the tumor reactivity of autologous TILs and allogeneic γδ T cells via tumor organoid–immune cell coculture

The efficacy of conventional αβ T cell-based immunotherapies is often limited by tumor immune evasion. γδ 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 γδ 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 Vγ9Vδ2 T cell. T-cell activation was quantified by comparing CD137 expression on γδ T cells versus CD4+ and CD8+ T cells via flow cytometry after coculture. The cytotoxicity of Vγ9Vδ2 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-γ, granzyme B, perforin) were measured from coculture supernatants using a cytometric bead array. Baseline analysis revealed that γδ 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 γδ 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 γδ T cells was more than double that of CD8 T cells (12.55% vs. 5.66%, p = 0.0467). Allogeneic Vγ9Vδ2 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-γ ( > 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 γδ T cells, establishing them as a primary reactive subset capable of bypassing MHC-restriction bottlenecks. The potent dismantling of tumor architecture by allogeneic Vγ9Vδ2 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. A patient-derived breast cancer organoid (BCO)–immune cell coculture system is established to mimic the tumor microenvironment. Autologous γδ T cells exhibit superior baseline reactivity and achieve preferential tumor-induced activation (24.85% CD137+) after BCO coculture. Allogeneic Vγ9Vδ2 T cells mediate potent, dose-dependent cytotoxicity against BCOs (maximal efficacy at 10:1 E:T ratio). Cytotoxicity of Vγ9Vδ2 T cells is driven by IFN-γ, granzyme B, and perforin secretion. The coculture system serves as a preclinical tool for evaluating γδ T cell-based immunotherapies.