Detecting Tumor Reactivity of Autologous and Allogeneic γδ T Cells via Tumor Organoid-Immune Cells Co-Culture

Abstract Background: Cancer immunotherapies primarily target conventional αβ T cells, yet their clinical effectiveness is frequently undermined by immune evasion mechanisms inherent to tumors. γδ T cells, a distinct class of unconventional lymphocytes, recognize tumor-associated antigens independently of MHC molecules, potentially overcoming these limitations. However, comprehensive evaluations of γδ T cell functionality within the tumor microenvironment (TME) remain scarce. Methods: We developed an innovative patient-derived tumor organoid (PDTO) and immune cell co-culture platform, integrating autologous tumor-infiltrating lymphocytes (TILs) and healthy donor-derived allogeneic Vγ9Vδ2 T cells. Utilizing fluorescent cell-tracing, multiplex cytokine analyses, and flow cytometry, we assessed γδ T cell infiltration, activation, and cytotoxicity within breast cancer organoids, closely mimicking physiological tumor conditions. Results: Our findings reveal that autologous γδ T cells demonstrate significantly enhanced tumor-specific activation compared to conventional αβ T cell subsets, as indicated by elevated CD137 expression. Additionally, allogeneic Vγ9Vδ2 T cells exhibited robust cytotoxic activity against breast cancer organoids, with cytotoxic efficacy correlating with higher effector-to-target ratios and increased secretion of IFN-γ, granzyme B, and perforin. Conclusion: The established PDTO–immune cell co-culture platform provides a physiologically relevant model for evaluating γδ T cell reactivity and cytotoxicity. These results underscore the potential of γδ T cells as effective candidates for cancer immunotherapy, offering significant promise for developing novel therapeutic strategies against solid tumors.