Background Glioblastoma (GBM) is a clinical unmet need that could benefit from targeted immunotherapy, such as adoptive transfer of engineered T cells. Gamma-delta (γδ) T cells are a promising cellular substrate as they naturally possess the ability to target GBM using endogenous cytotoxicity receptors. Yet, treatment of GBM with γδ T cells has revealed little evidence of clinical utility. We hypothesize that genetic engineering will enable the production of γδ T cells with optimized anti-GBM activity and overcome the limited efficacy noted thus far. Here, we investigated γδ T cell anti-GBM activity and explored the role IL-15 plays in important effector functions, notably proliferation, to inform our engineering strategy.

Methods Human Vγ9Vδ2 (γδ) T cells were expanded from PBMCs using Zoledronate, IL-2 and IL-15. In some cases, the γδ T cells were engineered with lentiviruses encoding DAP12-associated synthetic antigen receptors (SARs). γδ T cells were co-cultured with primary patient-derived GBM cells, in the presence or absence of IL-15 to assess cytotoxicity, cytokine production and proliferation.

Results While non-engineered γδ T cells were able to robustly kill primary GBM cells through endogenous receptors, the γδ T cells failed to proliferate and produce cytokine. Engineering γδ T cells to express DAP12-associated SARs targeting GBM antigens (CD133, HER2 and IL13Rα2) greatly augmented cytotoxicity and production of inflammatory cytokines upon co-culture with GBM cells, but the SAR-engineered γδ T cells still failed to proliferate. Inclusion of IL-15 in γδ T cell/GBM co-cultures enhanced the cytotoxic capacity of γδ T cells, improved their survival, and promoted robust proliferation, which was further amplified by the presence of a SAR. Engineering γδ T cells with IL-15 transgene variations (secreted, membrane-bound) did not reproduce the enhancement observed with supplementary IL-15 due to insufficient transgene expression and impairment of the manufacturing process.

Conclusions T cell expansion is a major correlate of therapeutic efficacy. The inability of γδ T cells to proliferate following exposure to primary GBM cells explains the poor clinical activity, thus far. Recapitulating the role of IL-15 at the tumour site will be necessary to unleash the full potential of γδ T cells. While forced expression of IL-15 has proven successful in αβ T cells, it was insufficient in the context of γδ T cells. We are currently exploring the biological consequences of IL-15 supplementation, to identify engineering strategies that can recapitulate the beneficial impacts of the cytokine at the tumour site.

Acknowledgements This work was supported by the Samuel Family Foundation.

Ethics Approval This research was approved by the McMaster Health Sciences Research Ethics Board and all donors in this study provided informed written consent.