Costimulation of gamma delta TCR and TLR7/8 promotes V delta 2 T-cell antitumor activity by modulating mTOR pathway and APC function

Background Gamma delta (gamma delta) T cells are attractive effector cells for cancer immunotherapy. V delta 2 T cells expanded by zoledronic acid (ZOL) are the most commonly used gamma delta T cells for adoptive cell therapy. However, adoptive transfer of the expanded V delta 2 T cells has limited clinical efficacy. Methods We developed a costimulation method for expansion of V delta 2 T cells in PBMCs by activating gamma delta T-cell receptor (gamma delta TCR) and Toll-like receptor (TLR) 7/8 using isopentenyl pyrophosphate (IPP) and resiquimod, respectively, and tested the functional markers and antitumoral effects in vitro two-dimensional two-dimensional and three-dimensional spheroid models and in vivo models. Single-cell sequencing dataset analysis and reverse-phase protein array were employed for mechanistic studies. Results We find that V delta 2 T cells expanded by IPP plus resiquimod showed significantly increased cytotoxicity to tumor cells with lower programmed cell death protein 1 (PD-1) expression than V delta 2 T cells expanded by IPP or ZOL. Mechanistically, the costimulation enhanced the activation of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt)-the mammalian target of rapamycin (mTOR) pathway and the TLR7/8-MyD88 pathway. Resiquimod stimulated V delta 2 T-cell expansion in both antigen presenting cell dependent and independent manners. In addition, resiquimod decreased the number of adherent inhibitory antigen-presenting cells (APCs) and suppressed the inhibitory function of APCs by decreasing PD-L1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) expression in these cells during in vitro V delta 2 T-cell expansion. Finally, we showed that human V delta 2 T cells can be expanded from PBMCs and spleen of humanized NSG mice using IPP plus resiquimod or ZOL, demonstrating that humanized mice are a promising preclinical model for studying human gamma delta T-cell development and function. Conclusions V delta 2 T cells expanded by IPP and resiquimod demonstrate improved anti-tumor function and have the potential to increase the efficacy of gamma delta T cell-based therapies.