γδ T cells cultured with artificial antigen-presenting cells and IL-2 show long-term proliferation and enhanced effector functions compared with γδ T cells cultured with only IL-2 after stimulation with zoledronic acid

Background aims: Immunotherapeutic approaches using gamma delta T cells have emerged as the function of gamma delta T cells in tumor surveillance and clearance has been discovered. In vitro expansion methods of gamma 9 delta 2 T cells have been based on phosphoantigens and cytokines, but expansion methods using feeder cells to generate larger numbers of gamma delta T cells have also been studied recently. However, there are no studies that directly compare gamma delta T cells cultured with phosphoantigenswith those cultured with feeder cells. Therefore, this study aimed to compare the expansion, characteristics and effector functions of gamma delta T cells stimulated with K562-based artificial antigen-presenting cells (aAPCs) (aAPC-gamma delta T cells) and gamma delta T cells stimulated with only zoledronic acid (ZA) (ZA-gamma delta T cells). Methods: Peripheral blood mononuclear cells were stimulated with ZA for 7 days, and aAPC-gamma delta T cells were stimulated weekly with K562-based aAPCs expressing CD32, CD80, CD83, 4-1BBL, CD40L and CD70, whereas ZA-gamma delta T cells were stimulated with only IL-2. Cultured gamma delta T cells were analyzed by flow cytometry for the expression of co-stimulatory molecules, activating receptors and checkpoint inhibitors. Differentially expressed gene (DEG) analysis was also performed to determine the difference in gene expression between aAPC-gamma delta T cells and ZA-gamma delta T cells. In vitro cytotoxicity assay was performed with calcein AM release assay, and in vivo anti-tumor effect was compared using a U937 xenograft model. Results: Fold expansion on day 21 was 690.7 +/- 413.1 for ZA-gamma delta T cells and 1415.2 +/- 1016.8 for aAPC- gamma delta T cells. Moreover, aAPC-gamma delta T cells showed continuous growth, whereas ZA-gamma delta T cells showed a decline in growth after day 21. The T-cell receptor V gamma 9(+)delta 2(+) percentages (mean +/- standard deviation) on day 21 were 90.0 +/- 2.7% and 87.0 +/- 4.5% for ZA-gamma delta T cells and aAPC-gamma delta T cells, respectively. CD25 and CD86 expression was significantly higher in aAPC-gamma delta T cells. In DEG analysis, aAPC-gamma delta T cells and ZA-gamma delta T cells formed distinct clusters, and aAPC-gamma delta T cells showed upregulation of genes associated with metabolism and cytokine pathways. In vitro cytotoxicity revealed superior anti-tumor effects of aAPC-gamma delta T cells compared with ZA-gamma delta T cells on Daudi, Raji and U937 cell lines. In addition, in the U937 xenograft model, aAPC-gamma delta T-cell treatment increased survival, and a higher frequency of aAPC-gamma delta T cells was shown in bone marrow compared with ZA-gamma delta T cells. Conclusions: Overall, this study demonstrates that aAPC-gamma delta T cells show long-term proliferation, enhanced activation and anti-tumor effects compared with ZA-gamma delta T cells and provides a basis for using aAPC-gamma delta T cells in further studies, including clinical applications and genetic engineering of gamma delta T cells. (C) 2021 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.

Automated summary

This study compared the expansion, characteristics, and effector functions of gamma delta T cells stimulated with K562-based artificial antigen-presenting cells and gamma delta T cells stimulated with zoledronic acid. The results showed that aAPC-gamma delta T cells exhibited long-term proliferation, enhanced activation, and superior anti-tumor effects compared to ZA-gamma delta T cells.