Optimization of Ex Vivo Gamma Delta T (GDT) Cell Expansion from Patients with Solid Tumor.
Authors: Morley Jacqueline S. , Wang Qiao , So Hang-Fai , Mattarollo Stephen R. , Hagi Tomomi , Tokuyama Hirotake , Truong Vy , Nieda Mie , Yokokawa Kiyoshi , Nicol Andrew 
Published: October 2019

Abstract
Aminobisphosphonates such as Zoledronic acid have anti-tumor activities in a range of malignancies in addition to their well established role in diminishing skeletal complications and controlling hypercalcaemia. A significant component of this anti-tumor activity may relate to the potent expansion and activation of Vg9Vd2 gamma delta T (GDT) cells induced by these agents, coupled with the capacity of zoledronic acid to sensitize malignant cells to GDT mediated killing. Based on these observations we aimed to determine culture conditions suitable for generation of large numbers of activated GDT cells for clinical use as an adoptive immune therapy. In addition we aimed to characterize the cytotoxic potential of these cells and to determine whether it was possible to predict which patients had the greatest expansion capacity and hence be most suitable for this type of therapy. Mononuclear cells from peripheral blood collected by venesection or steady state leukapheresis from patients (n=37) with a range of malignancies were cultured in large volume flasks (200ml) with zoledronic acid, high concentrations of IL-2 (700 IU/ml) and AB plasma for 7–14 days. Before, during and after culture GDT cells were counted and assessed phenotypically using 4 color flow cytometry (CD45RA, CD27, Pan Gamma and CD3) to determine absolute and relative numbers of different subsets. Cytotoxic activity of GDT cells after culture was assessed using a flow cytometry based assay for apoptosis (AnnexinV-7AAD) following co-culture of the GD T cells with a range of malignant targets, including B cell lymphoma, with and without pretreatment with zoledronic acid. Responders (arbitrarily considered suitable for adoptive immune therapy) were defined as those that could generate sufficient GDT cells from a single leukapheresis to produce a series of 8 treatments, each with > 2.50E+08 GDT cells. Using this definition our results were that 46% of patients were responders (n=37), producing from 2.10E+09 to 2.40E+10 (median = 6.20E+09) total GDT cells/leukapheresis. The pattern of expression of CD45RA and CD27 prior to culture clearly distinguished responders from non-responders. Responders had high relative numbers of T central memory (CD45RA-CD27+) and T effector memory (CD45RA-CD27-) (range 69–99%, median 89%) cells, whilst non-responders had baseline GDT cell populations low in these subsets (range 9–50%, median 28%) (n=15). After culture, the majority of the GDT cells were T effector memory cells (range 73–97%, median 85%) and had high expression of the activation marker CD69 and of IFN gamma. IFN-g expression peaked from day 6 to 10. GDT cells from all responders evaluated demonstrated cytoxicity of up to 65% with an effector:target ratio of 5:1 against malignant targets, including the B lymphoma cell line Daudi. We conclude that large numbers of cytotoxic, GDT cells suitable for adoptive immune therapy can be generated from a substantial proportion of subjects with malignancy and that potentially suitable subjects can be reliably predicted by flow cytometric assessment of peripheral blood.