In Vitro Expansion of Blood γδ T Cells in Patients with Myeloma/Lymphoma and Anti-Tumor Cytotoxicity of the Expanded γδ T Cells.
Authors: Saito Anri , Narita Miwako , Watanabe Norihiro , Tochiki Nozomi , Hiroi Yumi , Shirasaki Takayoshi , Takizawa Jun , Furukawa Tatsuo , Toba Ken , Aoki Sadao , Aizawa Yoshifusa , Takahashi Masuhiro 
Published: October 2019

Abstract
In order to establish an efficient anti-tumor cellular immunotherapy using blood In order to establish an efficient anti-tumor cellular immunotherapy using blood γδ T cells, we investigated the in vitro expansion of γδ T cells in the patients with myeloma and lymphoma by the culture of PB-MNC with bisphosphonate and a low dose of IL-2 and we demonstrated the cytotoxic activity of the expanded γδ T cells against myeloma/lymphoma cells. Simultaneously we explored the potent methods for enhancing the anti-tumor cytotoxic activity of γδ T cells by both directions of activating the expanded γδ T cells and making target tumor cells sensitive to γδ T cells. For the activation of γδ T cells, expanded γδ T cells were exposed with type I IFN, monocyte-derived dendritic cells (mo-DC), or plasmacytoid dendritic cell like cell line PMDC05 (leukemia cell line established from CD4+ CD56+ acute leukemia in our laboratory) for 2 days. For the enhancement of sensitivity of target tumor cell to γδ T cells, we aimed to increase the content of IPP (the potent pyrophosphate antigen for γδ T cells) in tumor cells by decreasing the metabolic downstream of IPP. For decreasing the downstream of IPP, we tried to suppress FPP synthetase, which is involved in downstream metabolism of IPP, by using nitrogen-containing bisphosphonate. In addition, the expression of stress-induced molecules such as MICA/B on target tumor cells was evaluated in association with the level of cytotoxicity of γδ T cells against the tumor cells. Compared with normal control, the patients with myeloma (n=8) demonstrated decreased percentage and counts of PB γδ T cells. Patients with lymphoma (n=7) showed a wide range of values in PB γδ T cells, covering a normal range. Amplification rate of PB γδ T cells by culture with zoledronate and IL-2 varied markedly from patient to patient up to 120 times in myeloma and 90 times in lymphoma. Expanded γδ T cells generated in patients with myeloma/lymphoma were demonstrated to possess the cytotoxic activity against myeloma/lymphoma cells by 51Cr-release assay and CFSE-labeled target cell. The cytotoxic activity of expanded γδ T cells was enhanced by the exposure of γδ T cells with type I IFN (IFN-α and IFN-β). The activation of γδ T cells, which was evaluated by the elevation of CD69 expression, was observed by the exposure of γδ T cells with type I IFN, mo-DC, or PMDC05 for 2 days. The sensitivity of target myeloma/lymphoma cells to γδ T cells was enhanced by the exposure of the target cells to bisphosphonate such as zoledronate. The expression level of MICA/B on target tumor cells was demonstrated to be associated with the potency of cytotoxicity of γδ T cells against the tumor cells. The present study demonstrated that γδ T cells expanded from myeloma/lymphoma patient’s blood are cytotoxic to myeloma/lymphoma cells. There are two methods practically available for enhancing the cytotoxic activity of expanded γδ T cells against myeloma/lymphoma cells, one of which is activating γδ T cells and the other is elevating the sensitivity of target cells by using bisphosphonate.