Anti-Tumor Cytotoxicity of Blood γδ T Cells Expanded from Leukemia Patients Against Autologous Leukemia Cells—Enhancement of the Anti-Tumor Cytotoxicity by Type I IFN.
Authors: Saito Anri , Narita Miwako , Watanabe Norihiro , Yokoyama Ayumi , Sekiguchi Asuka , Tochiki Nozomi , Alldawi Louie , Takizawa Jun , Hashimoto Shigeo , Furukawa Tatsuo , Toba Ken , Aizawa Yoshifusa , Takahashi Masuhiro 
Published: April 2020

Abstract
In order to establish an efficient anti-tumor cellular immunotherapy using blood γδ T cells, we investigated the cytotoxic activity of γδ T cells expanded from patients with leukemia against autologous leukemia cells and explored the potent methods for enhancing the anti-tumor cytotoxic activity of γδ T cells. We clarified that γδ T cells generated from leukemia patients possess the cytotoxic activity against autologous leukemia cells. Besides, anti-tumor cytotoxic activity of expanded γδ T cells was enhanced by the short-term culture of γδ T cells with type I IFN (IFN-α and IFN-β). The sensitivity of target leukemia cells to γδ T cells was enhanced by the exposure of the target cells to bisphosphonate such as zoledronate, which is one of the antigens recognized by γδ T cells and elevates the content of potent antigen for γδ T cells, isoprenyl pyrophosphate (IPP), in tumor cells. Blood γδ T cells were expanded from anti-CD3 microbead-separated T cells or anti-γδ TCR microbead-separated γδ T cells in the patients with acute myelogenous leukemia by the culture with zoledronate and a low concentration of IL-2 for 1–2 weeks. For the activation of expanded γδ T cells, cultured γδ T cells were exposed with type I IFN for 1–3 days. The supernatant prepared from the culture of type I IFN-activated γδ T cells was assayed for cytokine (IFN-γ, TNF-α, IL-4, IL-5, IL-10) concentration by cytometric bead array. Anti-tumor cytotoxicity of γδ T cells was evaluated by 51Cr-release assay by using purified γδ T cells as effector cells and autologous leukemia cells as target cells. In most patients with acute leukemia, γδ T cells could be markedly expanded by the culture with zoledronate and IL-2 and almost all the expanded γδ T cells possessed Vδ2 TCR. Expanded and purified γδ T cells derived from the patients with leukemia were demonstrated to be cytotoxic against autologous leukemia cells. By the culture of expanded γδ T cells with type I IFN, the expression of the activation marker CD69 and the apoptosis molecule Trail was enhanced at the concentration dependent of type I IFN especially IFN-β. The expanded γδ T cells were shown to produce a remarkable amount of IFN-γ and a considerable amount of TNF-α and the cytokine production was increased by the addition of type I IFN. In addition, the cytotoxic activity of γδ T cells was enhanced by incubating target leukemia cells with zoledronate for 1–2 days. The present study demonstrated that γδ T cells expanded from patient’s blood are cytotoxic to patient’s leukemia cells. It is also demonstrated that there are two methods practically available for enhancing the cytotoxic activity of expanded γδ T cells against leukemia cells, one of which is activating γδ T cells by using type I IFN, and the other is elevating the sensitivity of target cells by using bisphosphonate. These findings implied the possibility that type I IFN-activated γδ T cells could be efficiently applied for cellular immunotherapy in the patients with hematological malignancies who is being administered with bisphosphonate. Moreover, in vivo administration of bisphosphpnate, a low dose of IL-2 and type I IFN could be effective for tumors as γδ T cell-based cellular immunotherapy.