Dendritic cells (DCs) are potent antigen presenting cells and possess a direct anti-tumor cytotoxic ability. Nevertheless, the mechanism of anti-tumor cytotoxicity by DCs and the methods for its evaluation are not fully elucidated. In order to clarify this mechanism of cytotoxicity, we examined the ability of DCs 1) to suppress [3H] thymidine (3H-TdR) uptake by tumor cells; 2) to induce cytolysis on 51Cr-labeled tumor cells; 3) and to induce DNA fragmentation on 3H-TdR labeled tumor cells (JAM test). Cytolysis and DNA fragmentation are markers of necrotic and apoptotic mechanisms of cytotoxicity in vitro, respectively. DCs inhibited approximately 38.6% to 54.8% of the growth of B4D6, NB4, U937, and Daudi cells as evaluated by the uptake of 3H-TdR. However no cytolysis was verified by 51Cr-release assay. On the other hand, cytotoxicity rates found using the JAM test ranged from 3 to 81% depending on the cell line and the effector to target cell ratio. The discrepancy of cytotoxicity between 51Cr-release assay and the JAM test may be due to the phagocytosis of apoptotic tumor cells or the absorption of released 51Cr by DCs surrounding the target cells. In conclusion, the JAM test was more sensitive than the 4-h and the 10-h 51Cr-release assay to investigate cytotoxicity mediated by DCs toward hematopoietic tumor cell lines in vitro.
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.
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