Presentation Authors: Teruki Shimizu*, Osaka, Japan, Masatsugu Miyashita, Atsuko Fujihara, Fumiya Hongo, Osamu Ukimura, Eishi Ashihara, Kyoto, Japan
Introduction: In clinical trials, Zoledronic acid (ZOL) pretreatment in cancer cells is widely used to improve the efficacy of human Î³Î´T cell immunotherapy.We herein provide new strategies for the enhancement of Î³Î´T cell function by anticancer pretreatment in urinary bladder cancer (UBC) cells. The aim of this study was whether pretreatment of cancer cells with standard anticancer agents would increase the cytotoxicity of Î³Î´T cells.
Methods: The surface expression of NKG2D, TCRVÎ³9, TCRVÎ´2 and the intracellular levels of perforin and GranzymeB were examained. In in vitro assays, UBC cell lines T24, TCCSUP, and UMUC3 were used. Flow cytometric analysis of CFSE /propidium iodide (PI) staining was used in in vitro cytotoxicity assays. Imaging of cancer cells lysed by human Î³Î´T cell was captured by laser microscopy. UBC cells were treated with various standard anticancer agents at sub-lethal concentrations, including cisplatin (CDDP), gemcitabine (GEM), methotrexate (MTX), vinblastine (VBL), adriamycin (ADR), and mitomycin C (MMC). The expressions of MICA/B, ULBP1, and ULBP2/5/6 in UBC cells were investigated. In in vivo experiments, the efficacy of intravesical administration of ex vivo-expanded Î³Î´T cells was examined in an orthotopic xenograft model using In Vivo Imaging System (IVIS).
Results: Ex vivo-expanded Î³Î´T cells expressed NKG2D receptor on the cell surface and both TCRVÎ³9, TCRVÎ´2 lineage were well expanded. Activation of ex vivo-expanded Î³Î´T cell was confirmed by intracellular staining of granules, containing perforin and GranzymeB. We examined the synergistic effects of anticancer agents and Î³Î´T cells in vitro. We found that these anticancer agents upregulated the expressions of MICA/B and ULBP family in UBC cells, which Î³Î´T cells use to recognize cancer cells and resulted in the increased cytotoxicity of Î³Î´T cells. These effects were abrogated by induction of siRNA against MICA/B. Also, these findings were confirmed by MICA/B or NKG2D blocking experiments. We also found that intravesical administration of Î³Î´T cells showed potent cytotoxicity using an orthotopic xenograft. Finally, we revealed that low dose gemcitabine pretreatment synergistically increased the cytotoxicity of Î³Î´T cells in vivo.
Conclusions: These results indicated that Î³Î´T cell adoptive immunotherapy in combination with standard anticancer agents is an effective strategy and may be a promising approach to the treatment of UBC.
Source of Funding: MEXT, 15K10604; MEXT, 26461436; MEXT, S1511024L