The expression levels of PD-L1 in cancer cells affect γδT cell cytotoxicity

[Speaker] Mako Tomogane:1
[Co-author] Teruki Shimizu:1,2, Masatsugu Miyashita:1,2, Yusuke Sano:1, Daiki Shimizu:1, Keigo Amari:1, Ryosuke Wakabayashi:1, Yuki Toda:1, Kazuyuki Takata:1, Eishi Ashihara:1
1:Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Japan, 2:Department of Urology, Kyoto Prefectural University of Medicine, Japan

[Background] T cells are divided into two major populations distinguished by their surface expression of αβ and γδT cell receptors. αβT cells recognize non-self peptide fragments restricted by major histocompatibility complex (MHC), whereas γδT cells display MHC-unrestricted cytotoxicity. γδT cells recognize isopentenyl pyrophosphate (IPP) produced through the mevalonate pathway in cancer cells. Zoledronic acid (ZOL) inhibit farnesyl pyrophosphate (FPP) synthase in the mevalonate pathway. By inhibiting FPP synthase, ZOL treatment accumulates IPP in cancer cells, and γδT cells exert anti-tumor effect on cancer cells. Programmed cell death-1 (PD-1)/PD ligand1 (PD-L1) axis prevents αβT cell cytotoxicity against cancer cells, and anti-PD-L1 monoclonal antibody (mAb) treatment restores the cytotoxicity. However, the role of this axis in γδT cell cytotoxicity is not elucidated clearly so far. In this study, we clarified the association between PD-L1-positive cancer cells and γδT cells for developing γδT cell immunotherapy.
[Methods] Peripheral blood mononuclear cells (PBMCs) from healthy donor were isolated by density gradient centrifugation and cultured for 11 days by adding ZOL and rhIL-2. The expression levels of PD-1 on γδT cells and PD-L1 on cancer cells were analyzed using a flow cytometer. γδT cells were used as effector cells in cytotoxicity assay against cancer cells using carboxyfluorescein diacetate succinimidyl ester (CFSE) and propidium iodide (PI). γδT cell cytotoxicity against CFSE-labeled cancer cells were investigated using a flow cytometer.
[Results] The percentage of CD3+/TCRγδ+ cells after ex vivo expansion was approximately 70% detected. γδT cells increased upto 450-900 folds and expressed low levels of PD-1. Various levels of PD-L1 were expressed in 17 cancer cell lines. γδT cells showed effective cytotoxicity against ZOL-pretreated cancer cells. Anti-PD-L1 mAb treatment enhanced the anti-tumor effects against a part of cancer cell lines. Then, we analyzed the correlation between the expression intensity of PD-L1 in cancer cells and the changes of the γδT cell cytotoxicity. We revealed that anti-PD-L1 mAb treatment enhanced the anti-tumor effects against cancer cells that expressed higher levels of PD-L1.
[Conclusions] Combination with γδT cells and targeting the PD-1/PD-L1 axis could be useful as a cancer immunotherapy against cancer cells with high expression levels of PD-L1.

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