Expansion of γδT Cells By New Generation IMiDs and Their Cytotoxicity Against Myeloma Progenitors
Authors: Miki Hirokazu , Harada Takeshi , Cui Qu , Oda Asuka , Bat-Erdene Ariunzaya , Tenshin Hirofumi , Amachi Ryota , Sumitani Ryohei , Uemura Munenori , Takahashi Mamiko , Iwasa Masami , Fujii Shiro , Teramachi Jumpei , Nakamura Shingen , Kagawa Kumiko , Ozaki Shuji , Abe Masahiro 
Published: October 2019

Abstract
Although a therapeutic outcome with extended survival has been improved in patients with multiple myeloma (MM) owing to implementation new anti-MM agents, MM eventually relapses and still remains incurable. MM-initiating cells or MM progenitors are generally accepted to contribute to disease relapse through their drug-resistant nature. In order to overcome this drug resistance, various forms of immunotherapies have been studied. One such approach is the application of γδT cells. Vγ9Vδ2γδT (Vδ2 γδT) cells are important effectors in the first-line defense and tumor surveillance, and can be activated and expanded from peripheral blood mononuclear cells (PBMCs) upon treatment with zoledronic acid (Zol) and IL-2. New generation IMiDs, namely lenalidomide (LEN) and pomalidomide (POM), not only exert direct anti-MM actions, but also activate immune cells surrounding MM cells. In the present study, we aimed to clarify the effects of new generation IMiDs, LEN and POM, on activation and expansion of Vδ2 γδT cells in combination with Zol and the cytotoxic effects of ex-vivo expanded Vδ2 γδT cells against MM progenitors. After PBMCs isolated from normal donors were incubated for one week with Zol at 1 microM and LEN at 1 microM in combination, Vδ2 γδT cells were robustly expanded to be a majority of CD3-positive T cells as observed with Zol at 1 microM and IL-2 at 100 U/ml in combination. The expanded Vδ2 γδT cells expressed intracellular IFN-γ but not Foxp3 along with increased surface expression of NKG2D and DNAM-1, molecules known to be associated with NK or cytotoxic T cell-mediated cytotoxic activity, indicating induction of Th1-like Vδ2 γδT cells. In the same way as LEN, POM at 0.01, 0.1 and 1 microM was able to expand Vδ2 γδT cells from PBMCs isolated from all 10 normal donors. The expansion efficiency was slightly better with POM over LEN in some donors. The expansion efficiency with Zol plus these IMiDs appeared to be weaker in PBMCs from a majority of patients with MM than those from normal donors, as previously reported with Zol plus IL-2. Intriguingly, these IMiDs further expanded upon treatment with Zol plus IL-2 Vδ2 γδT cells from PBMCs from some donors. However, these IMiDs alone only marginally expanded Vδ2 γδT cells in the absence of Zol, suggesting costimulation of antigen-primed Vδ2 γδT cells by these IMiDs. Thus, LEN or POM appears to be able to expand Th1-like Vδ2 γδT cells in the presence of Zolwithout exogenous addition of IL-2. Such expanded Th1-like Vδ2 γδT cells exerted potent cytotoxic activity against MM cells. Interestingly, the expanded Vδ2 γδT cells also markedly minimized the sizes of side populations in RPMI8226 and KMS-11 cells, and suppressed their clonogenic capacity as determined by in vitro colony formation and tumorigenic capacity in SCID mice, suggesting targeting a drug-resistant clonogenic MM cells. Panobinostat, a pan-HDAC inhibitor, upregulated surface expression of NKG2D ligands, ULBPs and MICA/B, on MM cells, while impairing MM cells. Pretreatment with panobinostat may further potentiate the cytotoxic activity of Vδ2 γδT cells against MM cells. Combination with novel anti-MM agents warrants further study in terms of further enhancement of anti-MM effects with the ex-vivo expanded Th1-like Vδ2 γδT cells. The precise underlying mechanisms for ex-vivo expansion of Th1-like Vδ2 γδT cells should be clarified to make the best use of these new generation IMiDs with this unique and interesting phenomenon.

Disclosures
No relevant conflicts of interest to declare.