Recently, immune cell therapy has attracted attention as a therapeutic alternative to refractory cancer. In particular NK cells are promising tool that can be used both autologous and allogeneic without further genetic modification. In order to apply NK cells to clinical application, it is essential to culture high-purity and high-efficacy NK cells in large quantities. For this reason, it is very important to develop an optimal strategy for large-scale NK cell expansion. In this study, we investigated the role of peripheral blood mononuclear cells (PBMCs) as feeder cells, which are key technology for ex vivo NK cell expansion, and key factors, which are important for NK cell proliferation. In addition, we have developed a powerful feeder cells that can replace PBMC feeder cells and apply for large-scale NK cell expansion.
We confirmed that $CD4^+ T$ cells directly contribute to NK cell proliferation among a variety of cells present in PBMC. And we have also identified that OX40, 4-1BB, and transmembrane $TNF-\alpha(mTNF-\alpha)$, which are inducible after NK cell activation, play an important role as NK cell receptors involved in NK cell proliferation. Especially, blocking of OX40 and 4-1BB signals completely inhibited proliferation and survival of NK cells in NK cell expansion.
To expand NK cells in large quantities, it is necessary to stimulate feeder cells repeatedly, thus a large amount of PBMCs are used as feeder cells for this purpose. We assessed NK cell proliferative potential using seven kinds of T cell lines, which possess T cellcharacteristics, to develop feeder cells capable of replacing PBMCs and selected the $CD4^+ T$ cell line, HuT 78. HuT 78 cells exhibited equal to or higher NK cell proliferative capacity than PBMCs. To develop powerful feeder cells, we generated genetically engineered feeder cell line by transducing one to four gene combinations of OX40L, 4-1BBL, $mTNF-\alpha$ and membrane bound IL-21 (mbIL-21) into HuT 78 cells and assessed NK cell expansion. HuT 78 cells expressing 4-1BBL, $mTNF-\alpha$, and mbIL-21 was selected as feeder cells for ex vivo expansion of NK cells. 4-1BBL/$mTNF-\alpha$/mbIL-21- HuT 78 feeder cells induced millions of folds of NK cell proliferation through repeated stimulation of them, and expanded NK cells retained in vitro efficacy equivalent to several thousand-fold of expanded NK cells. In addition, NK cells expanded with PBMCs and 4-1BBL/$mTNF-\alpha$/mbIL-21-HuT 78 cells were exhibited equivalent anti-tumor efficacy despite the big difference in NK cell proliferation.
In conclusion, we have shown that $CD4^+ T$ cells play an important role as feeder cells for NK cell cultivation and that OX40, 4-1BB, and $mTNF-\alpha$ signals are essential for NK cell proliferation. In addition, PBMC feeder cells can be replaced with genetically modified HuT 78 cells, which were transduced with 4-1BBL, $mTNF-\alpha$ and mbIL-21 (eHuT 78 cells), and we have been able to expanded clinically useful NK cells in large quantities using this eHuT 78 cells.