Depletion of CD3-positive cells enables to generate purified and expanded human natural killer cells from peripheral blood mononuclear cells showing strong killer activity

摘要

PURPOSE: Natural killer (NK) cells play a crucial role of the innate immune system, and clinical studies to treat advanced malignancies using NK cells are emerging worldwide; however, overall purity and numbers of NK cells and their antitumor potencies are unsatisfactory as to obtain clinical outcome. We therefore attempted to establish an efficient method to purify and expand the highly activated NK cells ex vivo. METHODS: Under approval of the institutional ethical committee, peripheral blood mononuclear cells (PBMC) were collected from healthy volunteers and patients with advanced malignancies. CD3+-cells were depleted by magnetic beads, and CD3-depleted PBMC (CD3-PBMC) could be expanded in the presence of high concentration hIL-2 and 5% human autoserum for 14days to expand NK cells. The characteristics of these CD3-/CD56+ NK cells were subjected to the expression of surface markers, CD107a mobilization and cell-mediated cytotoxicity against K562 cells in vitro compared with those of primary NK cells. RESULTS: CD3-PBMC could be expanded in the presence of high concentration hIL-2 and the majority of the expanded cells were CD3-/ CD56+ (>90%) after 14days culture. The expanded NK cells expressed various killercell immunoglobulin-like receptors (KIRs), natural cytotoxicity receptors (NCRs), CD69, NKG2D, and CD16. CD107a mobilization assay and cytotoxicity assay revealed that expanded NK cells could kill K562 target cells more efficiently than that seen by primary NK cells. CONCLUSION: We have established new methods to deal with the current issues underlying NK-based immunotherapy. Overall purity of expanded NK cells was sufficiently expressing various receptors, including KIRs and NCRs. Moreover, these cells showed strong cytotoxic activity against K562; and importantly, we could obtain these NK cells from PBMCs from patients with advanced malignancies using this method. Therefore, this novel method may contribute to provide a new tool for clinical cancer immunotherapy.