Expansion and Activation of Natural Killer Cells using Autologous Feeder Layer and Anti-CD۳ Antibody for Immune Cell Therapy of Hepatocellular Carcinoma

Introduction: Adoptive cell therapy using natural killer cells is one of the promising approaches for treatment of some cancers. Various methods have been investigated for ex vivo expansion of NK cells in large-scale, but most of them involved cancer or genetically modified cells as feeder layer and also some of them have the risk of T cell contamination and graft-versus-host disease. Methods: In this study, irradiated autologous peripheral blood mononuclear cells with an anti-CD۳ monoclonal antibody were used as feeder layer. For activation and expansion of NK cells, human recombinant IL۲ and IL۱۵ were used. After co-culturing of expanded NK cells (eNKC) and isolated NK cells (iNKC) with hepatocellular carcinoma (HCC) cells, the viability of eNKC compared to iNKC were analyzed by CCK-۸ assay, degranulation of NKCs was assayed by measuring CD۱۰۷a expression, secretion of IFN-γ and TNF-α from NKCs was measured by Enzyme-Linked Immunosorbent Assay and the expression level of human Perforin and Granzyme B genes in the NKCs were analyzed with Real Time PCR. Results: This method strongly expanded highly purified NK cells with powerful cytotoxicity against HCC cells. The expanded NK cells showed high level of expression of degranulation marker and human Perforin and Granzyme B genes, and also was secreted larger amounts of TNF-α and IFN-γ compared with fresh isolated NK cells. Conclusion: we proposed an effective method for expansion of cytotoxic NK cells using irradiated autologous PBMC as feeder layer for more successful transfer of allogeneic NK cell in immuno cell therapy of HCC.

Keywords:

Natural Killer cell expansion , immune cell therapy , Hepatocellular Carcinom