Natural Killer Cell Expansion with Autologous Feeder Layer and Anti-CD3 Antibody for Immune Cell Therapy of Hepatocellular Carcinoma.

Faezeh Hosseinzadeh,Farnaz Dabiri Oskouei,Abbas Hajifathali,Somayeh Ebrahimi-Barough,Samad Muhammadnejad,Iman Seyhoun,Mahdi Shadnoush,Javad Verdi,Jafar Ai,Fatemeh Hosseinzadeh

doi:10.31557/apjcp.2019.20.12.3797

Abstract

Background:one of the promising approaches for treatment of some cancers is adoptive cell therapy using natural killer (NK) cells. 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 (GVHD). Method:In this study, irradiated autologous peripheral blood mononuclear cells (PBMCs) as feeder layer with an anti-CD3 monoclonal antibody (mAb) were used. For activation and expansion of NK cells, human recombinant IL2 and IL15 were used. After co-culturing of expanded NK cells (eNKC) and isolated NK cells (iNKC) with hepatocellular carcinoma (HCC) cells, the viability of eNKC in compared to iNKC were analyzed by CCK-8 assay and degranulation of NK cells after co-culturing was assayed by measuring CD107a expression. Enzyme-Linked Immunosorbent Assay (ELISA) assay was used for the ability of NK cells to secretion of IFN-γ (interferon-γ) and TNF-α (Tumor Necrosis Factor-α) after co-culture with HCC cells. Real Time PCR analysis was used for expression of human Perforin and Granzyme B genes in the NK cells exposed to target HepG2 cells. Result: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.

Highlights

Up to 20 percent of human peripheral blood lymphocytes was composed natural killer (NK) cells that defined as CD3−CD56+ cells (Cooper et al, 2001)
The isolated NK cells were cultured at the presence of autologous irradiated peripheral blood mononuclear cells (PBMCs) as feeder layer, OKT3 monoclonal antibody (mAb), rhIL-2 and rhIL-15, under specific conditions
Immune-cell therapy of hepatocellular carcinoma (HCC) with NK cells is an attractive approach in recent years due to high frequency and cytotoxicity of hepatic NK cells (Ishiyama et al, 2006; Tian et al, 2013) and for unique property of NK cells faced with surface major histocompatibility complex (MHC)-I gene expressed on the normal cells that led to inhibition of NK cells and prevention of graft-versus-host disease (GVHD) (Ljunggren and Malmberg, 2007)

Summary

Introduction

Up to 20 percent of human peripheral blood lymphocytes was composed NK cells that defined as CD3−CD56+ cells (Cooper et al, 2001). Different studies have investigated on the ex vivo expansion of NK cells from various sources such as PBMC, bone marrow, cord blood and embryonic stem cells (Cheng et al, 2013; Koepsell et al, 2013). In these efforts, cancer cell-based feeder cells such as genetically modified K562 cells (Fujisaki et al, 2009; Gong et al, 2010), or Epstein–Barr virus-transformed lymphoblastoid

Methods

Results

Conclusion