Expansion of Human NK Cells Using K562 Cells Expressing OX40 Ligand and Short Exposure to IL-21.

Soonho Kweon,Junsang Doh,Seokho Kim,Alaa Kassim Ali,Jinho Kim,Seung-Hwan Lee,Hongbi Yu,Sang-Ki Kim,Jaemin Lee,Duck Cho,Sejong Chun,Minh-Trang Thi Phan

doi:10.3389/fimmu.2019.00879

Abstract

Background: Natural Killer (NK) cell-based immunotherapy used to treat cancer requires the adoptive transfer of a large number of activated NK cells. Here, we report a new effective method to expand human NK cells ex vivo using K562 cells genetically engineered (GE) to express OX40 ligand (K562-OX40L) in combination with a short exposure to soluble IL-21. In addition, we describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction.Methods: K562-OX40L cells were generated by lentiviral transduction and were used as feeder cells to expand and activate NK cells from PBMCs in the presence of IL-2/IL-15. Soluble IL-21 was also added in various concentrations only once at the beginning of the culture. NK cells were expanded for 4–5 weeks, and the purity, expansion rate, phenotype and function (cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine production, CD107a degranulation) of these expanded NK cells were compared to those generated by using K562 feeder cells.Results: The culture of NK cells with K562-OX40L cells in combination with the transient exposure to IL-21 highly enhanced NK cell expansion to approximately 2,000-fold after 4 weeks of culture, compared to a 303-fold expansion using the conventional K562 cells. Mechanistically, the OX40-OX40L axis between the feeder cells and NK cells as well as the homotypic interaction between NK cells through the OX40-OX40L axis were both necessary for NK cell expansion. The short exposure of NK cells to IL-21 had a synergistic effect with OX40 signaling for NK cell expansion. Apart from their enhanced expansion, NK cells grown with K562-OX40L feeder cells were similar to those grown with conventional K562 cells in regard to the surface expression of various receptors, cytotoxicity, ADCC, cytokine secretion, and CD107 degranulation.Conclusion: Our data suggest that OX40 ligand is a potent co-stimulant for the robust expansion of human NK cells and the homotypic NK cell interactions through the OX40-OX40L axis is a mechanism of NK cell expansion.

Highlights

The adoptive transfer of natural killer (NK) cells is a promising adjuvant approach for cancer immunotherapy, obtaining sufficient numbers of activated NK cells is important for an effective NK cell-based immunotherapy
To determine whether the overexpression of OX40L on K562 feeder cells would enhance NK cell expansion ex vivo, we generated a K562 cell line that expresses OX40L by using lentiviral transduction
Messenger RNA levels of the OX40 ligand were detected on the transduced cells, but not on parental K562 cells (Figure 1A)

Summary

Introduction

The adoptive transfer of natural killer (NK) cells is a promising adjuvant approach for cancer immunotherapy, obtaining sufficient numbers of activated NK cells is important for an effective NK cell-based immunotherapy. Several soluble cytokines such as IL-2 and IL-15 have been used to expand NK cells, but IL-2 alone or in combination with IL-15 results in minimal NK cell expansion [1]. The mechanism of NK cell expansion through the interaction between GE feeder cells expressing co-stimulatory factors and NK cells has not been elucidated. We describe a possible mechanism of the NK cell expansion through the OX40 receptor-OX40 ligand axis which is dependent on NK cell homotypic interaction

Methods

Results

Conclusion