Analysis of NK cell clones obtained using interleukin-2 and gene-modified K562 cells revealed the ability of "senescent" NK cells to lose CD57 expression and start expressing NKG2A.

Maria A Streltsova,Dean A Lee,William G Telford,Leonid M Kanevskiy,Alexander M Sapozhnikov,Sofya A Erokhina,Elena I Kovalenko,Niklas K Björkström

doi:10.1371/journal.pone.0208469

Abstract

In this work, we analyzed the phenotype and growth of human NK cell clones obtained by the stimulation of individual NK cells with IL-2 and gene-modified K562 feeder cells expressing membrane-bound IL-21 (K562-mbIL21). We generated clones from NK cells at distinct differentiation and activation stages, determined by CD56, CD57 and HLA-DR expression levels. Less differentiated CD56bright NK cell subsets showed higher cloning efficiency compared with more differentiated CD56dim subsets, especially with the CD57bright subset. However, clones from the CD56dimCD57– subset lived longer on average than other subsets. Moreover, several clones with the highest cell numbers were derived from CD56dimCD57–HLA-DR−cells. Most of the clones including those derived from more differentiated CD56dimCD57+/–NKG2A– NK cells showed a less-differentiated NKG2A+ phenotype. Also, CD57– cells were frequently observed in clones derived from CD57+ NK cells suggesting the loss of CD57 during the cloning process. On the other hand, KIR surface expression once detected for a clone never disappeared entirely, confirming irreversibility of the KIR expression. In summary, we have demonstrated that in specific conditions terminally differentiated CD57+ human NK cells are able to acquire the CD57– phenotype that was previously not observed and, thus, was considered impossible.

Highlights

The phenotype of NK cells changes during differentiation and activation, forming subsets with various functionalities [1,2]
Basing on the phenotypic analysis of the growing clones, we have found the ability of terminally differentiated NK cells to change, acquiring CD57–NKG2A+ phenotype, that is typical for less mature cells
NK cells circulating in the peripheral blood may differ in their ability to respond to the proposed stimuli, e.g. IL-2 and K562-mbIL21

Summary

Introduction

The phenotype of NK cells changes during differentiation and activation, forming subsets with various functionalities [1,2]. CD56bright and CD56dim NK cells can differ in their expression patterns of NKG2A, KIR and other surface markers during differentiation process [1,7] that leads to additional variations in their functional activity and response to stimuli. We have chosen the combination of IL-2 and K562 feeder cells, genetically modified to express membrane-bound IL-21 and other surfaceassociated molecules (K562-mbIL21) as an initial stimulation for cloning [9] These stimuli were shown to lead to the steady proliferation of activated NK cells for 6 weeks and significant cell expansion [9]. IL-2 used in this model at initial stage and for weekly clone restimulation is one of the powerful inducers of NK cell proliferation [13] It stimulates the differentiation of NK cells and increases their functional activity [14]. Basing on the phenotypic analysis of the growing clones, we have found the ability of terminally differentiated NK cells to change, acquiring CD57–NKG2A+ phenotype, that is typical for less mature cells

Results

Discussion

Methods