Abstract
A pattern of natural killer cell (NK cell) heterogeneity determines proliferative and functional responses to activating stimuli in individuals. Obtaining the progeny of a single cell by cloning the original population is one of the ways to study NK cell heterogeneity. In this work, we sorted single cells into a plate and stimulated them via interleukin (IL)-2 and gene-modified K562 feeder cells that expressed membrane-bound IL-21 (K562-mbIL21), which led to a generation of phenotypically confirmed and functionally active NK cell clones. Next, we applied two models of clone cultivation, which differently affected their phenotype, lifespan, and functional activity. The first model, which included weekly restimulation of clones with K562-mbIL21 and IL-2, resulted in the generation of relatively short-lived (5–7 weeks) clones of highly activated NK cells. Levels of human leukocyte antigen class II molecule—DR isotype (HLA-DR) expression in the expanded NK cells correlated strongly with interferon-γ (IFN-γ) production. The second model, in which NK cells were restimulated weekly with IL-2 alone and once on the sixth week with K562-mbIL21 and IL-2, produced long-lived clones (8–14 weeks) that expanded up to 107 cells with a lower ability to produce IFN-γ. Our method is applicable for studying variability in phenotype, proliferative, and functional activity of certain NK cell progeny in response to the stimulation, which may help in selecting NK cells best suited for clinical use.
Highlights
The phenotype of natural killer cells (NK cells) substantially changes during their differentiation and activation, forming heterogeneous subpopulations with various expression of surface receptors, effector molecules, and signal proteins, as well as different functional activity [1,2]
Several combinations of cytokines and feeder cells, including K562 cells and modified K562-mbIL21 cells, were tested for NK cell cloning in order to maximize the efficiency of clone generation (Figure 1B,D)
IL-2 was essential for NK cell clone generation
Summary
The phenotype of natural killer cells (NK cells) substantially changes during their differentiation and activation, forming heterogeneous subpopulations with various expression of surface receptors, effector molecules, and signal proteins, as well as different functional activity [1,2]. The mechanisms of NK cell differentiation are not completely clear During this process, due to certain epigenetic changes, NK cells lose the expression of the NKG2A/CD94 receptor and begin to express inhibitory killer-cell immunoglobulin-like receptors (KIRs), as well as cluster of differentiation (CD), the cell maturation marker. Clonally expanded NK cells were used to study distribution of CD94/NKG2A and CD94/NKG2C heterodimers [5]; changes in CD94/NKG2 receptor expression depended on the functional activity of NK cells [6]. It was shown that heterogeneity in NK cell cytotoxic activity was related to various expression levels of different activating and inhibitory receptors in NK cells. Functional tests utilizing NK cell clones may help in defining cytotoxicity in NK cells for different tumor variants [8]
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