Human Natural Killer (NK) Cells: Differential Expression of Phosphatase and Tensin Homologue Deleted On Chromosome Ten (PTEN) During NK Cell Development Regulates Its Cytolytic Activity Against Leukemic Target Cells

Abstract 254

Human NK cells are CD56(+)CD3(-) large granular lymphocytes characterized by the ability to directly kill virally infected or malignantly transformed cells. Five stages of human NK cell development can be identified in secondary lymphoid tissue. Stage 4 NK cells are immature CD56^bright and have poor cytolytic activity against sensitive leukemic target cells at rest while stage 5 NK cells are mature CD56^dim and have potent cytolytic activity against the same leukemic cells at rest. Both stage 4 CD56^bright and stage 5 CD56^dim NK cells can be found circulating in blood. We sought to determine the mechanism responsible for this different cytolytic activity by exploring changes in gene expression between CD56^bright NK cells and CD56^dim NK cells. We first observed that CD56^bright NK cells expressed ∼5 fold greater amounts of PTEN protein over CD56^dim NK cells by western blot (n=5, p < 0.04). Given that PTEN is a lipid phosphatase that opposes the PI3K/AKT pathway, we hypothesized that it may negatively regulate NK cell cytolytic activity. In order to test this, we used lentiviral infection to overexpress PTEN in the human NK cell line NK-92. Relative to cells infected with an empty lentiviral vector, NK-92 cells with overexpression of PTEN showed decreased cytotoxicity against sensitive leukemic target cells by at least 25% at all effecter:target ratios (n=4, p < 0.02). Next, we infected primary human NK cells with the same vector and showed an approximately 30% reduction in degranulation of cytolytic mediators as determined by CD107a mean fluorescent intensity (M.F.I.) when cultured with sensitive leukemic target cells (n=4, p<.08). Consistent with this, murine NK cells were isolated from FVB mice with a heterozygous germline deficiency in Pten (Pten+/−) and showed at least a 25% increase in cytotoxicity against sensitive lymphoma target cells (n=4, p<.0001). At the molecular level, when NK-92 cells overexpressing PTEN were bound to their sensitive leukemic target cells, they showed decreased activation along the AKT and ERK1/2 signaling pathways, which are known as positive regulators of NK cell cytotoxicity. Collectively, we identify a functional role for the tumor suppressor PTEN in normal human NK cell development. We demonstrate differential expression of PTEN in immature and mature human NK cells, show that it is a negative regulator of NK cell cytolytic activity, and suggest that this differential expression may in fact account for the difference in baseline cytolytic activity found in the CD56^bright and CD56^dim human NK cell subsets, possibly via the AKT and ERK1/2 pathways.