Efficient and Reproducible Retroviral Infection of Primary Human Natural Killer Cells.

Molecular characterization of human natural killer (NK) cells will require targeted gene delivery to inhibit and activate specific signaling pathways, yet to our knowledge, an effective means to deliver such products for long-term gene expression without disrupting normal cellular processes has not been described. In this study we have developed a retroviral strategy to effectively express gene products in the NK cell, whereby its effector functions of cytotoxicity and cytokine production remain intact. Using an EBV/retroviral hybrid vector PINCO, we demonstrate infection of human peripheral blood NK cells with simultaneous expression of a marker for infection - the enhanced green fluorescent protein (EGFP) - along with various genes of interest. This technique results in successful infection of the CD56^dim NK population that predominates among human peripheral blood NK and is the effector of antibody-dependent cellular cytotoxicity (ADCC) and natural killing. In addition, we demonstrate infection of the CD56^bright NK subset as well as the NK-92 and NK-L cell lines. Finally, we modify PINCO to express a cytoplasmically truncated murine CD8 molecule in place of GFP. The resulting vector enables us to transduce NK cells with multiple genes of interest simultaneously and provides an alternative purification method to FACS by using magnetic beads. In summary, we have devised an efficient and highly reproducible methodology for the targeted delivery of gene products to human NK cells that should now provide opportunities to dissect the molecular processes critical to normal NK cell physiology and to genetically manipulate NK cell populations prior to their administration in cancer therapy.