Two-color flow cytometric detection of serial cell-mediated cytotoxicity by natural killer (NK) lymphocytes

Abstract

Abstract Serial killers are much more productive than one-hit wonders, especially when it comes to killing cancer. Natural killer lymphocytes are significant cells in many anti-cancer immunotherapies, such as monoclonal antibody based- and adoptive cell transfer therapies. However, current data show that less than 30% of peripheral blood NK cells are capable of sequentially killing more than one cell, i.e., serial killing. These serial killers are not only capable of killing up to 6 target cancer cells each, but are also reported to deliver lytic hits at a faster rate and with more killing power than their counterpart NK cells. Current methods to identify serial killer NK cells, such as time-lapse cinematography, are laborious, expensive, and require highly skilled expertise and equipment. In order to enrich and grow serial NK cells, an inexpensive, simple assay is needed to detect and isolate them from blood for subsequent in vitro growth. Here we developed a flow cytometric method to identify serial killers via 2-color detection of the cell surface marker CD107a, expressed on the NK cell surface following killing. The NK-92 cell line was incubated with target Raji B leukemia cells for 2-hours, then labeled with one color, AF647, anti-CD107a mAb. Cells were then re-incubated for an additional 4-hours and subsequently labeled with a second color, PE, anti-CD107a mAb. Serial killers labeled double positive for both colors at 6 hours. We found that NK-92s are capable of killing at least 14 target Raji cells each and about 18% of the NK-92s can be identified as serial killers via two-color CD107a mAb labeling.