CD95/Fas-ligation and resting NK or IL-2 activated NK (LAK) attack limit NK-92 efficacy.

Abstract

Abstract Adoptively transferred NK-92 cells and genetically modified variants must undergo irradiation to prevent proliferation, since NK-92 cells are themselves tumors. This malignant quality also makes NK-92 cells susceptible to attack by patients’ NK cells. Here, we investigated the effect of gamma-irradiation and/or Fas-ligation on NK-92 killing against K562 tumor cells, as well as the effect of irradiation on the susceptibility of NK-92 cells to attack by primary NK cells. To evaluate killing, we used a 51Cr-release assay with multiple effector to target (E:T) K562 tumor cell ratios at 4- and 8-hour time points, 10 Gy irradiation and IgM antibody ligation of Fas. (K562 cells lack detectable Fas). NK-92 cells, maintained in 1000 U/ml of recombinant IL-2, retained high cytotoxic activity immediately after irradiation, but lost >50% activity one day after 10 Gy irradiation. NK-92 cells were highly resistant to death initiated by CD95-ligation, whether irradiated or not. However, the surviving NK-92 cells suffered loss of cytotoxicity following Fas-ligation. This loss of cytotoxicity was more pronounced when Fas-ligation and irradiation were combined. Irradiation also made NK-92 cells more sensitive to attack by resting NK cells and IL-2 activated NK (LAK) cells. These findings demonstrate that Fas-ligation and irradiation are detrimental to NK-92 functionality and support the recommendation that genetic removal of CD95/Fas may increase cytotoxicity by NK-92 cells and potentially, other cell lines used for adoptive immunotherapies. This research was supported in part by a UNR Foundation Award, an NIH P30 GM110767 for the Cytometry Center, a Nevada Undergraduate Research Award, and by the Nevada INBRE program (NIH GM103440) for research scholarship and supplies that helped support the project.