Abstract Introduction: NK cells are currently being explored as a treatment modality for many oncologic diseases. However, the small number of NK cells in the circulation, approximately 5-15%, may limit the effectiveness of host NK cell immunity against cancer. Several methods to expand NK cells for subsequent adoptive infusion in cancer patients have been explored, including culturing NK cells in vitro with interleukin-2 (IL-2). Unfortunately, IL-2 stimulation alone results in only a small increase in NK cell proliferation. Recently, a novel in vitro method for large scale NK cell expansions using irradiated EBV-LCL feeder cells has been developed and is currently being utilized in an NHLBI clinical trial. Purpose: The aims of this study included: a) to identify differences in susceptibility to apoptotic pathways between fresh, overnight IL-2 activated, and expanded NK cells by comparing the death receptors DR4, DR5, and Fas; b) to determine when Fas expression is acquired on IL-2 activated NK cells; and c) to determine if Fas expression differs on NK cells expanded with different feeder cell populations. Methods: NK cells from healthy donors were isolated using CD56+ magnetic selection beads. Using flow cytometry, we examined Fas, DR4, and DR5 expression on fresh, overnight IL-2 activated, and NK cells expanded with either EBV-LCL or K562 41bb ligand transfected feeder cells for 7-14 days. To assess susceptibility to Fas mediated apoptosis, each NK cell group was cultured with recombinant FasL (rhFasL) for 12 hours with Annexin V expression by flow cytometry used to measure apoptosis. In addition, Fas expression was examined over multiple days for both IL-2 activated NK cells and expanded NK cells. Results: Fas expression was similar on overnight IL-2 activated NK cells compared to fresh NK cells. In contrast, NK cells maintained in IL-2 for more than 2 days and expanded NK cells had a substantial increase in Fas expression. Similar increases in Fas expression were seen in NK cells expanded with either EBV-LCL or K562 41bb ligand transfected feeder cells. Enhanced Fas expression was associated with increased susceptibility to rhFasL mediated apoptosis; the fold increase in Annexin V induced by rhFasL at 12 hours was significantly higher amongst expanded NK cells (5.6 fold increase; p = 0.0001) compared to either fresh NK cells (1.2 fold increase; p = 0.32) or overnight IL-2 activated NK cells (1.0 fold increase; p = 0.62). In contrast to Fas, surface expression of the TRAIL death receptors DR4 and DR5 were similar amongst all NK cell populations analyzed. Conclusion: These data show for the first time that expanded NK cells are more susceptible to Fas mediated apoptosis compared to fresh and overnight IL-2 activated NK cells. Whether expanded NK cells are more susceptible to apoptosis mediated by tumor cells expressing Fas Ligand in vitro and in vivo is currently being investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1271.