In vitro expanded natural killer (NK) cells are more susceptible to Fas mediated apoptosis compared to fresh or overnight IL-2 activated NK cells

NK cells, whose ability to spontaneously kill target cells without the need for antigen recognition, differentiation, or co-stimulation, are currently being explored as a treatment for cancer. NKs comprise only 5--15% of peripheral blood lymphocytes, which limit the effectiveness of host NK cell immunity against cancer. Methods to expand NKs have been explored, including culturing NKs in-vitro with interleukin-2 (IL-2). IL-2 stimulation alone results in only a small increase in NK proliferation. A novel in-vitro method for large scale NK expansions using EBV-LCL feeder cells has been developed. In vitro cytotoxicity assays have shown that expanded NKs have greater anti-tumor activity compared to fresh NKs, although no information exists as to whether these activated cells differ in their ability to survive in-vivo..;The aims of this study were to: identify differences in susceptibility to apoptosis, or programmed cell death, between fresh, IL-2 activated, and expanded NKs by comparing Death Receptors (DR) 4, DR5, and Fas; determine the susceptibility of Fas-mediated apoptosis on all groups; and determine whether blocking Fas receptors on expanded NKs will decrease the percentage of apoptosis. Surface expression of Fas, DR4, and DR5 was measured on all three groups. All were cultured with recombinant FasL (rhFasL) to assess susceptibility to Fas-mediated apoptosis. Once apoptotic susceptibility was determined, this was repeated blocking the Fas receptor with anti-Fas monoclonal antibody prior to culture with rhFasL.;Fas expression on expanded NKs was substantially higher than fresh and IL-2 activated NKs. Enhanced Fas expression was associated with increased susceptibility to rhFasL; the fold increase in Annexin V induced by rhFasL was significantly higher amongst expanded NKs (6.1 fold; p<0.001) compared to fresh (1.3 fold; p=0.03) or IL-2 activated NKs (1.5 fold; p=0.24). Surface expression of DR4 and DR5 were similar amongst all groups. Blocking Fas receptor on expanded NKs reduced the percentage of apoptosis induced by rhFasL to baseline.;These data show for the first time that expanded NKs are more susceptible to Fas-mediated apoptosis. In an attempt to overcome expanded NK cell susceptibility to Fas-mediated apoptosis, an shRNA to genetically knock down Fas expression is currently being explored.