In Vitro Expanded NK Cells Have Increased Natural Cytotoxity Receptors, TRAIL and NKG2D Expression, and Superior Tumor Cytotoxicity Compared to Short-Term IL-2 –Activated NK Cells.

Abstract 463

IL-2 activates NK cell enhancing their capacity to lyse tumor cells. To date, most clinical studies of adoptive NK cell transfer have utilized short-term (12-16 hours) IL-2-activated NK cells. Because IL-2 alone is ineffective in expanding NK cells in vitro, the relatively low numbers of NK cells obtained for infusion following short term IL-2 activation may limit the full therapeutic impact of this approach. To obtain larger numbers of NK cells, novel ex vivo expansion protocols that utilize irradiated EBV-LCL or K562 feeder cells have recently been developed. However, concerns exist that extensive ex vivo expansion might significantly reduce the in vivo proliferative potential and long-term viability of adoptively transferred NK cells. Here we investigated for differences in phenotype, tumor cytotoxicity and in vivo persistence between short-term IL-2 activated and long-term expanded NK cells. CD56+/CD3- NK cells were isolated from normal donors by immuno-magnetic bead selection and were either activated with IL-2 (500U/ml) for 12-16 hours or were expanded in vitro over 14 days using irradiated EBV-LCL feeder cells in IL-2 containing media (500U/ml). Short-term IL-2 activated NK cells did not expand in number in contrast to EBV-LCL stimulated NK cells which expanded 400-1000 fold by culture day 14. Flow cytometry analysis revealed no differences in expression of DNAM-1, 2B4, LFA-1 or granzyme B between short-term activated and expanded NK cells. However, expanded NK cells had significantly higher expression of TRAIL, NKG2D, and the natural cytotoxicity receptors NKp30, NKp44 and NKp46 and a slight increase in KIR3DL1 and KIR2DL2/3. A 4-hour ⁵¹Cr-release assay showed expanded NK cells were significantly more cytotoxic against K562 cell compared to short-term IL-2 activated NK cells; at a 1:1 effector to target ratio, 67±6%, 26±1%, and 9±1% of K562 cells were killed by expanded, short term IL-2 activated and fresh NK cells respectively (p<0.05). Increased TRAIL expression on expanded NK cells was also associated with increased lysis of TRAIL-sensitive tumor cells (RCC tumors treated with bortezomib); at a 1:1 E:T ratio, 55±3% and 5±2% of bortezomib-treated RCC tumors were killed by expanded and short-term IL-2 activated NK cells respectively (p<0.05). We next assessed for differences in the in vivo longevity of these NK cell populations when transferred into immuno-deficient mice. Two million NK cells were labeled with a near infrared-dye (DiR; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide) and injected intra-peritoneal (i.p.) into CB.17 SCID-beige mice. Mice were administered IL-2 (100,000U/ml bid i.p.) for five days then underwent bioluminescent imaging using the IVIS100 system. Although FACS analysis of NK cells performed immediately prior to injection showed increased DiR fluorescent intensity in short-term IL-2 activated vs. expanded NK cells, fluorescence signal in vivo was slightly higher in the first 24-96 hours in mice that received expanded NK cells; fluorescence intensity was 5-41% (p=0.003) stronger in recipients of expanded NK cells compared to mice receiving short-term IL-2 activated NK cells. We next evaluated the in vivo anti-tumor effects of activated vs. expanded NK cells. CB.17 SCID-beige mice were injected i.p. with luciferase transduced 526 human melanoma cells three days prior to receiving an i.p. injection of short term IL-2 activated vs. expanded NK cells (+ bid i.p. IL-2). Bioluminescent imaging measuring tumor flux to calculate tumor burden and tumor doubling time showed no difference in tumor progression between both NK cell cohorts.

In conclusion, these results demonstrate that ex vivo expanded NK cells are phenotypically and functionally different than short-term IL-2 activated NK cells. Expanded NK cells have increased expression of natural cytotoxicity receptors, NKG2D and TRAIL and have greater TRAIL-mediated tumor cytotoxicity compared to IL-2 activated NK cells. Importantly, despite extensive ex vivo proliferation, expanded NK cells appear maintain similar longevity in vivo as non-expanded short term IL-2 activated NK cells.