22: Ex-vivo expansion (EvE) of previously cryopreserved cord blood (CB) into natural killer (NK) cells with enhanced AML and neuroblastoma cytotoxicityPotential role of CB NK cells in adoptive cellular immunotherapy (ACI)

CD56+ NK subsets exhibit differential NK receptors (NKR) such as cytotoxicity profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors (NCR) involved with tumor target recognition (Farag et al Blood, 2002). In particular, AML and Neuroblastoma are NK sensitive pediatric tumors. CB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after UCBT (Cairo et al Transfusion, 2005). We demonstrated the ability to EvE CB in short-term culture with IL-2, IL-7, IL-12 and anti-CD3 with increased CD3-/16+/56+dim and bright subsets expressing KIR3DL1, KIR2DL2, KIR2DL1/S1 and CD94/NKG2a with increased NK and LAK cytotoxicity (Ayello/Cairo et al BBMT, 2006). In this study, we compared short-term culture (48 hrs) with prolonged cultures (4-10 days) on expansion of NK cells expressing NCR, KIR, NKG2, lytic ability and mechanisms of tumor lysis. Rethawed CB cells were cultured 2-10 days with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY]. NKR expression (KIR2DS4, NKG2D, CD94, NKp46), intracellular granzyme B and LAMP-1 receptor (CD107a) expression were determined by flow cytometry. Cytotxcity of EvE effector CB cells was measured by europium release assay at 20:1 E:T ratio with tumor targets K562 (NK), Daudi (LAK), Kasumi-1 (AML) and SYSY5Y (neuroblastoma). KIR2DS4 was significantly increased at day 10 vs 2 in ABCY in both CD3-/16+/56+bright and dim subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9±0.2%, p<0.001, respectively). C-lectin receptor CD94/NKG2D expression was increased at day 7 vs 2 (41.4±0.43 vs 23.7±2.0%, p<0.001). NCR expression in CD3-/16+/56+dim NKp46 subset was increased at day 7 vs 2 (10.1±0.06 vs 2.62±0.8, p<0.001). Granzyme B expression was increased from day 2 to 10 (25.8±1.7 vs 45.1±1.7%, p<0.001). CD107a expression was significantly increased at day 7 vs 2 (12.9 ±1.4 vs 69.3±2.2%, p<0.001). Additionally, increased cytotoxicity was demonstrated at day 7 vs 2 with tumor targets K562 (71.5±0.81 vs 53.8±3.9%, p<0.001), Daudi (63.9±0.73 vs 38±1.1%, p<0.001), Kasumi-1 (56.6±0.4 vs 31.8±1.8, p<0.001) and SYSY5Y (59.5±5.35 vs 32.6±4.9%, p<0.01). In summary, CB MNC may be thawed at time of transplantation, recryopreserved, rethawed at a later date, expanded and activated up to 10 days to yield viable NK subsets which appear to be cytolytic against AML and Neuroblastoma and could be potentially used as ACI post UCBT.