110: Enhanced Natural Killer (NK) and NK T Cell Activation, Expansion and Cytokine Protein Production following Ex-Vivo Engineering (EvE) of Previously Cryopreserved Cord Blood (CB): Potential for CB NK and NK T Cells in Adoptive Cellular Immunotherapy (ACI)

CB is limited by the absence of available donor effector cells following UCBT. We demonstrated the immaturity of CB by reduced expression and production of IL-15, IL-12 and IL-18 in activated CB (Qian/Cairo, Blood, 1997; Lee/Cairo, Blood 1996; Satwani/Cairo, Br J Haem, 2005) may contribute to diminished CB cellular immunity and delayed immune reconstitution after UCBT. NKCD3-/56+ effector cells recognize target cells and express activating and inhibitory receptors. NKTCD3+/56+ cells may also play a role in allograft and tumor cytotoxicity. We reported the ability to EvE cryopreserved, thawed, recryopreserved, rethawed CB with increased NKbright/dim subsets expressing KIRs and NCR with enhanced in-vitro and in-vivo cytotoxicity (Ayello/Cairo. BBMT, 2006). In this study, we compare 2 vs 7 d expansion and activation of NK and NKT subsets expressing NKRs and the production of IL-15, IL-18 and IFN-g. Rethawed CB cells were cultured with anti-CD3, IL-2, IL-7 and IL-12; 2–7 days. CD3, CD16, CD56, CD94, NKG2A, NKG2D, NKp46, KIR2DS4, KIR3DL1, LAMP-1 and granzyme B expression were determined by flow cytometry; IL-15, IL-18 and IFN-g protein by ELISA. Non-adherent total cells were significantly increased (6.2 ± 214 × 107 vs 5.8 ± 57 × 106, p < 0.001) with no change in NKdim subset; but significant increased NKT subset (71.8 ± 6.0 vs 2.97 ± 0.3%, p < 0.001). NKbright cells were decreased (3.3 ± 1.1 vs 13.4 ± 1.4%, p < 0.01); whereas, NKTbright subset was significantly increased (10.6 ± 1.4 vs 0.7 ± 0.1%, p < 0.05). NK KIRdim/bright subsets were not significantly increased, but NKT KIR3DL1dim subset was increased (17.1 ± 1.2 vs 1.1 ± 0.5%, p < 0.05). CD94/NKG2A expression was decreased (7.8 ± 1.3 vs 22.7 ± 1.0%, p < 0.001.) while CD94/NKG2D was increased (41.4 ± 0.4 vs 23.7 ± 2.0%, p < 0.001). NK and NKT KIR2DS4 subsets were increased (24.8 ± 0.1 vs 3.1 ± 0.4%, p < 0.001; 19.0 ± 0.6 vs 1.0 ± 0.1%, p < 0.001, respectively), as well as CD107a (65.3 ± 2.2 vs 12.95 ± 1.5%, p < 0.001), granzyme B (33.6 ± 0.61 vs 25.8 ± 1.79%, p < 0.01), and IL-18 and IFN-g protein production (730 ± 4.7 vs 183 ± 8.8 pg/ml, p < 0.001; 37.3 ± 7.6 vs 21 ± 1.4 pg/ml, p < 0.05). In summary, CB MNC may be thawed at time of UCBT, recryopreserved, rethawed, and activated for up to 7 d to yield increased NK and NKT subsets with increased KAR expression (KIR2DS4, CD94/NKG2D), NK activation (CD107a), and production of IL-18 and IFN-g, suggesting that cryopreserved CB cells may be EvE for potential use as ACI for DLI after UCBT.