CB use is limited by the absence of available donor effector cells (NK, and NKT cells) for SCT (Cairo, et al Transfusion, 2005). The immaturity of CB, characterized by reduced expression and production of IL-15, IL-12 and IL-18 in activated CB (Qian et al, Blood, 1997; Lee at al, Blood, 1996; Satwani et al. Br J Hem, 2005), may contribute to reduced CB cellular immunity and delayed immune reconstitution after UCBT. In innate immunity, NK cells (CD3/56+) are regulated by various NK receptor (NKR) expression. NK(CD56dim) cells are cytotoxic, constitute 90% of the NK population while 10% (CD56bright) are cytokine producing (Shereck/Cairo, Ped Bld Can, 2007). NKT cells, (CD3+/CD56+), may play a role in allograft and tumor cytotoxicity. We demonstrated the ability to EvE (48 hrs; with anti-CD3 (50ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY]) c ryopreserved, t hawed, re c ryopreserved, re t hawed, (CTCTE) CB with increases in NKbright/dim subsets expressing NKRs and enhanced NK in-vitro and in-vivo cytotoxicity (Ayello et al. BBMT, 2006). We compared day 2 vs 7 EvE CTCTE CB expansion and activation (LAMP-1) of NK and NKT cells expressing NKRs, IL-15, IL-18 and IFN-g protein levels and mechanisms of cytotoxicity. Rethawed nonadherent CB cells were EvE with ABCY. NKRs (CD3, CD16, CD56, CD94, NKG2A, NKG2D, NKp46, KIR2DS4, KIR2DL2), intracellular perforin and LAMP-1 expression were determined by flow cytometry. IL-18, IL-15 and IFN-g protein measured by ELISA. Non-adherent total cell number were significantly increased at day 7vs2 (6.28±214.7×107 vs 5.87±56.9×106, p<0.001, respectively) with no change in the NKdim (CD3/16+/56+dim) subset (40.4±2.1 vs 30.3±8.0%, p=NS, respectively); yet, NKT subset was significantly increased (71.85±6.03 vs 2.97±0.29%, p<0.001). NKbright cells were significantly decreased at day 7 vs 2 (3.3.±1.1 vs 13.4±1.4%, p<0.001) and no change in NKdim KIR inhibiting receptors subset. Further, NK activating receptor (KIR2DS4) in both NK and NKT subsets was increased at day 7 vs 2 (24.8±0.08 vs 3.1±0.43%, p<0.001; 18.9±0.6 vs 1.1±0.06%, p<0.001, respectively). Similarly, both NKdim and NKT NKp46 were increased at day 7 vs 2 (11.4±0.4 vs 2.1±0.2%, p<0.001; 35.9±0.6 vs 7.5±1.1%, p<0.001, respectively). Activating NCR (CD94/NKG2D) expansion was significantly increased at day 7 vs 2 (41.4±0.4 vs 23.7±2%, p<0.001) and NCR inhibitory receptor (CD94/NKG2A) subset was decreased (7.83±1.34 vs 22.7±0.98%, p<0.001). Perforin expression was decreased at day 7 vs 2 (55.7±1.8 vs 84.3±1.3%, p<0.001) yet LAMP-1 expression was increased (65.3±2.2 vs 12.95 ±1.5%, p<0.001). IL-18 and IFN-g protein production was increased at day 7 vs 2 (730±4.7 vs 183±8.8 pg/ml, p<0.001; 37.3±7.6 vs 21.6±1.4 pg/ml, p<0.05) as well as increased NK cytotoxicity (K562:71.5±0.8 vs 54±3.9%, p<0.001; Kasumi-1(AML):56.7±0.5 vs 38±1.2%, p<0.001). In summary, CB MNC may be thawed at time of UCBT, recryopreserved, rethawed at a later date, EvE and activated for up to 7 days to yield increased NK and NKT subsets with increased KAR expression (KIR2DS4, CD94/NKG2D, Nkp46), NK activation (LAMP-1), production of IL-18 and IFN-g and cytotoxicity without an increase in NK inhibitory receptor expression. These data suggest that cryopreserved CB cells may be EvE for potential use as ACI for DLI after UCBT.

Author notes

Disclosure:Research Funding: The Pediatric Cancer Research Foundation funds research.