Abstract • 170

We investigated methods to ex vivo expand and activate cord blood (CB) dendritic cells (DC), CB T-cells and peripheral blood (PB) T-cells. Briefly, CB was collected from term vaginal deliveries and MNCs were separated by Ficoll-hypaque (FH) density gradient and adherent cells were resuspended in AIM-V with 10% autologous CB plasma and GM-CSF (100 ng/ml), TNF-α (2.5 ng/ml), Flt3 ligand (25 ng/ml), IL-4 (5 ng/ml) in flasks for 14 d. There was a 22 ± 1.2 fold increase (p<0.01) in cells expressing CD1a+/CD80+/CD86+ and a significant 4.3±0.2 fold reduction in CD14+ cells. Ex vivo expanded DC stimulated allogeneic T-cells by MLR at 1:25 and 1:5 APC to T-cell ratio as measured by MTT proliferation assay (160±13% and 350±50% respectively, p<0.01). CB MNC were isolated by FH then cultured with 5% CB plasma and 5% XLCM™ (kindly provided by Hemosol, Inc. Ontario, Canada). Cell cultures were analyzed and resuspended on days 4, and 7. There was a dramatic 173±58 fold expansion (p<0.05) with an increase cells expressing CD4+/CD25+ (60±6% vs. 0.17±.07%, p<0.0001) and CD8+/CD25+ (33±5% vs. 0%, p<0.0001). Similarly, cytokine activation has been performed with PBMNC which were isolated by FH and non-adherent MNC were cultured in AIM-V serum-free medium with anti-CD3 (50 ng/ml), IL-12 (5-100 U/ml), and IL-2 (5-1000 U/ml) over 10 days with demidepletion on days 4, 7, and 10. On day 7 there was 6.9±0.6 fold expansion of PBMNC, a significant increase in cells expressing CD3+ (98±0.4 vs. 76±2, p<0.05) and CD8+ (71±3% vs. 33±2%, p<0.05). Similarly, there was an increase in MHC Class II expression (39±7 vs. 17±2%, p<0.05), CD45RO+ (61±13 vs. 36±3.4%, p<0.05), and CD25+ (54±12% vs. 0.4±0.1%, p<0.01). Cell culture (IL-2 at 50U/ml) supernatant levels of INF-γ were significantly increased by day 4 to 313±16.7ng/ml vs. 0.1±0.1ng/ml of control (p<0.001), and TNF-α levels were significantly increased to 700±75pg/ml vs. 71±40pg/ml for control (p<0.05). In summary, CB DC can be generated using autologous CB plasma, Flt-3L, GM-CSF, TNF-α, and IL-4, and a significant expansion of CB T-cells can be generated using XLCM™. The combination of anti-CD3, IL-2, and IL-12 significantly expands PB CTL subpopulations and leads to functional activation. Future studies are under way to develop optimal protocols for combining ex vivo expansion of DC and CTL.