Ten Fold Ex-vivo Expansion of Allostimulatory Dendritic Cells (DC) from Cord Blood by Flt-3 Ligand, GM-CSF, TNF-α, IL-4 and Autologous Plasma: Implications for Strategies for Neonatal Vaccinations and/or Post Cord Blood Transplant Immunotherapy ♦ 1378

Abstract

Dendritic Cells (DC) are potent antigen presenting cells and are considered important cellular components for immunoregulation. Cord blood (CB) has recently been recognized as an alternative source of stem cells however transplant recipients often experience delayed immune reconstitution. DC may be useful for enhancing immunity against infection and/or malignant disease. In this study, we explored the use of 10% autologous plasma to generate DC from CB adherent MNC. CB was collected from scheduled Cesaren-section or vaginal deliveries and CB MNC were collected by Ficoll-hypaque density centrifugation. Plasma was recovered from the supernatant and heat inactivated at 56°C. The plastic adherent population was cultured in AIM-V with 10% CB plasma supplemented with GM-CSF (100 ng/ml) TNF-α, (2.5 ng/ml), Flt3-Ligand (25 ng/ml) and IL-4 (5 ng/ml). Control without cytokines and/or 10% plasma were also cultured. Cytokine cultured cells developed characteristic DC morphology with many slender, elongated, and/or numerous fine membrane projections. By immunohistochemistry these morphologically distinct cells were CD1a+ and CD14-. Control cells maintained macrophage appearance of large adherent cells without membranous projections and were CD14+ but CD1a-. Cytokine cultured cells had significant phenotypic changes(confluent culture vs. day 0, mean ±SEM) consistent with DC expression of CD1a+CD80+ (7.5±1.4% vs. 0.9±0.2%, p<0.01, n=6) (8-fold) and CD1a+CD80+CD86+ (5.3±1.2% vs. 0.6±0.2%, p<0.01, n=6)(10-fold) with a fall of CD14+ (5.6±1.6% vs. 33.2±5.7, p<0.01, n=6). Allogenic primary Mixed Lymphocyte Reaction (MLR) with T-cells enriched by column was significantly increased over CB adherent cells by 351±49% (p=0.001, n=3) at ratio of 1:5 and 160±13% (p=0.002, n=3) at ratio of 1:25 (DC:T-cell). In summary, DC can effectively be generated with 10% autologous plasma from CB adherent MNC, acquire co-stimulatory molecules and are powerful stimulators of T-cell alloreactivity.