Implication of delayed TNF-alpha exposure on dendritic cell maturation and expansion from cryopreserved cord blood CD34+ hematopoietic progenitors

Most currently used systems for dendritic cell (DC) production from progenitors entail tumor necrosis factor alpha (TNF-alpha) at the onset of cell culture, based on the notion that TNF-alpha might be required in the early stages of DC development. To optimize conditions for DC expansion from cryopreserved cord blood (CB) CD34+ hematopoietic progenitors, we took a dynamic approach to define the timing of TNF-alpha exposure to the culture. We cultured cord blood CD34+ cells in RPMI-1640 with 10% human AB plasma, stem cell factor (days 1-6), granulocyte-macrophage colony-stimulating factor (days 1-18), interleukin-4 (days 6-18) and varying schedules of TNF-alpha (0-144 h after thawing). Expression of the DC-associated markers, including CD83/CD1a, HLA DR/CD86/CD80, CD14/CD40, was monitored every 3 days. Our data demonstrate that delayed TNF-alpha exposure by 48-72 h after thawing gave rise to two- to three-fold increase in the yield of CD83+ DCs that were highly active in stimulating allogeneic T-cell proliferation compared to immediate TNF-alpha exposure. Thus, the immediate exposure of cryopreserved cord blood CD34+ cells to TNF-alpha, potentially compromising DC expansion, should be avoided. This finding should be of significant consideration when using cryopreserved CD34+ progenitor cells as a source of immunologically competent DCs in a clinical setting.