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J Immunol Methods. 1996 Sep 27;196(2):121-35.

Improved methods for the generation of dendritic cells from nonproliferating progenitors in human blood.

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Rockefeller University, New York 10021, USA.


We have investigated an improved method for generating sizable numbers of mature dendritic cells from nonproliferating progenitors in human blood. The procedure uses 1% human plasma in the place of 10% fetal calf serum and involves two steps. The first step or 'priming' phase is a 6-7 day culture of T cell depleted mononuclear cells in medium supplemented with GM-CSF and IL-4. The second step or 'differentiation' phase requires the exposure to macrophage conditioned medium. This medium cannot be replaced by several known cytokines such as TNF-alpha, IL-1, IL-6, IL-12 and IL-15, and cannot be inhibited with neutralizing antibodies to IL-1, TNF-alpha, IL-6 or IL-12 alone, or in combination. Using this two-step approach, we obtain substantial yields. About 1-3 x 10(6) mature dendritic cells are generated from 40 ml of blood vs. < 0.1 x 10(6) from noncytokine treated blood. The dendritic cells derive from progenitors found primarily in a radioresistant population of CD14+ and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to plastic, and very strong T cell stimulatory activity. Strong APC function was evident for both the proliferation of allogeneic T cells in the MLR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted markers is also expressed, including CD83, p55, and perinuclear CD68. All of these dendritic cell properties are retained for at least 3 days when the cytokines are removed, suggesting that these populations are stable and terminally differentiated. We suggest that these cells will be effective in vivo as adjuvants for active immunotherapy.

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