IMPROVED METHODS FOR THE GENERATION OF DENDRITIC CELLS FROM NONPROLIFERATING PROGENITORS IN HUMAN BLOOD

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 ca lf 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 suppl emented with GM-CSF and IL-4. The second step or 'differentiation' pha se 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 ant ibodies to IL-1, TNF-alpha, IL-6 or IL-12 alone, or in combination. Us ing this two-step approach, we obtain substantial yields. About 1-3 x 10(6) mature dendritic cells are generated from 40 mi 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 CDl 4(+) and adherent blood mononuclear cells and have all the features of mature cells. They include a stellate cell shape, nonadherence to pla stic, and very strong T cell stimulatory activity, Strong APC function was evident for both the proliferation of allogeneic T cells in the M LR, and the generation by syngeneic T cells of class I restricted, CTL responses to influenza virus. A panel of dendritic cell restricted ma rkers is also expressed, including CD83, p55, and perinuclear CD68. Al l 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.