Clinical grade of gerneration of dendritic cells for immunotherapy

In order to develop a protocol for clinical grade generation of dendritic cells (DCs) for cancer immumotherapy, aphereses were performed with the continuous flow cell separator and materials were derived from 10 leukemia patients that had achieved complete remission. Peripheral blood monocytes were cultured in vitro with GM-CSF, IL-4 for 6 days, then TNF-alpha (the TNF-alpha group) or TNF-alpha, IL-10, IL-6, PGE2 (the cytokine mixture group) were added to promote maturation. Cell number was counted by hematology analyzer, and phenotype study (CD1a, CD14, CD83) was carried out by flow cytometry, and the function of DCs was examined by mixed lymphocyte reaction. The results showed that (0.70 +/- 0.13)x10(7)/mL (the TNF-alpha group) and (0.79 +/- 0.04)x 10(7)/mL (the cytokine mixture group) DCs were generated respectively in peripheral blood obtained by leucapheresis. The phenotypes were as follows: CD1a(+) (74.65 +/- 4.45)%, CD83(+)(39.50 +/- 4.16)%, CD14(+) (2.90 +/- 1.76)% in TNF-alpha group, and CD1a(+) (81.86 +/- 5.87)%, CD83(+) (81.65 +/- 6.36)%, CD14(+) (2.46 +/- 1.68)% in the cytokine mixture group. It was concluded that leucapheresis may be a feasible way to provide large number of peripheral blood monocytes for DC generation, and combined administration of TNF-alpha, IL-1 beta, IL-6, and PGE(2) may greatly promote maturity.