Optimizing Preparation of Normal Dendritic Cells and bcr-abl + Mature Dendritic Cells Derived from Immunomagnetically Purified CD14 + Cells

Abstract

The goal of this work was to optimize dendritic cell (DC) preparations obtained from patients suffering from chronic myeloid leukemia (CML) and compare them with DC prepared from normal CD14⁺ mononuclear cells (MNC). We studied normal DC and bcr-abl ⁺ leukemic DC (CML-DC) yields, expression of membrane molecules, differentiation status, and ability to stimulate T cells. We isolated DC precursors from PBMC by CD14-specific immunoadsprotion and cultured them for 7 days in GM-CSF and IL-4, followed by a 3-day incubation to fully differentiate the cells. We evaluated cultures of CML-DC using RPMI 1640 medium supplemented with FBS and X-VIVO 15 medium containing human AB serum. In contrast to cells matured in RPMI 1640, virtually all cells incubated in X-VIVO 15 expressed CD83, a marker of mature DC. CML-DC and normal DC were indistinguishable in expression of CD83, resulting in the highest percentage reported so far. The yields of normal DC and CML-DC from CD14⁺ cells were indistinguishable. The percentage of bcr-abl ⁺ cells in PBMC varied among patients between 65% and 97% and the final CML-DC preparations were >98% bcr-abl ⁺ the highest purity of bcr-abl ⁺ cells to date. Normal DC and CML-DC were equally effective in stimulating proliferation of allogeneic and autologous T cells. These techniques provide highly enriched, mature, functional CML-DC.