Objective To induce human mononuclear cell of cord blood into CD 3 activating killing (CD 3AK) cells with anti-CD 3 monoclonal antibody (CD 3McAb) and recombinant human interleukin-2 (rhIL-2), so that their proliferative activity, activity of killing action, phenotypes and level of secretory cytokines can be observed dynamically. Methods The increase of the number of cells was counted by Tapan-blue staining. The killing action can be measured by using methyl -thiazolyl-tetrazolium-array. The phenotypes of cells were analysed by using indirect immunofluorescence assay. The levels of IL-6, interferon-? (IFN-?) and tumor necrotic factor-? (TNF-?)in culture supernatants were analysed by using enzyme-linked irnmunosorbent assay(ELISA). Results The increase of the number of CD 3AK cells from cord blood was the highest amounting to 78.56 times in the second week. The killing action reached the peak on day 12, and all target cells (malignant cell lines) could be killed significantly. The heterogeneous phenotypes of CD 3AK cells showed that the number of cells with CD 3+, CD 8+, CD 25+, CD 38+, CD 16+ and CD 56+ increased significantly on day 7,14 compared with those of pre-culture (P

OBJECTIVETo establish a serum-free culture system of dendritic cells (DCs) from chronic myeloid leukemia (CML) cells so that DCs vaccine may be applied to the adoptive immunotherapy of CML in the near future.

METHODSFetal calf serum, serum-free medium and autologous serum were used for culture of DCs. The usage of cytokines was classified into two groups: group A (stem cell factor, granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4) and group B (granulocyte/macrophage colony-stimulating-factor, tumor necrosis factor-alpha and interleukin-4). The phenotypes of DCs were analyzed by using indirect immunofluorescence and flow cytometry. Mixed leukocyte responses were performed by methyl thiazolyl tetrazolium (MTT) assay. Chromosome analysis of DCs can be achieved by displaying G banding. T cells from CML patients were stimulated with autologous DCs and T-cell cytotoxicity was measured by (MTT) assay.

RESULTSCD34(+) cells or mononuclear cells were obtained from peripheral blood or bone marrow samples of eight patients of chronic-phase CML. Group A of serum-free medium was better than group B in expansion of total cell numbers and the rate of DCs. These results of serum-free medium were not significantly different from those of fetal calf serum medium, but the results of autologous serum medium were inferior to two groups above. The expression of major histocompatibility complex class II antigen on the surface of DCs was notable (> 50%), but the expression of CD83 and the costimulatory molecules CD86 was not noticeable (10% - 50%). Although CD1a(+)/CD14(-) DCs were potent stimulators of allogeneic lymphocytes, expansion of T cells from normal volunteers were not significant (average 27.2 fold at DCs: T cells ratio of 1:10). At day 12, CD1a(+) cells from three patients were studied by displaying G banding and Ph(+) cells in these populations were 100%, 98% and 60%, respectively. At an effector: target ratio of 40:1, 32% to 45% cytotoxicity was noted with DC-stimulated T cells against autologous leukemia cells.

CONCLUSIONSA stable serum-free culture system of CML-DCs was established. The expression of CD83 and CD86 on the surface of CML-DCs and DCs' potent stimulation of allogeneic lymphocytes were not notable. DCs in CML patients can be derived from the malignant clone and these malignant DCs could induce anti-leukemic reactivity in autologous T lymphocytes without the necessity for additional exogenous antigens.

Cells, Cultured ; Culture Media, Serum-Free ; Cytotoxicity, Immunologic ; Dendritic Cells ; physiology ; Humans ; Immunotherapy, Adoptive ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; immunology ; therapy ; T-Lymphocytes ; immunology