[The immune effects of rituximab on dendritic cells derived from patients with primary immune thrombocytopenia].

OBJECTIVE: To explore the changes of surface antigen and function of rituximab on dendritic cells derived from patients with Primary immune thrombocytopenia (ITP) to further understand the effective mechanism of immunotherapy. METHODS: The peripheral blood mononuclear cells (PBMCs) were isolated from remission patients with ITP before and after low-dose rituximab infusion, and the PMNCs were stimulated for 5 days by rhGM-CSF and rhlL-4 in 5% CO2 air at 37°C incubator. Then all of DCs were cultured with TNF-α for 48 hours. The morphology of DCs was monitored under inverted microscope daily, and the surface antigens of the DCs were analysed by flow cytometry, meanwhile the levels of IL-12p70 and TGF-ß1 in supernatants were detected by ELISA, mix lymphocyte reaction was performed by MTT assay. RESULTS: (1) Rituximab-treated-DCs showed no obvious tree-like protruding compared with untreated-DCs. The former cells were small and most of nucleus were centric. (2) The expressions of HLA-DR, CD80, CD83 and CD86 on rituximab-treated-DCs \[56.37 ± 3.95)%, (36.41 ± 2.82)%, (30.45 ± 4.61)% and (41.98 ± 4.17)%, respectively\] were significantly lower than those untreated-DCs \[(73.71 ± 7.61)%, (55.14 ± 7.30)%, (80.91 ± 7.09)% and (59.03 ± 3.43)%, respectively\](all P < 0.05), the concentration of IL-12p70 was significantly lower, \[(66.87 ± 4.29)% vs (50.17 ± 14.52)%\], while that of TGF-ß1 \[(9.70 ± 0.31)%\] higher than the untreated-DCs \[(2.70 ± 0.36)%\] (P < 0.05). (3) The abilities to activate T cells proliferation of rituximab-treated-DCs reduced compared with untreated-DCs. CONCLUSION: The surface antigen of ITP-DCs and the concentration of IL-12p70 reduced after the low-dose rituximab infusion. The abilities to activate T cells proliferation reduced while the concentration of TGF-ß1 increased. Rituximab may achieve its therapeutic effect on ITP by downregulating the immunoreactivity of DCs.

[In vitro inducing differentiation of bone marrow mononuclear cells of chronic myeloid leukemia].

BACKGROUND & OBJECTIVE: Interferon-alpha (IFN-alpha), an important immunoregulatory cytokine, has been widely used in treating virus hepatitis, lymphoma, and chronic myeloid leukemia (CML), and showed evident effect, but the mechanism is unclear. Dendritic cells (DCs), specialized antigen-presenting cells (APCs), play a pivotal role in activating initial T cells, and maintaining cell immune responses. Does the efficiency of IFN to CML relate to the DCs induced by IFNy What kind of effect do DCs have on IFN therapy for CMLy Up to now, few researches are available. This study aimed to observe whether the DCs were induced through culturing bone marrow mononuclear cells (BMMNCs) of CML in vitro, investigate the mechanism of IFN-alpha therapy for CML, and then provide a new strategy for clinical therapy. METHODS: BMMNCs were obtained from blood of CML patients by Ficoll-Paque density gradient centrifugation, and induced with IFN-alpha and granulocyte-macrophage colony-stimulating factor (GM-CSF) (IFN-alpha/ GM-CSF group), or interleukin-4 (IL-4) and GM-CSF (IL-4/ GM-CSF group), or IFN-alpha, GM-CSF, and IL-4 (IFN-alpha/GM-CSF/IL-4 group) for 7 days. Morphology of BMMNCs was observed under transmissional and optical microscope. The phenotypes [CD1a, CD83, CD86, human leukocyte antigen (HLA)-ABC, HLA-DR, CD54] were assayed by flow cytometry (FCM). The mixed lymphocyte reaction(MLR) of DCs was evaluated by MTT assay. RESULTS: After inducements, BMMNCs showed typical dendritic projections, and highly expressed CD1a, CD83, CD86, HLA-ABC, HLA-DR, and CD54. Positive rates of HLA-ABC and HLA-DR were higher in IFN-alpha/ GM-CSF group and IFN-alpha/GM-CSF/IL-4 group than in IL-4/ GM-CSF group (P<0.05). Positive rate of CD86 and MLR were the highest in IFN-alpha/GM-CSF/IL-4 group (P<0.05). Positive rates of DC antigens and MLR in IFN-resistant group were significantly lower than those in newly diagnosed group and IFN-sensitive group (P<0.05), but positive rate of CD86 and MLR have no significant difference among 3 groups in the presence of IFN-alpha/GM-CSF/IL-4 (P>0.05). CONCLUSIONS: The BMMNCs of CML cultured in the presence of IFN-alpha and other cytokines can be induced into DCs with morphologic and immunophenotypic characteristics, overexpresses major histocompatibility complex (MHC) molecules, co-stimulatory molecules, and adhesion molecules, and have enhancing MLR. The possible mechanism of IFN-alpha therapy for CML may be relate to DCs.

[Induction of anti-leukemic immunity of dendritic cells derived from multidrug resistant leukemia K562/A02 cells with high expression of P-glycoprotein and sensitive K562 cells].

This study was aimed to investigate and compare the anti-leukemic effect mediated by dendritic cells (DC) derived from multidrug resistant (MDR) leukemia K562/A02 cells with high expression of p-glycoprotein (P-gp) and sensitive K562 cells. Multidrug resistant K562/A02 cell line and sensitive K562 cell line from chronic myeloid leukemia (CML) were induced for differentiating to DC in complete RPMI 1640 culture medium supplemented with GM-CSF (1 000 U/ml), IL-4 (500 U/ml) and TNF-alpha (100 ng/ml) for 14 days. The morphologic features of DC were observed by means of optical microscopy and the phenotype of DC was detected by flow cytometry. T-cell stimulating activity was determined by allogeneic lymphocyte reaction (allo-MLR). Cytotoxic activity was measured by MTT assay. The results indicated that DC derived from K562/A02 cells and K562 cells similarly showed the typical morphology of dendritic cell and expressed the surface differentiation antigens and costimulatory molecules CD1a, CD83, HLA-DR, CD80 and CD86 of DC. In allo-MLR, K562/A02-DC had a higher capacity to induce lymphocyte proliferation, compared with K562-DC (P < 0.05). K562/A02-DC and K562-DC could similarly generate specific cytotoxic activity against K562/A02 cells or K562 cells respectively, but low reactivity against HL-60 cells. More importantly, the cytotoxic activity mediated by K562/A02-DC was stronger than that by K562-DC against K562/A02 cells or HL-60/VCR cells (P < 0.01, respectively). It is concluded that functional DC can be differentiated from multidrug resistant leukemia K562/A02 cells as well as sensitive K562 cells in the presence of GM-CSF, IL-4 and TNF-alpha. Especially, DC derived from K562/A02 cells can induced a p-glycoprotein specific anti-leukemic immunity.