The Effect Of The Dendritic Cells Derived From Acute Myeloid Leukemia And Induced By PA-MSHA On CD4~+CD25~+ Regulatory T Cells

Objective:CD4+CD25+ Treg cells which have the nature of immune suppression and immune incompetence are about 5%-10% of peripheral CD4+ T cells in healthy people, and participate in process of immune suppression. The numbers of Treg cells are higher in peripheral blood and tumor tissues of a variety of cancer patients and are responsible for inducing and maintaining peripheral tolerance. Treg cells have two subsets, natural Treg and adaptive Treg. Adaptive Treg cells can be differentiated from CD4+ T by the change of expression on the costimulatory molecules of APC or the change of the cytokines. Treg have been suggested to play an important immunopathological role in human cancer by lowering the intrinsic T-cell immunity towards tumor-associated antigen, resulting in tumor immune evasion. Some reports have indicated that Treg depletion can be useful in tumor biotherapy. Treg depletion would eliminate immune-suppression mediated by Treg leading to enhance T-cell activity. Another study has confirmed that immature DC can induced CD4+T cells to adaptive Treg. The current strategies of cancer immunotherapy focus on breaking immune tolerance and enhance the host T cell response. Pseudomonas aeruginosa-mannose sensitive hamemagglutination vaccine (PA-MSHA vaccine) is prepared from Pseudomonas aeruginosa which contains mannose-sensitive hemagglutinin pili. It has been widely used for anti-inflammatory treatment, even for anti-tumor treatment as immune modulator. According to reports, PA-MSHA vaccine could improve clinical efficiency as adjuvant therapy for a variety of cancer such as leukemia, malignant lymphoma, lung cancer, breast cancer, gastric cancer, liver cancer and so on. This vaccine can increase the antigen presenting function of dendritic cells (DCs) or Th1/Th2 proportion, and regulate unbalance between cellular immune and humoral immune to achieve the purpose of effective treatment of tumors. However, the specific mechanism is still not very clear. Acute myeloid leukemia (AML) is a malignant hematopoietic disorder characterized by proliferation of immature myeloid precursors with considerable impairment of the immune system. Compared with healthy controls, AML patients had a higher proportion of CD4+CD25+ T cells in peripheral blood.Professor Sun reported PA-MSHA vaccine can improve immune function in patients with acute leukemia. The level of IL-2, the activity of NK cells, the ratio of CD4+/CD8+ in PA-MSHA vaccine treatment group are significantly higher than those in control group.But whether PA-MSHA vaccine can take effect on anti-tumor through reducing the number and function of the Treg cells or not, has not been reported. This study will detect the effect of the dendritic cells derived from acute myeloid leukemia and induced by PA-MSHA on CD4+CD25+ regulatory T cells, and is to investigate the immune regulatory fuctions of PA-MSHA to acute myeloid leukemia.Methods:The buffy coat separated from peripheral blood cells of untreated newly diagnosed patients with acute myeloid leukemia by leukapheresis was diluted 1:1 with phosphate buffered saline(PBS). Lymphocytes were isolated using Ficoll lymphocyte separating medium, and the cells were incubated in RPMI1640 complete mediums for 3h. Then after the suspended cells were removed, recombinant human granulocyte-macrophage colony-stimulating factor(rhGM—CSF)and recombinant human interleukin-4(rhlL-4) were added into medium and the culture were incubated for 7 days. Then the cells were devided into three groups. As the control group, the first group was not given any treatment. The second group was added with PA-MSHA. Tumor necrosis factor-a(TNF-a) added in the third group, which continued to incubat 24 hours. The morphological feature was observed with inverted microscope everyday. The phenotypes of cells were detected by flow cytometry, and the proliferation effect of three groups of AML-DCs on Allogeneic T lymphocytes was investigated by a mixed lymphocyte reaction system which was analyzed by MTT assay. In the 8th day, the CD4+ T cells separated from peripheral blood cells of healthy adult by magnetic cell sorting (MACS) system were respectively incubated with the DCs from the three groups for 7 days.Then the supernatant concentration of IL-10, TGF-p was detected by ELISA kit. The cells of all the groups were harvested respectively and further tested for the expression of CD4+CD25+ T cells by flow cytometry. The RT-PCR method was used to examine Foxp3mRNA expression.Results:The morphology of dendritic cells in PA-MSHA group and TNF-a group were irregular with slender synapses on their surface, and the expressions of CDla,CD80, CD83, CD86and HLA-DR on mature dendritic cells in PA-MSHA group and TNF-a group were significantly higher on day 8 than the control group, and proliferation indexes of the two groups was also higher than the control group as the more numbers of stimulating cells. In addition, the levels of IL-10 and TGF-β, the expressions of CD4 and CD25 on Treg, and the expression of Foxp3mRNA in PA-MSHA group and TNF-a group are all higher than the control group.Conclusion:PA-MSHA can further promote the maturation of AML-DC, and thus inhibit the differentiation from CD4+ T cells to Treg. So PA-MSHA can enhance anti-tumor activity for acute myeloid leukemia patients.