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The Change and Mechanism Research on the CD4~+CD25~+ Regulatory T Cells in Malignancy Hematopathy Patients

Author: ChengYuSheng
Tutor: WangGuiQin
School: Shanxi Medical
Course: Medical Immunology
Keywords: Acute leukemia CD4 CD25 regulatory T cells Immunosuppressive C57BL / 6 mice Foxp3 mRNA Immunotherapy
CLC: R733
Type: Master's thesis
Year: 2009
Downloads: 133
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Abstract

In 1995, Sakaguchi et al first reported about 5% to 10% of the cells in the peripheral blood of normal mice, CD4 T cells express CD25 molecules (IL-2 receptor α chain) sustained high subsets of cells low expression of the CD45RB molecule, called CD4 ~ CD25 ~ regulatory T cells (CD4 ~ CD25 ~ regulatory T cell, CD4 of ~ CD25 ~~ Treg for short). CD4 ~ CD25 ~ Treg through the \Many tumor-associated antigen is normal autologous component, rather than abnormal product of the mutant gene, showed that the tumor immunity to a certain extent is an autoimmune, therefore, CD4 ~ CD25 ~ Treg having a role in inhibition of tumor immunity. Leukemia is a clonal malignancy originated in hematopoietic stem cells, immunotherapy of leukemia in recent years has gradually become the hot spot in leukemia treatment, tumor antigen specific immunotherapy effect is not very satisfactory, may be through with tumor cells some mechanism to evade the immune system's attack. It has been confirmed that the CD4 ~ CD25 ~ Treg cells can suppress tumor-specific immune response, plays a negative regulatory role of immunotherapy, is another mechanism of tumor immune suppression. In order to explore the hematologic malignancies develop in CD4 ~ CD25 ~ Treg negative regulatory role. This topic is intended to study the following: (1) detection of CD4 ~ CD25 ~ Treg content in peripheral blood of patients with acute leukemia. (2) mice in vitro observation of tumor can induce CD4 ~ CD25 ~ Treg increased proportionately. Part I: patients with hematologic malignancies CD4 ~ CD25 ~ detection purposes of regulatory T cells in peripheral blood: detection of changes in acute leukemia patients with peripheral blood CD4 ~ CD25 ~ Treg subsets, and its significance in the pathogenesis of hematologic malignancies. Methods: Department of Hematology, the Second Hospital of Shanxi Medical patients with acute leukemia (AL) 32 patients from September 2007 to May 2008, both the initial issuance of the hospitalized patients before chemotherapy, 20 males and 12 females, with an average age 53.6 years. All patients with acute leukemia were confirmed by clinical, bone marrow cytology, immunophenotyping diagnosed. The control group of healthy subjects in hospital 16 cases, male 10 cases and 6 females, average age 48.2 years. Peripheral blood CD4 ~ CD25 ~ Treg cell levels detected by flow cytometry. Measured by ELISA patients and healthy controls serum TGF-β and IL-10 levels, and analysis of IL-10 and TGF-β levels and CD4 ~ CD25 ~ Treg correlation in the serum of patients with acute leukemia. Lymphocyte percentages are expressed as mean ± SD, between the two groups were compared using the t test for independent samples. The results: the proportion of CD4 T cells, CD4 ~ CD25 ~ Treg cells in the peripheral blood of patients with acute leukemia compared with normal control (3.96 ± 1.86)% vs (2.08 ± 1.63)% (P lt; 0.01), consistent with the literature. CD4 ~ CD25 ~ Foxp3 Treg cells in peripheral blood of patients with acute leukemia accounts for the proportion of CD4 T cells compared with normal controls (3.22 ± 1.01)% vs (0.83 ± 0.72)% (P lt; 0.01), in patients with acute leukemia in peripheral blood CD4 Foxp3 T cells was significantly higher than that in normal controls (14.9 ± 2.92)% vs (5.68 ± 1.21)% (P lt; 0.001). ELISA method for detection of acute leukemia patients with serum TGF-β1 and IL-10 were 26.9 ± 5.2ng/ml and 74.6 ± 8.7 pg / ml. Peripheral blood CD4 ~ CD25 ~ Treg cell levels and serum of TGF-β1 (r = 0.508, P lt; 0.05), IL-10 (r = 0.556, P lt; 0.05) levels were positively correlated. Conclusions: CD4 ~ CD25 ~ Treg cells in the peripheral blood of patients with acute leukemia, the proportion was significantly higher than normal controls, suggesting that CD4 ~ CD25 ~ Treg increased the proportion of the anti-tumor immune dysfunction in patients with leukemia, the tumor is easy growth and metastasis. ② acute leukemia patients with peripheral blood CD4 ~ CD25 ~ Treg cell levels of serum IL-10 and TGF-β expression levels were positively correlated, indicating that IL-10 and TGF-β in the inhibitory effect of CD4 ~ CD25 ~ Treg cells play an important role . These studies will provide useful inspiration for the target immune regulation strategies CD4 ~ CD25 ~ Treg. Part II: tumor cells in murine regulatory T cells to CD4 ~ CD25 ~ purpose: whether the observed experimental murine tumor cells secrete soluble substances can induce CD4 ~ CD25 ~~ of Treg differentiation and proliferation, the purpose of this study mechanisms in patients with hematologic malignancies CD4 ~ CD25 ~ Treg elevated preliminary exploration. : RPMI1640 culture medium containing 10% fetal bovine serum (FBS) cultured EL-4 lymphoma cell line derived from C57BL / 6 mice and erythroleukemia tumor cell lines FBL3 of, 5% CO2, at 37 ° C, saturated humidity conditions under cultivation, the culture supernatant of the preparation of tumor cells. Selected from the group of 6-8 weeks old male of C57BL / 6 mice conventional cervical dislocation sacrificed, the spleen was removed aseptically, using a 100 mesh stainless steel mesh Preparation of spleen cell suspension, adjusting the concentration of the living cells of 1 × 10-7/ml. Take 6-well plate divided into four groups: the first group to the blank control: 0.5 ml mouse spleen to lymphocytes 1ml1640 culture medium. The second group: 0.5 ml of mouse spleen lymphocytes 1ml tumor cell culture supernatants. Third group: 0.5ml of mouse spleen lymphocytes CD3 monoclonal antibody (final concentration of 2μg/ml) 1ml 1640 culture liquid. The fourth group: 0.5 ml of mouse spleen lymphocytes CD3 mAb (final concentration 2μg/ml) 1 ml of tumor cell culture supernatant. Group CD4 ~ CD25 ~ Treg content detected by flow cytometry after 72 hours of incubation; RT-PCR was used to detect CD4 CD25 Treg cell-specific marker expression of Foxp3 mRNA, the test was repeated three times. Results: The results showed that the EL-4 cell culture supernatant induced group CD4 ~ CD25 ~ Treg accounted lymphocytes was significantly higher than that of the control group (11.4 ± 0.51%, 6.3 ± 0.34%, P lt; 0.05); CD3 mAb and EL-4 culture supernatant co-induction group CD4 ~ CD25 ~~ of Treg accounted for the proportion of lymphocytes is a significant difference compared with the control group CD3 mAb (12.8 ± 0.59%, 8.1 ± 0.41%, P lt; 0.05); same FBL3 supernatant induced group CD4 ~ CD25 ~ Treg accounted for the proportion of lymphocytes was significantly higher than the control group (11.8 ± 0.93%, 7.03 ± 0.75%, P lt; 0.05); CD3 mAb and FBL3, culture supernatant common induction of CD4 ~ CD25 ~ Treg was significantly higher than CD3 mAb control group (13.1 ± 0.56%, 9.19 ± 0.38%, P lt; 0.05). RT-PCR analysis showed significantly increased Foxp3 mRNA expression of specific marker molecules join tumor cells (EL-4 and FBL3) culture supernatant induced. That there may be certain immunomodulatory factors prompted the mouse spleen CD4 ~ CD25 ~ Treg cells increased proportionately in these tumor cell supernatant. Conclusion: The tumor cell culture supernatant can promote mouse lymphocyte CD4 ~ CD25 ~ Treg proportion increased, at the same time its specific marker molecules of Foxp3 mRNA expression was significantly increased. This study In vitro tests proved the tumor microenvironment induced proliferation of CD4 ~ CD25 ~ Treg cells, indicating that the tumor promoting CD4 ~ CD25 ~ Treg increased. In summary the results show that (1) acute leukemia patients CD4 ~ CD25 ~ Treg cells increased the proportion may be so impaired immune function in patients. ② tumor cells secrete soluble substances can induce the proliferation of CD4 ~ CD25 ~ Treg cells, the tumor can promote CD4 ~ CD25 ~ Treg increased. These findings not only can better explain hematologic malignancies in patients with immune dysfunction and the sick host the the fact poor prognosis, and prompted CD4 ~ CD25 ~ Treg cells can become a new target for the treatment of cancer. The study revealed the mechanism of hematologic malignancies in patients with impaired immune function, and laid the foundation for the pathogenesis of clinical hematological malignancies and tumor immunotherapy.

The Change and Mechanism Research on the CD4~+CD25~+ Regulatory T Cells in Malignancy Hematopathy Patients

Abstract

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