The Change And Mechanism Research On The CD4~+CD25~+ Regulatory T Cells In Malignancy Hematopathy Patients

In 1995 Sakaguchi etal were the first to found that a subset of CD4+ lymphocytes in peripheral blood of normal mice highly expressed the CD25(IL-2R-a) and lowly expressed the CD45RO,which constitute 5-10% of peripheral CD4+T cells and reffered as to CD4⁺CD25⁺ regulatory T cells (CD4+ CD25+ Treg). Accumulating evidences show can suppress the immune response to self and non-self antigen in active way and contribute to the maitainance for self-immune tolerance. Many tumor associated antigens expressed on the normal cells and not the product of gene mutation and indicate the tumor immunity is a autoimmunity to some extent.As such, Treg cells may also block autitumor immune responses. Leukemia is a hematologic neoplasm in which malignant cells are present in the born marrow and the blood.Now the immunotherapy became the hot spot for the leukemia treatment.But the effect of antigen specific immunotherapy was anything but satisfactory,because the tumor cells may escape from the immune surveillance through some kind of mechanism. CD4⁺CD25⁺ Treg can suppress tumor specific immune response and make the tumor cells grow and metabasis easily. The investigation for the immunocyte in leukemia patients will benefit to learn the mechanism of invasion and metastasis of tumor and explore the new treatment. we drew up the research content:To test the change of CD4⁺CD25⁺ Treg in peripheral blood of acute leukemia patients; To explore if the tumor can induce the up-regulation of CD4⁺CD25⁺ Treg by the experiment in vitro with mice.This study aimed to approach the negative regulatory factors and the mechanism of the development in malignancy hematopathy and look for the new way for the immunotherapy of malignancy hematopathy ultimately.Partâ… :The test of CD4⁺CD25⁺regulatory T cells in peripheral blood of malignancy hematopathy patientsPurpose:To analyze the change of CD4⁺CD25⁺ Treg in peripheral blood lymphocyte of leukemia patients and to investigate their significance in the leukemia progress mechanism.Methods:We collected 32 leukemia patients in the second hospital of shanxi medical university from September 2007 to May 2008.All of them were first invasion and not receive the chemotherapy.Amone which had 20 male,12 famale,the mean age of the leukemia patients was 53.6 .These leukemia patients make a definite diagnosis by clinical symptom,bone marrow cell and immunity typing.The corresponding healthy controls come from the health examination centre.All of 16 ones have 10 male and 6 famale. The mean age is 48.2.The frequency of CD4⁺CD25⁺ Treg in the peripheral blood(PB) was determined by flow cytometry. The serum level of TGF-βand IL-10 was measured by the ELISA method.Correlation analysis between the proportion of CD4⁺CD25⁺ Treg and the level of TGF-βand IL-10 was done in leukemia patients.The data were summarized as the mean±standard error.Statistical significance was accepted at the P<0.05 level.All the statistical analysis were performed using the SPSS statistical software package.Result:The mean proportion of CD4⁺CD25⁺ Treg of all CD4+T cells in acute leukemia patients was significantly higher than that of the normal control (3.96±1.86) % vs (2.08±1.63) % (P<0.01).We acquire the proportion of CD4⁺ CD25⁺ Foxp3⁺ Treg of all CD4+T cells in acute leukemia patients is notably higher when compare with the normal control (3.22±1.01)% vs(0.83±0.72)% (P<0.01).The proportion of CD4⁺Foxp3⁺T cells increased notablely in leukemia patients compare with the normal contro(l14.9±2.92)% vs (5.68±1.21)% (P<0.001). the level of acute leukemia patients blood serum TGF-β1 and IL-10 is 26.9±5.2ng/ml and 74.6±8.7 pg/ml by ELISA.A position correlation is found between the number of CD4⁺CD25⁺ Treg and the serum level of TGF-β(r=0.508,P<0.05)and IL-10. (r=0.556, P<0.05).Conclusion:â‘ We provided the evidence for an increased pool of CD4⁺CD25⁺ Treg in the peripheral blood in acute leukemia patients.These data suggest the increased number of this cell population correlate with the failure of antitumor immunity and make the tumor grow and metastasize easily.â‘¡we found both the TGF-βand IL-10 played a key role for the immunosuppression in CD4⁺CD25⁺ Treg.These investigation will provide the beneficial enlightenment for the immunoregulation targeting CD4⁺CD25⁺Treg.Partâ…¡: The impact of tumor cells on CD4⁺CD25⁺regulatory T cells in vitro with micePurpose:This study was aimed to explore if the the supernatant from cultured tumor cells tumor induced the proliferation of CD4⁺CD25⁺ Treg by the experiment in vitro with mice. This study investigate the mechanism of CD4⁺CD25⁺ Treg increased in the onset of malignancy hematopathy.Method: The lymphoma cell line EL-4 and the erithroleukemic cell line FBL₃ origin from the C57BL/6 mice were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum,5%CO2,37℃,saturate humidity. We prepared the supernatant from cultured tumor cells. Then the single-cell suspensions of splenocytes were prepared sterile after execute the normal C57BL/6 mice. We grinded the spleen gently with steel cage of 100 meshes and suspended the cells in PBS(1×10⁷cells/ml). The experiment separated four groups.The first group was black control:0.5ml mouse spleen lymphocytes+1ml RPMI 1640 medium;The second group:0.5ml mouse spleen lymphocytes+1ml the supernatant from cultured tumor cells;The third group:0.5ml mouse spleen lymphocytes+CD3 McAb(the final concentration was 2μg/ml)+ 1ml RPMI 1640 medium;The forth group: 0.5ml mouse spleen lymphocytes+ CD₃ McAb(the final concentration was 2μg/ml)+1ml the supernatant from cultured tumor cells.After cultured 72 hours CD4⁺CD25⁺ subset were analyzed by flow cytometry and the gene expression level of the specific marker Foxp3 were tested by RT-PCR. The experiment repeated three times. The statistical analysis were the same as the Part I.Result: In vitro experiment indicated the proportion of CD4⁺CD25⁺ Treg in all lymphocyte induced by the supernatant derived from EL-4 was significantly higher than that of the black control group(11.4±0.51%,6.3±0.34%,P<0.05).The proportion of CD4⁺CD25⁺ Treg in all lymphocyte induced by the supernatant derived from EL-4 and CD3-McAb has statistics difference than that of the CD3-McAb control group(12.8±0.59%,8.1±0.41%,P<0.05).The proportion of CD4⁺CD25⁺ Treg induced by the supernatant derived from FBL3 was significantly higher than that of the black control group(11.8±0.93%,7.03±0.75%,P<0.05).The proportion of CD4⁺CD25⁺ Treg induced by the supernatant derived from EL-4 and CD3-McAb was obviously higher than that of the CD3-McAb control group(13.1±0.56%,9.19±0.38%,P<0.05).The level of corresponding Foxp3 mRNA also increased after added the tumor cells supernatant by RT-PCR. These data suggested that in the supernatant may occur immune regulatory factors which upregulated the number of CD4⁺CD25⁺ Treg cells.Conclusions: The experiment indicated that the tumor cells supernatant may occur immune regulatory factors which increased the number of CD4⁺CD25⁺ Treg cells.Our study certified the tumor can induce the up-regulation of CD4⁺CD25⁺ Treg.In a word,these results indicateâ‘ The increased number of CD4⁺CD25⁺ Treg may damage the immune function in acute leukemia patients.â‘¡the solubility factors from tumor cells can induce the increase of CD4⁺CD25⁺ Treg.It indicate the tumorigenesis can facilitate proliferation of CD4⁺CD25⁺ Treg.These data can not only explain preferably the reason for the dysfunction of the immune system and poor prognosis in malignancy hematopathy patients but also show that CD4⁺CD25⁺ Treg may become the new target of immunotherapy for cancer. Our experiment reveal the mechanisms for the dysfunction of the immune system in malignancy hematopathy patients.These results established the foundation for the researchment about the pathogenesis and the tumor immunotherapy in clinical malignancy hematopathy.