CD₄～+CD₂₅～+ Regulatory T Cells Increasing In Tumor Patients: Possible Involvement In Disease Progression And Immunotherapy

Part â… . CD₄⁺CD₂₅⁺Regulatory T cells in Patients with Breast Cancer: Possible Involvement in Disease Progression Purpose: T cells constitutively expressing CD4 and CD25 markers are essential for maintenance of self-tolerance and have been referred to as regulatory T cells (Tregs). Tregs also have been reported to be potent inhibitors of an antitumor immune response. The current study was designed to determine whether cancer patients exhibit an expanded Tregs pool. Method: The frequency of Tregs in the peripheral blood (PB) of 49 patients suffering from breast cancer, 14 patients with benign disease and 9 healthy volunteers was determined by flow cytometry. The PB of breast cancer patients was collected before surgery, after surgery, and after chemotherapy. The serum level of TGF-P and IL-10 was measured by the ELISA method. In order to analyse the relationship between Tregs and lymph node (LN) status, the frequency of Tregs in LN was determined. And the expression level of TGF-P and IL-10 gene was measured by quantitative RT-PCR method. Result: In breast cancer patients (n=49), benign patients (n=14), and normal donors (n=9), the prevalence of Tregs in CD₃⁺ T cells was 5.29%±2.29%, 3.84%±1.40% and 3.11%±0.68% respectively. The prevalence of Tregs was significantly higher in breast cancer patients when compared with carcinoid patients and normal controls. The number of CD₄⁺CD₂₅⁺ cells in the PB correlated with tumor stage (P<0.05), tumor size (r=0.562, P<0.05) and LN status (P=0.025). A positive correlation was found between the number of Tregs and serum level of TGF-β and IL-10. The number of Tregs decreased after surgery, but chemotherapy seemed induced an increase in this cell population. The percentage of CD₄⁺CD₂₅⁺ cells in tumorinfiltrating lymph node(LN) was 21.98%±4.85%, which is increased comparing with the non- infiltrating LN (PO.05) . In tumor infiltrating LN the expression level of TGF-p increased and INF-y decreased. The percentage of CD4+CD25+ cells in LN was related with TGF-P and IL-10 expression level. Conclusion: We provided evidence for an increased pool of CD4+CD25+ regulatory T cells in the peripheral blood of cancer patients with potent immunosuppressive features. The number of this cell population correlated with tumor progression. These findings should be considered for the design of immunomodulatory therapies such as dendritic cell vaccine.Part II. The Mechanism of CD4+CD25+ regulatory T CellsExpansion in Tumor PatientsAbstract Purpose: The number of CD4+CD25+ regulatory T cells of tumor patients increased comparing with normal controls. Tregs also have been reported to be potent inhibitors of an antitumor immune response. The mechanism of CD4+CD25+cells differentiation in tumor patients is a matter of debate. Methods Separate the PBMCs into CD4+CD25+ and CD4+CD25'cells. Detect the CD4+CD25+ cells expansion and CD4+CD2s" cells differentiation after co-culturing with tumor cells or denditic cells loaded with tumor antigen. Infuse of CFSE labeled CD4+ cells into tumor bearing nude mice or mice injected with dendritic cells. 72hrs later, the CD4+CD25+Foxp3+ cells in the peripheral blood of mice were detected by flowcytometry. Results CD4+CD25+ cells could not expansion whether they werestimulated with tumor cells or dendritic cells. But under the similar condition, CD/CD25* cells could become CD25+ and potent suppressor cells. When CD4+ cells were applied simultaneously with MCF-7 or dendtitic cell, the percentage of CD4+CD25+Foxp3+ CFSE labeled cells in peripheral blood of nude mice increased. And the number of CD4+CD25+Foxp3+ cells was higher in the region that fluorescence of CFSE was stronger. Conclusion Tumor cells can educate CD/CD25" cells to develop suppressive activity. It may be helpful to explain the phenomenon that the Tregs increased in tumor patients.Part III. The Influence of CD4+CD25+ Regulatory T cells on Cytokine-Induced Killer (CIK) CellsAbstract Purpose Identify the influence of CD4+CD25+ regulatory T cells on the proliferation and cytotoxicity of CIK cells and the effect of dendrtitic cells (DC) on the immune regulatory function of Tregs. Methods Remove CD4+CD25+ cells from PBMC and induce CIK in vitro (CIK-Tregdel). Compare the proliferation and cytotoxicity of CIK-Tregdel and regular CIK. Detect the activity after adding Tregs at the ratio of 20 '. K 10 : K 5 '. 1 into CIK-Tregdel. Analyze the function of CTLA-4, GITR, TGF-p and IL-10 molecules of the regulatory T cells in CIK. DCs were harvested on day 7 and cocultured with the CIK. The frequency of Tregs in CIK was determined by flow cytometry. The cytotoxicity was examined by LDH assay. The level of TGF-p, IL-10, IFN- Y , IL-2 and IL-6 was analyzed by ELISA method. Results The percentage of 7AAD"BrdU+ cells in CIK-Tregdel and...