Regulatory T Cells In Patients With Diabetes On The Expression Of Peripheral Blood And Its Expansion Model

Objective To analyze the percentage of FOXP3+regulatory T cells in peripheralblood from patients with diabetes.Methods Three groups included: type1diabetes （n=43cases）, type2diabetes （n=16cases） and healthy control （n=19cases）. ZnT8A, GADA and ICA were detectedby radioligand binding assay. The proportions of regulatory T cells in peripheralblood were determined by flow cytometric analyses.Resultsâ‘ In the peripheral blood, the percentage of CD4⁺CD25⁺T cells in CD4⁺Tlymphocytes from patients with type1diabetes or type2diabetes were lower thanthe percentage of healthy control.â‘¡In the peripheral blood, patients with type1diabetes showed lower ratio of CD4⁺CD25⁺FOXP3+T cells compared with healthycontrol and patients with type2diabetes, no significant difference was detectedbetween healthy control and patients with type2diabetes.â‘¢There was no obviouscorrelation between the duration of diabetes and the percentage of peripheralFOXP3+regulatory T cells.Conclusion In the peripheral blood, there was a deficiency in the FOXP3+regulatoryT cells population from patients with type1diabetes, type2diabetes had smallinfluence to percentage of FOXP3+regulatory T cells. There was no significantrelationship between the percentage of FOXP3+regulatory T cells and duration ofdiabetes. Objective To isolate CD4⁺CD25⁺CD127^dim/-TT cells from human peripheral bloodand research their functional characteristics. To decide the possibility ofCD4⁺CD25⁺CD127^dim/-TT cells as starting cells for expansion of Tregs in vitro.Methods PBMC were obtained from1U buffycoat by Ficoll density gradientcentrifugation. CD4⁺CD25⁺CD127^dim/-TT cells were isolated from PBMC bymagnetic-activated cell sorting （MACS）. The purity and phenotype ofCD4⁺CD25⁺CD127^dim/-TT cells were determined by flow cytometric analyses. Theexpressions of FOXP3within human PBMC, CD4-CD127highT cells andCD4⁺CD25⁺CD127^dim/-TT cells were examined by RT-PCR.Resultsâ‘ Purity of human CD4⁺CD25⁺CD127^dim/-TT cells sorting by the immunemagnetic beads was （87.4士2.6）%, with the （85.6士4.2）%expressions ofFOXP3;â‘¡FOXP3within Tregs can be detected by RT-PCR. Tregs expressed ahigher level of FOXP3than PBMC and CD4-CD127highT cells.Conclusion CD4⁺CD25⁺CD127^dim/-TT cells can be effective sorted out fromperipheral blood by two-step with MACS. CD4⁺CD25⁺CD127^dim/-TT cells express aphenotype of FOXP3. CD4⁺CD25⁺CD127^dim/-TT cells are reliable starting cells forexpansion of Tregs in vitro. Objective To establish a proper protocol for effective expansion ofCD4⁺CD25⁺CD127^dim/-TT cells in vitro and study factors that influence the efficiencyand purity of expansion of CD4⁺CD25⁺CD127^dim/-TT cells in vitro.Methods CD4⁺CD25⁺CD127^dim/-TT cells were plated at104cells per well in a96-wellplate and stimulated with anti-CD3/anti-CD28-beads. Different dose of IL-2andrapamycin were added to the culture.The cells were harvested at different time. Cellswere analyzed for survival and apoptosis by Annexin V and PI staining and evaluatedfor phenotype by FACS. The suppress function of expanded cells was analyzed bymixed lymphocyte culture.Resultsâ‘ After sorted by magnetic beads, CD4⁺CD25⁺CD127^dim/-TT cellsproliferated quickly due to anti-CD3/anti-CD28-beads joint with high concentrationsof IL-2, quantity of Tregs increased（67±5）folds at the end point of the first cultureweek and （20±3）folds at the end point of the second culture week.â‘¡Percentageof dead cells was（14.2±2.1）%after a week of culture, this Percentage increased to（16.6±3.2）%afteranotherculture week and27.4%afterthe third culture week.â‘¢Purity of cells was dropped from （87.4士2.6）%to（65.1±4.0）%over a14-dayculture period, but CD4⁺CD25⁺T cells percent remains close to90%.â‘£Results ofmixed lymphocyte culture suggested that expanded Tregs could effectively suppressproliferation of CD4⁺CD25-T cell in vitro.Conclusion Tregs can be effective expanded by anti-CD3/anti-CD28coatedmicrobeads joint with high concentrations of IL-2. Two rounds of stimulation of7days each were suitable. Expanded cells possessed regulatory functions in vitro.