Experimental Study On Regulatory T Cells Induced By Immature Dentritic Cells In Vitro

Regulatory T cells are an important part of maintaining self-tolerance andconsist of different T cell subsets. They mainly play a role in suppressiveimmunoregulation function. Regulatory T cells have broad perspective ininfection immunity, tumor immunity and transplantation immunity.Objective: To establish a stable culture system of dendrtic cell (DC) fromperipheral blood monocyte cell in vitro, immature and mature DCs culturedwith cord lymphocytes. To investigate the cell phenotype, intracellularcytokine and immunoregulatory function of regulatory T cells induced byimmature DCs from cord lymphocytes.Methods: The monocytes were isolated from human peripheral bloodmonouclear cells through attachment to the plastic surface. The acquiredmonocytes were subjected to GM-CSF and IL-4 for 5-7 days culture to induceimmature DCs. The immature DCs were then exposed to GM-CSF andTNF-α for another 5-7 days in order to maturation. The morphology of DCswere observed under the inverted phase contrast microscope and flowcytometry was used for phenotypic analysis of CD83. As metioned aboved, thecord lymphocytes were isolated from cord monouclear cells. Cordlymphocytes cultured with immature DCs for 5-7 days and then regulatory Tcells were induced, as experiment group. Cord lymphocyte cultured withmature DCs of 14 days to induce stimulator T cell, as negative control group.Flow cytometry was used for exoression analysis of CD25 molecule andintracellular cytokine IL-10,IFN-γ of two group's cells. The two grpup's cellsas different ratio were added in allogenic mixed lymphocyte culture system toassay responding activity. MTT was used to analyze the effect of them on lysisactivity of CTL tumor cells.Results: The cells became clustered after using GM-CSF and IL-4culture for 4-6 daysand semi-attached cells distributed in the culture plate.After adding TNF-α, the suspension cell with round-shape and clustered cellswere increased dramatically. At the same time, the cells with fur-likeprojections were also observed in the culture. By day 12, most of the cellsshowed obvious fur-like projections on the cell surface and reached peakaround day 14. The immature DC expressed low level of CD83 but mature DCexpressed high level of CD83. The expression of CD25 had on significantsignificant deviation after cord lymphocytes were induced by immature DCand mature DC. In experiment group the percentage of positive cells secretingIL-10 and double positive cells of CD4/IL-10 increased, while in negativecontrol group the percentage of positive cells secreting INF-γ and oublepositive cells of CD4/ INF-γ increased. Regulatory T cells of experimentgroup suppressed the proliferation of allo-lymphocyte in dose-dependent wayand specific lysis activity of CTL to tumor cell. Instead, stimulator T cell ofnegative control group showed strong response activity to allo-lymphocytesand have synergistic effect on lysis activity of CTL.Conclusion: The combination GM-CSF and IL-4 is capable ofinducting immature DC from monocytes in vitro and addition TNF-α toculture system stimulates DC maturation. Regulatory T cells induced byimmature DC have the similar property with producing IL-10 Tr1 celland posses suppressive immunoregulation function.