The Study Of Function And Mechanism Of DMBA Promoted Proliferation Of Thymus Derived Regulatory T Cells In The Early Carcinogenesis

ObjectiveRegulatory T cells(Regulatory T cells,Tregs)belong to CD4~+T lymphocytes which express the transcription factor Foxp3 and regulate other immune function of T cell subsets,the normal physiological function of Tregs in vivo is essential to maintain immune homeostasis.the development and function disorders of Treg are closely related to the pathophysiological process of some severe immune-related diseases.In the development of tumor,Treg-mediated immune suppression is a key mechanism of tumor immune escape.Studies have proved that Treg has an important role in promoting tumor development and the number or proportion of Treg in the early time of tumor formation is also crucial.Therefore,our study use the classic atmospheric pollutants DMBA-induced tumor model to induce skin cancer and analyze some changes of Treg proliferation,and further explore the molecular mechanism of carcinogen DMBA regulated Treg proliferation.Methods1.We use carcinogen DMBA to smear normal C57 mice as a experimental model of skin cancer,and smear skin with acetone as a control,smear C57 mice with DMBA from day1 to day6,at the sixth day,these mice are sacrificed to get their skin draining lymph nodes for preparation of single cells.Then we use flurenscence staining to analyze the changes of number and proportion of Foxp3~+T cells in the early time of skin cancer.2.Mice are manipulated with acetone and DMBA.Getting out of their skin draining lymph nodes for preparation of single cells,and staining,we oberve whether the proliferation of Treg is changed and which kind of Treg is associated with the difference and proliferation.3.Sorting sterile CD4~+Foxp3-GFP~+T cells and CD4~+Foxp3-GFP~-T cells by flow cytometry,labeling with eFluor450 in vitro and transfer them to C57BL/6 mice and then respectively smear acetone and DMBA,6 days later,we sacrifice these mice and get out of their skin draining lymphnodes for preparation of single cells,then we analyze the proportion and proliferation of CD4~+Foxp3-GFP~+T cells and CD4~+Foxp3-GFP~-T cells in the four different groups by flow cytometry.4.Analyzing the level of relative cytokines associated with Treg function and stability related molecules in the skin draining lymphnodes by flow cytometry six days later after smearing with acetone and DMBA.5.We smear Foxp3-GFP mice with acetone and DMBA respectively.six days later,we sort Treg of the two groups,and coculture with effector T cells labeling with eFluor450 in vitro.4 days later,we analyze the proliferation of effector T cells.smearing C57 mice with acetone and DMBA,six days later,transfer OTII T cells into mice,after three days,we analyze the proliferation of OTII T cells.6.Gene Chip measure the difference of Treg transciptional level in different groups and choose some genes related to the proliferation.By presuming some potential signal pathways we select the inhibitors of these signal pathways,then assess whether the inhibitors influence the proliferation of Treg as DMBA treated.Results1.The proportion of CD4~+Foxp3~+T cells in the skin draining lymph nodes of mice is elevated in the early time of DMBA treatment.The level of transcription of Foxp3 is significantly increased.This increase of the proportion in Treg is DMBA-dependent,non-cancer-causing agents such as Th1 adjuvant CpG and Th2 adjuvant papain can not lead to a similar phenomenon.2.DMBA treatment increase ki67 expression of Treg in skin-draining lymph nodes,indicating that the proportion of Treg is elevated.the increased proportion mainly is CD4~+Foxp3~+Helios~+T cell subset derived from thymus.3.CD4~+Foxp3-GFP~-T cells transfered into C57 mice did not translate into Foxp3~+T cells,whereas the transfered CD4~+Foxp3-GFP~+T cells,after DMBA treatment,the proliferation of this population was significantly increased than acetone group,proved that the proliferation of Treg is due to the proliferation of tTreg.4.After DMBA treatment,inhibitory molecule associated with CD4~+Foxp3~+Treg function,such as CTLA4,GITR and Foxp3 stability related molecules such as EOS,were significantly increased in skin draining lymph nodes.5.In vitro and vivo,Foxp3~+Treg can significantly suppress the proliferation of antigen-stimulated CD4~+T cells after DMBA treatment.6.The Genechip results show that transcription level of V-ATPase subunit ATP6v0d2in tTreg compared to acetone control group(ace),iTreg cells and effector T cells is significantly increased.the result of the inhibitors of V-ATPase and downstream protein AMPK/mTORC1 show that the proliferation of tTreg DMBA treated is reduced as hambering V-ATPase-AMPK signal pathway,but the inhibitor of mTORC1 does not influence the proliferation of tTreg DMBA treated,so we presume the proliferation of tTreg may be related with V-ATPase-AMPK signal pathway.ConclusionDMBA can induce tumor by causing DNA damage.the proportion and proliferation of tTreg in skin draining lymph nodes was significantly increased after DMBA treatment in the early time of skin cancer and it can cause suppressive immune response,the mechanism is likely due to the activated V-ATPase-AMPK metabolism pathway of tTreg.