Decreased Circulating CD4+Foxp3+Helios+Treg In Patients With ACS And Its Mechanism Study

PART I Decreased Helios Expression in Regulatory T Cells in Acute Coronary Syndrome Back ground: Regulatory T cells(Tregs)play an essential role in acute coronary syndrome(ACS).However,there is debate about which Treg subsets are truly critical to ACS.CD25 and Foxp3 were once regarded as the canonical markers for Tregs,however,it was revealed later that some cells expressing CD25 and Foxp3 are pro-inflammatory cytokine-producing effector T cells,but not Tregs with suppressing function.Helios,a transcription factor,was recently reported to be a bona fide marker for thymus derived Tregs or activated Tregs with a suppression function,but little is known about its role in ACS.Therefore we investigated the Helios expression in Tregs.Methods: We therefore examined Helios+ Tregs in patients with ACS,patients with stable angina,and control subjects.73 patients with ACS,30 patients with stable angina and 48 control subjects were enrolled.The frequencies and estimated absolute numbers of different Treg subsets,including CD4+CD25+,CD4+CD25+Foxp3+,and CD4+CD25+Foxp3+Helios+,CD4+Foxp3+,and CD4+Foxp3+Helios+ Tregs in peripheral blood were measured by flow cytometry.Plasma cytokine level of interleukin-6(IL-6)and Transforming growth factor beta 1(TGF-beta1)was measured by ELISA.The mRNA expression of Foxp3 and Helios in purified CD4+ T cells was determined by RT-PCR.Results: The frequency and estimated absolute numbers of CD4+Foxp3+Helios+ Tregs were both decreased significantly in patients with ACS,compared with those subjects in the SA group and the control group;whereas no significant difference was found in the frequency or estimated absolute numbers of CD4+CD25+,CD4+CD25+Foxp3+ cells among the three groups.The frequency and estimated absolute numbers of CD4+Foxp3+Helios+ Tregs were positively correlated with the circulating levels of IL-10 and TGF-beta1.The mRNA expression of Foxp3 and Helios was decreased in CD4+ T cells from ACS patients.Conclusions: In summary,CD4+Foxp3+Helios+ Tregs was downregulated in patients with ACS and it may play a role in ACS.PART II The Inhibition Capacity of Tregs Was Impaired by Helios Gene KnockdownBackground: We aim to downregulate the Helios expression in Tregs by RNA interference,and try to find out whether the expression deficiency of Helios would impact the suppression capacity of Tregs.Methods: Isolate the CD4+CD25+ Tregs and CD4+CD25-T cells from healthy volunteers by microbeads purification.Interference the Helios expression in CD4+CD25+ Tregs by Helios shRNA lentivirus transfection.The Tregs were grouped according to the different transfection methods:(1)The control group in which no transfection action was conducted.(2)The con V group in which Tregs were transfected with lentivirus without an effective Helios shRNA sequence.(3)The Helios shRNA group in which the Tregs were transfected with effective Helios shRNA lentivirus.Detect the m RNA and protein expression levels of Helios by RT-PCR and flow cytometry respectively.Dye the CD4+CD25-T cells with CFSE-FITC,coculture the dyed CD4+CD25-T cells with the CD4+CD25+ Tregs from the control group,the con V group transfected with empty lentivirus and the Helios shRNA lv group,evaluate the proliferation of CD4+CD25-T cells by flow cytometry and the IFN-gamma production by ELISA.Results: Compared with the Tregs in the control group and the con V group,the Tregs in Helios shRNA group showed significant decrease in the Helios expression in both RT-PCR and flow cytometry examination,the expression levels of cytokine IL-10 and TGF-beta1 were significantly decreased,too.Compared with the Tregs in the control group and the con V group,the Tregs in Helios shRNA group exhibited impaired suppressive function on the CD4+CD25-effector T cells.Conclusions: Helios deficiency resulted in an impaired suppressive function in Tregs.