Impairment Of Circulating Cd4+Cd25+Garp+Regulatory T Cells In Patients With Acute Coronary Syndrome And Its Mechanistic Study

PART â…  Impairment of Circulating CD4+CD25+GARP+Regulatory T Cells in Patients with Acute Coronary SyndromeBackground:Atherosclerosis (AS) is an inflammatory and immune disease. Regulatory T cells (Tregs) suppress the activation of T cells and have been shown to play a protective role during the pathogenesis of AS. However, specific markers for Tregs are lacking. Recently, glycoprotein A repetitions predominant (GARP) was discovered as a specific marker of activated Tregs, and we therefore utilized GARP as a specific surface marker for Tregs in the current study.Methods:To assess whether GARP+Tregs are downregulated in patients with acute coronary syndrome (ACS), we examined CD4+CD25+GARP+T cell frequencies as well as their associated cytokines and suppressive function. Additionally, we compared GARP expression to that of FOXP3, which may be more sensitive as a marker of activated Tregs in patients with ACS. Results:Patients with ACS demonstrated a significant decrease in circulating CD4+CD25+GARP+Tregs. Moreover, the suppressive function of Tregs and levels of related cytokines were also impaired in ACS patients compared to those with stable angina (SA) or normal coronary artery (NCA). Additionally, after TCR stimulation, peripheral blood mononuclear cells (PBMCs) from patients with ACS exhibited a decrease in CD4+CD25+GARP+Tregs.Conclusions:These fndings indicate that circulating CD4+CD25+GARP+Tregs are impaired in patients with ACS. Thus, targeting GARP may promote the protective function of Tregs in ACS. PART II Construction of HDV Encoding GARP and HDV Encoding shGARP and Their Effectiveness ScreeningAim:It has been documented overexpression of GARP endows naive T cells with partialphenotype and function of Treg. To explore the role of GARP in ACS, here,we aimed to construct HDV encoding GARP and that of encoding shGARP as tools to futher investigation of the exact role of GARP in the pathogenesis of atherosclerosis.Methods:We subcloned human GARP/LRRC32into a HIV-derived vector (HDV) that encodes green fluorescent protein (GFP) as a marker. We designed three different shRNAs against GARP and subcloned them into HDV separately. We used RTPCR to confirm the effectiveness of the construction products.Results:Activated CD4+CD25T cellstransduced with HDV encoding GARP showed GARP mRNA upregulation in comparison to that of transduced with control vectors. Two of the three shGARP reduced GARP mRNA expression in activated CD4+CD25+T cells.Conclusions:We successfully constructed two efficient shRNAs against GARP in activated CD4+CD25+T cells and one efficient HDV encoding GARP.