Effects And Mechanism Of Cellular Immunity Homeostasis In Diabetic Adipose Inflammation And Acute Coronary Syndrome

BackgroundCardiovascular events induced by atherosclerosis are the major cause of death in patients with diabetes.Diabetic atherosclerosis is characterized by more progressive,more severe but lower response to medical treatment.However,the underlying mechanism is not clear.Accumulative evidences manifest that atherosclerosis can be aggravated by chronic systemic inflammation under metabolic dysfunction.Therefore,it is reasonable to mitigate diabetic atherosclerosis via reducing systemic inflammation.Visceral adipose tissue(VAT)is the major source of chronic systemic inflammation in diabetes.Because of same evolutionary and developmental foundation,metabolism and immune function of adipose tissue are closely related.With the metabolic overload growing,insulin resistance occurs in VAT,and further mediates inflammatory response.Circulating inflammatory cytokines secreted from VAT afflicts distal organs including blood vessels.As a result,it is expectable to compromise metabolic systemic inflammation via controlling VAT inflammation.The specific mechanism linking metabolic abnormalities of visceral adipose tissue and adipose tissue inflammation may be an effective target for controlling diabetic adipose inflammation.CD4+ T lymphocyte in VAT is bridging the gap between metabolic dysfunction and immune disorder.On one hand,CD4+ T lymphocytes are the carriers of metabolic inflammation signals via converting metabolic disorder to balance of pro-and anti-inflammatory CD4+ T lymphocytes.CD4+ T lymphocyte could be irritated by dysfunctional adipocytes and adipose-derived stem cells(ADSCs)via direct contact and paracrine,and become inflammatory polarized,among which pro-inflammatory phenotype cells(Thl,Th17)increase,while anti-inflammatory phenotype cells(Treg)decrease.On the other hand,CD4+ T lymphocytes are the propagators of metabolic inflammation.The turbulence in balance of pro-and anti-inflammatory CD4+ T lymphocytes will further lead to distortion of pro-and anti-inflammatory cytokines,which promotes macrophage activation,M1 polarization and expansion of adipose inflammation.Therefore,it is promising to ameliorate diabetic adipose inflammation via rectifying CD4+ T lymphocyte polarization.Diabetic visceral ADSCs have ability to predispose CD4+ T lymphocyte to polarize to inflammatory phenotype.ADSCs are a cluster of cells with the potential of multi-lineage differentiation.Normal ADSCs have powerful effect of immune suppression by inhibiting immuocytes proliferation and polarization.It is reported that ADSCs from obese individuals lost normal function and predisposed CD4+ T lymphocyte to polarize to Th1 and Th17,leading to further activation of monocyte and macrophage.However,the biological features and effects on CD4+ T lymphocyte of diabetic ADSCs remain unknown.Protein tyrosine phosphatase non-receptor type 2(PTPN2)takes an important part in regulating CD4+ T lymphocyte polarization.PTPN2,also known as T cell protein tyrosine phosphatase(TCPTP),was first cloned from human cDNA library.Subsequent studies confirmed its expression in multiple tissue and cells.PTPN2 regulates intracellular signal transduction via reducing phosphorylation through the effect of phosphatase.PTPN2 exerts an important influence on immune homeostasisby affecting the development and function of T cells.It is responsible for maintaining the resting state of T cells by significantly inhibiting T cell receptor(TCR)and cytokine-mediated T cell activation.Ptpn2-/-mice were reported to manifest immune disorder and systemic inflammation,with growing Thl and Th17 cells and decreasing Tregs in circulation and intestine.Despite this,whether PTPN2 could affect CD4+ T lymphocyte polarization in adipose tissue in type 2 diabetes is not clear.PTPN2/p-STAT3 is the candidate pathway to regulate adipose CD4+ T lymphocyte polarization.Signal transducer and activator of transcription 3(STAT3)is one of the most classical transcription factor mediating important physical activities such as proliferation,differentiation and inflammation,of which the phosphorylated STAT3 is the active form.STAT3 is critical for CD4+ T lymphocyte polarization as it can positively promote Th1 and Th17 polarization.It was found that PTPN2 can regulate JAK-STAT pathway,of which phosphorylated STAT3(p-STAT3)is the major substrate of PTPN2.Whether PTPN2 could regulate CD4+ T lymphocyte polarization in diabetic visceral adipose tissue through the above mechanism needs to be further elucidated.In addition,previous screening revealed that transcription factors STAT3 and FoxP3 might bind to upstream non-coding sequences of Ptpn2 gene.Whether STAT3 and FoxP3 bind to Ptpn2 promoter and trans regulate PTPN2 transcription and expression has not been reported.In summary,we propose the hypothesis that metabolic and immune disorders occur in visceral ADSCs in type 2 diabetes,and the interaction between diabetic ADSCs with CD4+ T lymphocyte leads to lymphocyte polarization,in which increased phosphorylated STAT3 promotes pro-inflammatory Thl and Th17 cells while inhibits Treg cells.This pro-/anti-inflammatory balance fluctuation propagates successive macrophage M1 polarization,which contributes to exaggerate adipose inflammation.Overexpression of PTPN2 in CD4+ T lymphocyte decreases STAT3 phosphorylation and further suppresses lymphocyte inflammatory polarization,thus rectifies the T cell pro-/anti-inflammatory balance and block the inflammatory signal delivery from adipose tissue to macrophage.STAT3 and FoxP3 could trans regulate PTPN2 expression through binding to Ptpn2 promoter sequence.Objectives(1)To determine the immune characteristics of ADSCs from type 2 diabetic mice and its effect on inducing CD4+ T lymphocyte polarization.(2)To study whether PTPN2 can inhibit the expansion of adipose inflammation by reducing the polarization of CD4+ T cells and its specific mechanism.(3)To investigate the promoter sequence of Ptpn2 gene and the mechanism of STAT3 and FoxP3 trans regulating PTPN2 expression.Methods(1)Type 2 diabetic ApoE-/-mice were constructed through high-fat diet combined with small dose streptozotocin(STZ)intraperitoneal injection(75mg/kg).(2)CD4+ T cell infiltration and polarization in stromal-vascular fraction(SVF)was examined via flow cytometry.Cytokines in SVF was examined via enzyme-linked immunosorbent assay(ELISA).(3)ADSCs were separated after type Ⅱ collagenase digestion and plastic adhesion,and cultured in complete DMEM medium.(4)ADSCs growth type,surface markers,insulin sensitivity,pro-and anti-inflammatory indicators and pro-inflammatory pathway were examined through CCK-8,flow cytometry,ELISA and western blotting respectively.(5)Mouse CD4+ T cells were separated via magnetic activated cell sorting(MACS).(6)Mouse CD4+ T cells were co-cultured with diabetic ADSCs.CFSE staining and CCK-8 were applied to determine the suppressive effect of-ADSC on CD4+ T cell proliferation.Flow cytometry and ELISA was applied to determine CD4’ T cell polarization.(7)CD4+ T cells were transfected with adenovirus FM/Ad5-PTPN2 after antibody activation and centrifugation.GFP expression was examined to certify the transfection efficiency.(8)Transfected CD4+ T cells were co-cultured with diabetic ADSCs to determine whether PTPN2 was involved in CD4+ T cell polarization in diabetic adipose inflammation.(9)Co-cultured CD4+ T cells were reco-cultured with macrophage RAW264.7.Assays of adhesion,migration and phagocytosis were applied to evaluate macrophage function.Flow cytometry,western blotting and ELISA were applied to determine the expression of macrophage M1 and M2 indicators and pro-inflammatory cytokines to evaluate macrophage polarization state.(10)Normal ADSCs were cultured with supernatant from T cell-macrophage co-culture system.Western Blotting was applied to detect the inflammatory response of ADSCs and the effect after PTPN2 overexpression.(11)Western Blotting was applied to examine PTPN2 expression,STAT3 phosphorylation,and polarization-related transcription factor expression in CD4+ T cells.The binding strength of PTPN2 and p-STAT3 were examined via co-immunoprecipitation(Co-IP)assay.(12)Mouse Ptpn2 gene promoter sequence was searched via online database and cloned via PCR.Luciferase reporter vector pGL-3-1742 promoter was constructed via endonuclease digestion,ligation,transformation,and the promoter activity of this sequence was confirmed through dual luciferase reaction assay.(13)The possible transcription factor bingding to Ptpn2 promoter was analyzed via online software ALGGEN PROMO.Chromatin immunoprecipitation(ChIP)assay was applied to investigate whether transcription factor STAT3 and FoxP3 could bind to Ptpn2 promoter.Dual luciferase reaction assay was applied to determine the regulatory effect of transcription factor STAT3 and FoxP3 through binding to Ptpn2 promoter.Results(1)Type 2 diabetic ApoE-/-mouse models were successfully constructed according to the method above.(2)Compared with control group,the ratio of CD3+CD4+ T cells in diabetic SVF obviously increased.The ratio of pro-inflammatory CD4+ T cells(CD4+IFN-γ+,CD4+IL-17A+)obviously increased while anti-inflammatory CD4+ T cells(CD4+CD127)decreased in diabetic SVF.Concentration of IFN-y increased while TGF-P decreased in diabetic SVF supernatant.(3)ADSCs were successively separated and cultured from normal and diabetes mice.ADSCs of two groups both expressed mesenchymal markers CD29,CD 105 and Pref-1,while they did not express CD34 and CD45.Two groups of ADSCs did not manifest significant distinct in morphology and growth type.(4)Compared with control group,type 2 diabetic ADSCs showed no response to small dose of insulin,which indicated that insulin resistance occurred in type 2 diabetic ADSCs.Additionally,Pref-1 expression in diabetic ADSCs decreased.(5)Compared with control group,type 2 diabetic ADSCs secreted less anti-inflammatory cytokine PGE2,but expressed more pro-inflammatory molecules MHC-II,CD40 and CD80.After ADSCs were stimulated by small dose of insulin,phosphorylated Erk significantly decreased,while phosphorylated NF-κB p65 significantly increased in type 2 diabetic ADSCs.(6)Compared with control ADSCs,the restriction effect on CD4+ T cell proliferation has been obviously damaged in type 2 diabetic ADSCs.(7)Compared with CD4+ T cells co-cultured with control ADSCs,CD4+ T cells co-cultured with diabetic ADSCs showed obvious increase in the ratio of pro-inflammatory CD4’ T cells(CD4 IFN-γ’,CD4+1L-17A+),while decrease in anti-inflammatory CD4+ T cells(CD4+CD127-).Concentration of pro-inflammatory cytokines IFN-y and IL-17A increased and anti-inflammatory cytokine TGF-βdecreased in supernatant co-cultured with diabetic ADSCs correspondingly.(8)CD4+T cells overexpressing PTPN2 were resistant to diabetic ADSCs inducement,among which the ratio of pro-inflammatory CD4+T cells decreases and anti-inflammatory CD4+T cells increases.Concentration of pro-inflammatory cytokines IFN-y and IL-17A decreased and anti-inflammatory cytokine TGF-P increased in supernatant correspondingly.(9)Compared with CD4+T cells co-cultured with control ADSCs,CD4+T cells co-cultured with diabetic ADSCs activated more macrophages,among which the ratio of adhesion,migration and phagocytosis all increased.CD4+T cells overexpressing PTPN2 activated less macrophages compared with diabetic group.(10)Compared with CD4+T cells co-cultured with control ADSCs,CD4+T cells co-cultured with diabetic ADSCs predisposed more macrophages to polarize to M1 phenotype,with increase in the ratio of F4/80+CD11cs and increase in the expression of iNOS.Meanwhile,concentration of pro-inflammatory cytokine TNF-a increased.Compared with diabetic group,CD4+T cells overexpressing PTPN2 induced less macrophage to polarize to M1 phenotype.The concentration of TNF-a and IL-6 in the supernatant fell back correspondingly.(11)Compared with supernatant from control group,ADSCs cultured with supernatant from diabetic group showed activated inflammatory pathway.Phosphorylation of p65 significantly increased.Phosphorylation of Akt without insulin stimulating also increased.Compared with diabetic group,ADSCs cultured with supernatant from PTPN2 overexpression group showed lower phosphorylation of p38 and p65.(12)Compared with CD4+ T cells co-cultured with control ADSCs,CD4+ T cells co-cultured with diabetic ADSCs showed no difference in PTPN2 expression.Even so,the phosphorylation of STAT3 obviously increased in CD4+ T cells co-cultured with diabetic ADSCs,with the increase of T-bet and ROR-yT,and the decrease of FoxP3.The ratio of(T-bet+ROR-yT)/FoxP3 was increased.In CD4+ T cells overexpressing PTPN2,the phosphorylation of STAT3 obviously decreased,with the decrease of T-bet and the increase of FoxP3.The ratio of(T-bet+ROR-yT)/FoxP3 declined.(13)Co-IP assay confirmed that PTPN2 could bind the p-STAT3.Compared with control group,the ratio PTPN2/p-STAT3 decreased obviously,with the increased phosphorylated STAT3 in Input.After overexpressing PTPN2,the ratio PTPN2/p-STAT3 recovered,with the decrease of STAT3 phosphorylation in Input.(14)The upstream sequence of mouse Ptpn2 gene(-1676bp～+66bp)was successfully cloned by large-fragment PCR and was 1742bp in length.It was correctly aligned with the mouse genome sequence in online database.The reporter vector pGL-3-1742 promoter was successfully constructed and the promoter activity was confirmed via dual luciferase reaction assay.(15)The online promoter analysis software ALGGEN_PROMO was applied to analyze the upstream sequence of the Ptpn2 gene.The-1800 bp sequence upstream of the Ptpn2 gene was predicted to have intensive transcription factor binding sites,including a STAT3 binding site and five FoxP3 binding sites.ChIP assay showed that the transcription factor STAT3 could bind to the predicted site,while FoxP3 could bind to part of predicted sites.Dual luciferase reaction assay results confirmed that STAT3 could inhibit the transcription and expression of pGL-3-1742 promoter plasmid luciferase by binding to the promoter sequence.Dual luciferase reporter assay also showed that the transcription factor FoxP3 could also inhibit the activity of the pGL-3-1742 promoter plasmid luciferase.The binding strength of STAT3 to Ptpn2 promoter increased while strength of FoxP3 to Ptpn2 promoter decreased after cocultured with T2DM ADSCs in CD4+ T cells.Conclusions(1)Type 2 diabetic ADSCs undergo impaired metabolic function and metabolic inflammation.The anti-inflammatory effects are weakened and the pro-inflammatory effects are significantly enhanced,leading to the pro-inflammatory polarization of CD4+ T cells and mediating downstream macrophage M1 polarization,which exacerbates the transmission and expansion of metabolic inflammation.(2)Phosphatase PTPN2 inhibits CD4+ T cells pro-inflammatory polarization and downstream macrophage M1 polarization induced by type 2 diabetic ADSCs by decreasing the phosphorylation level of STAT3 in CD4+ T cells,thereby cutting off the metabolic Inflammation transmission and expansion.(3)Transcription factors STAT3 and FoxP3 negatively trans regulate the expression of PTPN2 at the transcriptional level through interaction with the Ptpn2 gene promoter sequence.BackgroundAcute coronary syndrome(ACS)is the acute-onset type of coronary artery disease(CAD),and is the major reason of cardiac death,including unstable angina pectoris(UAP),non-ST-segment elevation myocardial infarction(NSTEMI)and ST-segment elevation myocardial infarction(STEMI).Among all the reasons causing ACS,primary cardiac events related to culprit atherosclerosis plaque accounts for the most.With the plaque advanced,the vulnerability of the plaque rises.Additionally,coronary flow reserve is obviously reduced at the existence of severe lumen stenosis,which causes frequent angina due to distal ischemia.Therefore,clinical diagnosis and risk stratification of CAD are often based on the severity of coronary stenosis degree.Coronary artery imaging provides detailed evidence for coronary stenosis,of which coronary angiography(CAG)is gold standard.However,due to invasive,expensive and technology dependent,the examination is used as confirmatory test rather than routine screening test.Screening tests,such as electrocardiogram,dynamic electrocardiogram and echocardiography,do not directly reflect coronary stenosis.In recent years,the value of circulating biomarkers for predicting the degree of coronary lesion in CAD patients has gradually received attention.The establishment of these markers will help early screening and intervention of high-risk patients,and will be of great value in early prevention and treatment of CAD and ACS.The systemic inflammation caused by abnormal balance of pro-inflammatory and anti-inflammatory immune cells is an important cause of coronary lesion deterioration in ACS.Innate immune response of which mononuclear-macrophage polarization is representative takes part in the whole pathogenesis of ACS from plaque rupture to thrombogenesis.Acquired immune response of which T helper cells polarization is representative also promotes inflammation in ACS.Blood cell counts,ratios and inflammatory cytokines have been tested in previous studies to evaluate systemic inflammation and balance of pro-inflammatory and anti-inflammatory immune cells.Although they have ability to predict disease severity and prognosis,they do not show more advantages in reflecting balance of anti-/pro-inflammatory cells,nor do they have relationships with coronary stenosis.We hope to screen out a novel biomarker molecue,which could be located in specific subgroups of cells,and could reflect the function of anti-/pro-inflammation.The indicator is expected to have protruding effects on atherogenesis,and its expression would dynamically change with the progress of CAD and ACS.Expression rate of this molecule will provide evidence for early warning and diagnosis,will be helpful with stratifying and estimating the risk,and will guide to evaluate the treatment response,benefit,and prognosis of patients with acute coronary syndrome.T cell immunoglobulin and mucin domain 3(Tim-3)coincides with the criteria above,and is a promising candidate biomarker to predict coronary lesion severity.Tim-3 is detected to express on varieties of peripheral immune cells.At present,it is believed that Tim-3 exerts immunosuppressive effect on T cells and induces to immune exhaustion.During tumorigenesis Tim-3 is upregulated on T cells,which facilitates tumor progression and metastasis by weakening immune surveillance.Whereas in systemic inflammatory disease,such as acute lung injury,experimental autoimmune encephalomyelitis and autoimmune hepatitis,Tim-3 is often down regulated,causing T cell over reactive and exacerbating systemic inflammation.The inflammation could be mitigated after Tim-3 overexpression or stimulation.Tim-3 also plays important roles in other immune cells,but the function varies in different cells.Recent studies reported close relationship between Tim-3 and atherosclerosis.During atherogenesis,Tim-3 expression on multiple immune cells was upregulated.However,atherosclerosis was deteriorated after blocking Tim-3,which suggested the protective role of Tim-3.Ratio of circulating CD8+Tim-3+ increased in patients with atherosclerosis.CD8+Tim-3s cells had vasoprotective effect via secreting anti-atherosclerosis cytokines.These results indicated a conjunctive role of Tim-3 connecting atherosclerosis with systemic immune balance.Thus we speculated that it is promising to predict severity of coronary lesions via testing Tim-3 expression rate on immune cells.In this study,we will examine the expression of Tim-3 on peripheral blood mononuclear cells of patients with ACS,and explored whether Tim-3 is a candidate biomarker for evaluating severity of ACS based on the relationship between Tim-3 and coronary lesion degree and clinical diagnosis.Objectives(1)To investigate the relationship between the expression of Tim-3 on peripheral blood mononuclear cells and the severity of coronary artery lesion in patients with acute coronary syndromes.To study the possibility of Tim-3 on peripheral blood mononuclear cells as a novel biomarker in patients with acute coronary syndrome.(2)To study the related factors affecting Tim-3 expression in peripheral blood mononuclear cells in patients with ACS.Methods1.Inclusion and exclusion criteriaMedical Ethics Committee of Qilu Hospital of Shandong University checked all the procedures and approved the protocols.All patients involved were formally written informed and they permitted to use blood samples and medical record for research use.Hospitalized patients preliminarily diagnosed ACS according to diagnostic criteria by AHA/ACC and planning to take CAG in our hospital were included.However,patients with valvular heart disease,cardiomyopathy,severe infectious disease,tumor,immune disease and renal dysfunction,or not taking CAG were excluded.According to the cardiac troponin I(cTnI),patients were divided as 2 groups:patients whose cTnI>30ng/L were included in AMI group,while patients whose cTnI<30ng/L were included in UAP group.Patients whose maximal coronary stenosis<50%confirmed through CAG were also included in control group.2.Sample collectionVenous blood sample(3mL)was collected before CAG with EDTA tube.PBMCs were separated and fixed within 2 hours after venipuncture.3.Acquisition of patients’ medical informationPatients’ data were retrieved from medical record in hospital information system(HIS),including demographic information(age,gender),results of medical history(history of hypertension,diabetes,smoking,drinking and medical therapy),results of physical examination(HR,SBP and DBP)and lab examination(TC,LDL-c,HDL-c,TG,FBG,CK-MB and cTnI).4.Acquisition and evaluation of patients’ CAG resultsAngiography films were reviewed and evaluated by two cardiologists.Severity of coronary stenosis was described by Gensini score according to previous study,along with the most degree of stenosis,the number of stenosis and the number of artery involved.5.Detection of Tim-3 expression on PBMCsThe expression rates of Tim-3 on CD4+,CD8+,CD11c+,and CD 14+ subsets of PBMCs were detected by flow cytometry.6.Statistical analysisThe statistical processes were conducted with the software SPSS 24.0.One-way ANOVA and ANCOVA were applied to compare Tim-3 expression rate of PBMCs among different groups of patients;Spearman’s correlation,student t test and multiple linear regression were applied to find affecting factors of Tim-3 expression;Spearman’s correlation and multiple linear regression were applied to analyze the relationship between Tim-3 expression rate of PBMCs with coronary lesion severity;ROC curve was applied to analyze the value of Tim-3 expression rate of PBMCs in predicting coronary lesion severity.Results1.Demographic characteristics of patients enrolled in the study(1)According to the inclusion and exclusion criteria,we included 131 patients,of whom 29 patients were included in control group,77 patients were included in UAP group and 25 patients were included in AMI group.There were no differences in age and gender among groups.(2)Compared with control group and AMI group,SBP was significantly elevated in UAP group.FBG was significantly higher while HDL-c was significantly lower in AMI group compared with the other two groups.cTnI was significantly elevated in AMI group compared with the other two groups.Rate of nitrates use was significantly higher in UAP group compared with control group.(3)As for coronary lesion severity,compared with control group,the maximal coronary stenosis was significantly higher in two ACS groups(UAP and AMI),and the quantities of stenotic segments and stenotic arteries were larger in ACS groups.The Gensini score was significantly higher in ACS groups.Patients of AMI group were afflicted more severe coronary lesions compared with UAP group.2.Detection of Tim-3 expression on PBMCsTim-3 could be examined on PBMC subgroups(CD4+,CD8+,CD11c+,CD14+cells)via flow cytometry in all blood samples.3.Related affecting factors of Tim-3 expression rate on PBMCs(1)There were strong internal correlations of Tim-3 expression among different PBMC subgroups(CD4+,CD8+,CD11c+,CD 14+ cells).(2)HDL-c positively correlated with Tim-3 expression rate of CD4+ cells(r=0.267,p<0.01).TG and age positively correlated with Tim-3 expression rate of CD 14+cells(r=-0.188,p<0.05,r=-0.200,p<0.05).History of diabetes may negatively affected Tim-3 expression rate of CD4+ cells and CD8+ cells.Therapy with statins may positively affected Tim-3 expression rate of CD4+ cells(β=4.61,p<0.05).4.Comparison of PBMCs Tim-3 expression rate among different clinical diagnostic groups(1)After controlling relative covariants,Tim-3 expression rate of CD4+ cells was different among groups.Specifically,Tim-3 expression rate of CD4+ cells was significantly lower in AMI group and UAP group compared with control group.No significant differences have been found between AMI and UAP group.(2)Tim-3 expression rate of CD 14+ cells was distinct among groups.Specifically,Tim-3 expression rate of CD14+ cells was significantly higher in AMI group and UAP group compared with control group.No significant differences have been found between AMI and UAP group.(3)There were no differences in Tim-3 expression rate of CD8+ cells and CD11c+ cells among groups.5.Correlation between PBMCs Tim-3 expression and coronary lesion severity(1)Tim-3 expression rate of of CD4+ cells negatively correlated with maximal coronary stenosis and Genisini score(r=-0.193,p<0.05;r=-0.274,p<0.01).Tim-3 expression rate of of CD8+ cells negatively correlated with Gensini score,cTnI and CK-MB(r=-0.194,p<0.05;r=-0.220,p<0.05;r=-0.262,p<0.05).Tim-3 expression rate of of Cilicc cells did not correlate with any indicators of coronary lesions.Tim-3 expression rate of of CD14+ cells positively correlated with maximal coronary stenosis and cTnI(r=0.212,p<0.05;r=0.226,p<0.05).(2)Multiple linear regression suggested that CD4+Tim-3+ cells were atheroprotective(β-0.28,p=0.029),while CD14+Tim-3+ cells and history of diabetes were atherogenic(β=0.26,p=0.015;P=0.358,p=0.001).The contrary effects of two PBMC subsets still held after controlling confoundings.It was suggested that the atheroprotective role of CD4+Tim-3+ cell depended on CD14+Tim-3+ cell.6.The value of Tim-3 expression rate in predicting coronary lesion severity.(1)Tim-3 expression rate of CD4+ cells could be used to predict maximal stenosis less than 70%.The cut-off value was 20.65%,which meant the maximal stenosis was less than 70%when Tim-3 expression rate of CD4+ cells was over 20.65%.The sensitivity was 50%and the specificity was 81.3%.Tim-3 expression rate of CD4+ cells had no advantage in predicting maximal stenosis less than 50%and 100%,or the quantities of lesional segments and arteries.(2)Tim-3 expression rate of CD14+ cells could be used to predict maximal stenosis 100%.The cut-off value was 15.8%,which meant the maximal stenosis is around 100%when Tim-3 expression rate of CD14+ cells was over 15.8%.The sensitivity was 62.5%and the specificity was 73.4%.Tim-3 expression rate of CD14+cells had no advantage in predicting maximal stenosis over 50%and 70%,or the quantities of lesional segments and arteries.(3)We found that the value CD14+ Tim-3+%/CD4+Tim-3+%could be used to predict maximal stenosis 50%,70%and 100%.The cut-off value was 0,62,0.78 and 0.96,which meant the maximal stenosis reached 50%,70%and 100%when the value was over 0.62,0.78 and 0.96 respectively.This value had sensitivity over 60%and specificity over 70%.However,CD14+ Tim-3+%/CD4+Tim-3+%had no significance in predicting quantities of lesional segments and arteries.Conclusions(1)Tim-3 on PBMCs is a novel circulating biomarker in patients with acute coronary syndrome,in which the expression rate of Tim-3 on CD4+ cells and Tim-3 on CD 14+ cells are related to severity of coronary lesion and clinical diagnosis.The rate of Tim-3 expression on CD4+ cells,the rate of Tim-3 expression on CD4+ cells,and the ratio of CD14+Tim-3+/CD4+Tim-3+ in peripheral blood were predictive of the severity of coronary lesions.(2)Tim-3 on PBMCs could be affected by many factors,among which high density lipoprotein cholesterol,triglycerides,statins use,age,and diabetes history can influence the rate of Tim-3 expression in peripheral blood mononuclear cells.