Absolute Reduction Of Peripheral CD4~+regulatory T Cell Subset Of Patients With Systemic Sclerosis And Its Restoration By Short-term And Low Dose IL-2 Treatment

Objective:1、The aim of the present study was to explore the absolute number status of CD4~+T subsets in peripheral blood of patients with SSc using our modified flow cryometric method.2、To explore the relationship between absolute counts of CD4~+regulatory T(Treg)cells and Th17 cells in patients with SSc and their disease activity.3、Investigate whether the imbalance of Th17 and Treg cells can be corrected by supplementing low dose interleukin-2(IL-2).Methods:1、The peripheral CD4~+T subsets from 54 patients with SSc and 30 healthy control subjects were analyzed.The patients were divided into the untreated group(n=29)and treated group(n=25),also divided into the untreated group(n=29)and treated group(n=25).2、Calculate all patient’s modified Rodnan skin scores(m Rss),revised EUSTARIndex and Medsger severity score(MSS);according to whether EUSTAR index score more than 2.5 points,divide SSc patients into inactive group(n=23)and activities Group(n=31).3、The treated patients were divided into group A(n=28)who only used low-dose glucocorticoids(GCS),and group B(n=26)who also used immunosuppressant cyclophosphamide(CYC),methotrexate(MTX),leflunomide(LEF)or mycophenolate mofetil(MMF).4、In addition,21 patients from 54 patients,given a small dose of IL-2(50WIU)treatment for a 5-day course,divided into pre-treatment group and post-treatment group.Directly using the results from flow cytometry combined with internal standard beads,absolute number of peripheral CD4~+T subsets from the subjects in each group were calculated.5、Directly using the results from flow cytometry combined with internal standard beads,absolute number of peripheral CD4~+T subsets from the subjects in each group were calculated.Measured data using the Wilcoxon rank sum test on the premise of not meeting the normality and homogeneity of variance.And using Spearman’s coefficient for correlation analysis.Results:1、Although there were some changes among CD4~+T cell subsets in peripheral blood from these SSc patients,the major alteration was the reductions of Treg cell absolutenumber.Comparedwiththenormalcontrols,thenumberof CD4~+CD25~+FOXP3~+Treg cells were significantly decreased in all patients(p=0.001),in untreated group(p=0.039),in treated group(p=0.02),while there was no difference between untreated group and treated group(p>0.05).The ratio of Th17/Treg in total patients(p=0.008),in treated group(p=0.005)were significantly higher than that in normal control group,2、Compared with the healthy control group,the absolute counts of CD4~+CD25~+FOXP3~+Treg cells were significantly lower than that in inactive group(P=0.02)and in active group(P=0.037),and the percentage of Th2 cells was significant increased in inactive group(P=0.017)and in active group(P=0.032).In addition,ESR was positively correlated with skin mRSS scores,disease activity scores and severity scores,the correlation coefficients were(r=0.276 P=0.043),(r=0.679,P<0.001),(r=0.39,P=0.004),moreover,percentage of Treg was positively correlation with the duration of the disease(r==0.308,P=0.023)and disease severity(r=0.294,P=0.031).However,the number of Th17 and Treg cells as well as percentages of Th17and Treg cells were not associated with mRSS score,disease activity scores and severity scores.3、Compared with the normal controls,the number of CD4~+CD25~+FOXP3~+Treg cells were significantly decreased in group A(p=0.005)and in group B(p=0.004),while there was no difference between group A and group B(p>0.05).The ratio of Th17/Treg in group B were significantly higher than that in normal control group(p=0.001),in group A(p=0.037),indicating a more imbalance between Th17 and Treg cells in group B.Moreover,the number of Th17 cells in group B was significantly higher than that in group A(p=0.011).4、After low dose IL-2 treatment,the absolute number of CD4~+CD25~+FOXP3~+Treg cells(p<0.001)were significantly increased,while the number of Th2 and Th17 cells were slightly higher than those before treatment with IL-2(p>0.05).Since Th17 cells increases significantly lower than Treg cells,so their ratio decreases significantly(p=0.001)to get re-balance.Conclusion:1、The absolute number of peripheral CD4~+CD25~+FOXP3~+Treg cells decreased in SSc patients,indicating that this reduction arising from the disease itself.Our findings suggest that SSc progression is associated with the absolute decrease of peripheral Treg cells.2、Although there was no statistically significant different,absolute number of the Treg cells trended to decrease in patients treated with immunosuppressive agents.3、The use of low dose IL-2 can effectively up-regulate Treg cellst,which restores the balance of Th17/Treg cells,but the long-term effect needs further investigation.