1型糖尿病患者外周Treg细胞亚群频率及Helios、细胞毒性T淋巴细胞相关抗原4分子表型分析

Study on the frequency and expression of Helios and cytotoxic T lymphocyte antigen 4 in CD4 Tregsubsets of type 1 diabetic patients

目的探讨1型糖尿病患者外周血单个核细胞（PBMC）中CD4调节性T细胞（Treg）及其亚群频率和其表达Helios、细胞毒性T淋巴细胞相关抗原4（CTLA-4）分子的变化情况。 方法选取2016年4月至2017年4月在南京医科大学第一附属医院内分泌科就诊的40例T1DM患者为研究对象（T1DM组），同期医院体检健康人群67名作为对照组。密度梯度离心法分离出PBMC。以CD25和FoxP3标记CD4 Tregs （FoxP3＋CD25＋），以CD45RA及FoxP3标记区分Treg细胞亚群（初始型nTreg，CD45RA＋FoxP3＋；激活型aTreg，CD45RA-FoxP3hi；分泌型sTreg，CD45RA-FoxP3＋），并进一步标记Treg各亚群中Helios及CTLA-4分子，行多色流式细胞术检测。用双侧未配对t检验分析两组之间的差异情况。 结果与健康对照组相比，1型糖尿病组PBMC中的总Treg细胞及其亚群（nTreg、aTreg及sTreg）占CD4 T细胞的频率差异均无统计学意义（t=0.109 5~0.575 0，均P〉0.05），并且两组间nTreg、aTreg及sTreg亚群占总Treg细胞的频率差异亦无统计学意义（t=0.143 8~0.272 3，均P〉0.05）。进一步研究发现，Helios分子在两组间Treg细胞及其亚群（nTreg、aTreg及sTreg）上表达无统计学差异（t=0.178 4~1.124 0，均P〉0.05）。但1型糖尿病组总Treg及sTreg、nTreg细胞亚群CTLA-4分子的表达均显著减少，差异有统计学意义（t=2.662~3.435，均P〈0.05），而该分子在aTreg亚群的表达差异无统计学意义（t=1.728，P=0.0887，P〉0.05）。 结论本研究人群中，1型糖尿病患者PBMC中CD4 Treg及其亚群未发现频率的显著变化，但sTreg及nTreg细胞亚群抑制性表型CTLA-4表达下调，这可能是导致1型糖尿病患者Treg细胞抑制功能下降的原因之一。

ObjectiveTo investigate the frequency and phenotype Helios and cytotoxic T lymphocyte antigen 4 （CTLA-4） of circulating FoxP3＋CD25＋ CD4 Tregs and Treg subsets in type 1 diabetes mellitus （T1DM） patients. MethodsForty T1DM patients and sixty-seven healthy individuals were enrolled in this study. Peripheral blood mononuclear cells （PBMCs） were obtained by density gradient centrifugation using ficoll. Then PBMCs were stained with Foxp3 and CD25 for CD4 Treg （FoxP3＋CD25＋）, CD45RA and FoxP3 for Treg subsets （naive Treg, CD45RA＋FoxP3＋; activating Treg, CD45RA-FoxP3hi; secreting Treg, CD45RA-FoxP3＋）, and additional Helios and CTLA-4 for immune phenotypes of Treg subsets. The analysis was performed with BDFACSAriaII using FACSDiva software. All the statistic analyses were performed by unpaired Student t test. ResultsCompared with the frequencies of healthy individuals, the frequencies of total Tregs, and nTreg, aTreg, sTreg subsets in CD4 T cells of T1DM patients did not alter significantly （t=0.109 5–0.575 0, all P〉0.05, respectively）;Similarly, the frequencies of nTreg, aTreg, and sTreg subsets in total Tregs of T1DM patients did not alter significantly （t=0.143 8–0.272 3, all P〉0.05）. Further phenotype analysis revealed that the expression of Helios in total Tregs and Treg subsets did not change in T1DM patients （t=0.178 4–1.124 0, all P〉0.05）. However, the expressions of CTLA-4 in total Tregs and sTreg, nTreg subsets were significantly lower in T1DM patients （t=2.662–3.435, all P〈0.05）;the expressions of CTLA-4 in aTreg subsets were not significantly lower in T1DM patients （t=1.728, P=0.088 7）. ConclusionAlthough circulating CD4 Tregs and Treg subsets did not change in T1DM patients, expression of CTLA-4 decreased significantly in sTreg and nTreg subsets, which may play a role in Treg dysfunction of T1DM patients.