尖锐湿疣患者外周血CD4^＋CD25^＋Foxp3^＋调节性T细胞的检测

Detection of CD4^＋CD25^＋Foxp3^＋ regulatory T cells in peripheral blood of patients with condylomata acurninata

目的探讨外周血CD4^＋CD25^＋调节性T细胞（Treg细胞）表型和功能改变所致的细胞免疫抑制效应在尖锐湿疣发病机制中的作用。方法采用免疫荧光标染技术，经流式细胞仪检测46例尖锐湿疣患者和43例正常人外周血CD4^＋CD25^＋T细胞转录因子Foxp3及细胞毒T细胞相关抗原4（CTLA-4）、糖皮质激素诱导的肿瘤坏死因子受体家族相关基因（GITR）、程序性死亡1（PD—1）等抑制性膜分子的表达水平。同时，用免疫磁珠细胞分选技术从外周血中分选出高纯度的CD4^＋CD25^＋T细胞，经体外刺激后分析胞内分泌白介素10（IL—10）和转化生长因子β（TGF—β）阳性细胞数。结果尖锐湿疣患者外周血CD4^＋CD25^＋Foxp3^＋Treg细胞数量比正常人对照组显著增加（P〈0．01），CD4^＋CD25^＋T细胞表面抑制性膜分子CTLA-4、PD—1表达明显增强（P分别为〈0．05及〈0．01），胞内TGF—β阳性细胞增多（P〈0．001）。结论人乳头瘤病毒感染可诱导CD4^＋CD25^＋Treg细胞活化增殖，高表达负性共刺激分子和分泌抑制性细胞因子，通过多种机制抑制机体的抗病毒免疫应答。

Objective To investigate the possible roles of cellular immunosuppression induced by phenotypic and functional changes of peripheral CD4^＋CD25^＋ regulatory T cells （Tregs） in the pathogenesis of condylomata acuminata. Methods Three-color flow cytometry was performed to examine the expression of transcription factor Foxp3 in, along with several inhibitory membrane molecules, i.e. cytotoxic T lymphocyte associated antigen-4 （CTLA-4）, glucocorticoid-induced TNF receptor family-related gene （GITR） and programmed death-1 （PD-1 ） on peripheral CD4^＋CD25^＋ T cells from 46 patients with condylomata acuminata and 43 normal human controls. Meanwhile, high purity of CD4^＋CD25^＋ T cells were isolated from peripheral blood using immunomagnetic beads, and stimulated to produce intracellular suppressor cytokines such as interleukin-10 （IL-10） and transforming growth factor-beta （TGF-beta）, which were detected by flow cytometric analysis. Results The number of peripheral CD4^＋CD25^＋ Foxp3^＋ Tregs increased significantly in patients with condylomata acuminata than that in the normal controls （7.37% ±2.43% vs 5.96% ± 2.09%, P 〈 0.001 ）. The expressions of CTLA-4 and PD-1 were 1.86% ±1.13% and 2.41% ±1.12%, respectively, in the patients, which were significantly higher than those in the normal controls（ 1.36% ±0.90% and 1.70% ±0.97%, P 〈 0.05 and 0.01, respectively）. The number of TGF-beta-positive CD4^＋CD25＋ T cells from peripheral blood were increased in patients than that in the controls （1.57% ±0.91% vs 0.78% ±0.24%, P 〈 0.001）. Conclusions Human papillomavirus infection can induce the activation and proliferation of CD4^＋CD25^＋ Tregs, enhance the expression of negative costimulatory molecules and secretion of suppressor cytokines, and inhibit antiviral immune response through multiple mechanisms.