慢性乙型肝炎患者浆样树突状细胞体外诱导CD4^＋CD25^＋调节性T细胞的研究

Human plasmacytoid dendritic cells from patients with chronic hepatitis B induce the generation of CD4^＋CD25^＋Treg in vitro

目的观察慢性乙型肝炎患者、乙型肝炎恢复者和健康人浆样树突状细胞（pDCs）体外诱导CD4^＋CD25^＋调节性T细胞（CD4^＋CD25^＋Treg）能力的差异，为阐明HBV感染慢性化的机制奠定基础。方法采用免疫磁珠分离法体外分离46例慢性乙型肝炎患者、10例乙型肝炎恢复者和25名健康人外周血单个核细胞中pDCs，并将其分别与健康人CD4^＋CD45RA^＋初始T细胞共培养。采用HBcAg或破伤风毒素对去除CD25^＋细胞的外周血单个核细胞进行增殖刺激后，使用流式细胞仪及RT—PCR对pDCsT共培养细胞中CD4^＋CD25^＋Treg的数量、表型及FOXP3基因表达情况进行测定；采用酶联免疫吸附法对共培养细胞上清液中的白细胞介素-10和转化生长因子β1进行了进一步检测。两组数据比较采用Mann Whitney U-test。结果当细胞增殖刺激物为HBcAg时，细胞增殖幅度漫性乙型肝炎患者组为（7999．36±374．74）cpm，乙型肝炎恢复者组为（11282．56±1174．46）cpm和健康人组为（12304．58±1462．81）cpm，慢性乙型肝炎患者组细胞增殖幅度明显小于乙型肝炎阪复者组和健康人组，U=0～22．0，P值均〈0．05；乙型肝炎恢复者组和健康人组间增殖幅度差异无统计学意义。当增殖刺激物为破伤风毒素时，细胞增殖幅度与阳性对照组之间，差异无统计学意义。CD4^＋CD25^＋Treg比例慢性乙型肝炎患者组为5．99％±1．85％，乙型肝炎恢复者组为3．04％±0．79％，健康人组为3．01％±1．53％，慢性乙型肝炎患者组中的CD4^＋CD25^＋Treg比例明显高于乙型肝炎恢复者组和健康人组，U=6．0～71．5，P值均〈0．05。3组人群pDCsT共培养细胞的CD4^＋CD25^＋T细胞均检测到Fox p3 RNA，而在CD4^＋CD25^-T细胞中，均未检测到Foxp 3R NA。3组人群pDCs—T共培养细胞实验组上清液的白细胞介素10和转化生长因子β1含量均明显高于阳性对照组。结论pDCs以诱导CD4^＋CD25^＋Treg形式参与了乙型肝炎的慢性化。

Objective To compare the ability of plasmacytoid dendritic cells （pDCs） from chronic HBV patients and healthy controls to induce the the generation of CD4^＋CD25^＋Treg cells. Methods The pDCs were isolated from PBMCs of 46 chronic HBV patients, 10 resolved HBV patients and 25 healthy controls by magnetic cell sorting. Purified CD4＋CD45RA^＋ naive T cells were incubated with allogeneic pDCs from chronic HBV infected patients, resolved HBV patients or healthy controls. The cells were stimulated with HBcAg or tetanus toxin. The proportion of CD4^＋CD25^＋Treg in CD4^＋ T cells primed by pDCs was determined by flow cytometry, the expression of Fox p3 mRNA was detected by RT-PCR, IL-10 and TGF β 1 expression was quantified using ELISA kits. Results Compared with pDC isolated from healthy controls and the resolved HBV patients, pDC from chronic HBV patients was more effective in suppression of CD4^＋ T cells proliferation and interferon production when CD25-depleted PBMCs were stimulated with HBcAg,（7999.36 ± 374.74 vs 11 282.56±1174.46; 7999.36 ± 374.74 vs 12 304.58 ± 1462.81, P 〈 0.05 ）. Depletion of CD4^＋CD25^＋ Treg from CD4^＋ T cells primed by pDC led to the lose of capability to suppress HBV- specific T-cell responses. When CD25- depleted PBMCs were stimulated with purified tetanus toxin, there was no significantly difference in proliferation between CD25-depleted PBMC co-cultured with pDC-primed CD4^＋ T cells and CD25-depleted PBMC cultured without pDC-primed CD4^＋ T cells. A higher percentage of CD4^＋CD25^＋ Treg was detected within the population of CD4^＋ T cells primed by pDC from chronic HBV patients compared with healthy controls and resolved HBV patients （5.99% ± 1.85% vs 3.04% ± 0.79% 5.99% ± 1.85% vs 3.01% ± 1.53%, P 〈 0.05 ）. Accordingly, CD25^＋Treg from pDC-primed CD4^＋ T cells displayed a higher Fox P3 mRNA level. The IL-10 and TGF β 1 could be also detectable in the supematants of pDC-primed CD4^＋ T cells. Conclusion pDCs from chronic hepatitis B induce the generation of a higher proportion of CD4^＋CD25^＋ Treg compared with pDCs from healthy controls.