慢性乙型肝炎患者CD4＋CD25＋调节性T细胞免疫抑制功能的研究

Inhibition of CD4 ＋ CD25 ＋ regulatory T cells in chronic hepatitis B patients

目的探讨慢性乙型肝炎患者CD4＋CD25＋调节性T细胞（Treg）免疫抑制功能。方法收集北京大学人民医院22例慢性乙型肝炎（CHB）患者外周血单个核细胞（PBMC）以及18名健康对照的PBMC标本，以流式分析对PBMC中CD4＋CD25＋Treg的频率进行分析；5-溴脱氧尿嘧啶核苷（BrdU）掺入法评价CIM＋CD25＋Treg的免疫抑制功能；并同时通过磁珠分选去除CHB患者PBMC中的CIM＋CD25＋Treg，分别以MHC-肽-五聚体法和酶联斑点免疫法（Elispot）检测HBVcore18-27抗原肽刺激对HBV特异性的细胞毒性T淋巴细胞（CTLs）的频率以及IFN-γ的分泌。结果CHB患者外周血中CD4＋CD25＋Treg细胞群所占CIM＋T细胞群的比例明显高于健康对照（t=3．74，P〈0．01）；CHB患者CD4＋CD25＋Treg细胞可非特异抑制自身活化的CIM＋CD25。T细胞，并呈剂量依赖的特点，且抑制能力与健康对照相比无明显差异；在经HBVcore18-27抗原肽诱导条件下，去除CD4＋CD25＋TregCHB患者，HBVcore18-27特异性CTLs的频率以及CTLs分泌IFN-1的频数与未去除CD4＋CI）25＋Treg组比出现显著上调（t=4．75，t=7．828，P〈0．01）。结论CHB患者循环中CD4＋CD25＋Treg细胞频率升高。在体外去除CHB患者PBMC的CIM＋CD25＋Treg后可显著地增强HBV抗原诱导的抗HBV细胞免疫应答。

Objective To evaluate the inhibition of CD4 ＋ CD25 ＋ regulatory T cells （Treg） in the chronic hepatitis B patients. Methods Peripheral blood samples were collected from 22 patients with chronic hepatitis B （CHB） and 18 healthy blood donors to isolate the peripheral blood mononuclear cells （PBMCs）. Flow cytometry was used to analyze the proportion of CD4 ＋ CD127^loCD25^hi-int Tregs in the CD4 ＋ T cells so as to calculate the proportion of CD4 ＋ CD25 ＋ Tregs in the CD4 ＋ T cells. BrdU incorporation method was used to evaluate the immune inhibition of the CD4 ＋ CD25 ＋ Tregs. CD4 ＋ CD25 - ceils were isolated by magnetic bead sorting technique. The CD4- T ceils and CD4 ＋ CD25- T cells ere mixed and stimulated by HBVcore 18-27 peptide. The PBMCs of the CHB patients with the Treg depleted and Treg not depleted underwent detection of HBVcorelS-27 specific cytotoxic T lymphocytes （CTLs）. The IFN-γ secretion of the CTLs in the PBMCs of CHB patients with Treg depleted and Treg not depleted was detected by HLA-pentamer and enzyme-linked immunospet assay （Elispot）. Results The proportion of CD4 ＋ CD12^loCD25^hi-intTreg in the CD4 ＋ T ceils used to reflect the percentage of CD4 ＋ CD25 ＋ Tregs in the CD4 ＋ T cells of the CHB patients was 4. 3% ＋ 2.4%, significantly higher than that of the healthy controls （2.1% ＋ 1.3%, t = 3.74,P 〈0.01 ）. There was no significant difference in the inhibition of CD4 ＋ CD25 - T cells by autogeneous CD4 ＋ CD25 ＋ T cells between the CHB patients and healthy controls. The frequency of CTLs induced by HBV core 18-27 of the CHB patients with their CD4 ＋ CD25 ＋ cells in circulation depleted was 0.74% ＋0.31%, significantly higher than that of the patients whose CIM＋ CD25 ＋ cells in circulation were not depleted （0. 17% ＋0.08%, t =4.75,P 〈0.01）. The frequency of IFN-T secreting spots of HBVcore18-27-specific CD8 ＋ T cells of the CHB patients with their CIM ＋ CD25 ＋ cells depleted was （ 112 ＋33）, significantly higher than that of the CHB patients whose CIM ＋ CD25 ＋ cells in circulation were not depleted [（23＋14）, t=7.828,P〈0.01）]. Conclusion The proportion of CD4＋CD25＋ Treg in CHB patients is increased compared to the healthy blood donor. The proliferative capacity of CD4 ＋ CD25 - T cells is inhibited by the presence of CD4 ＋ CD25 ＋ Treg dose-dependently, and the inhibition of CD4 ＋ CD25 ＋ Tregs in the CHB patients is similar to the inhibition of CD4 ＋ CD25 ＋ Tregs in healthy donors. The elimination of Treg cells followed by stimulation with HBVeore18-27 peptide significantly improves the anti- virus CTL responses in CHB patients.