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

摘要

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CD4+ 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CD4+ 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CD4+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CD4+ 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+ Treg CHB患者,HBVcore18-27特异性CTLs的频率以及CTLs分泌IFN-γ的频数与未去除CD4+ CD25+ 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CD4+ 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 healyhy blood donors to isolate the peripheral blood mononuclear cells (PBMCs).Flow cytometry was used to analyze the proportion of CD4+ CDl27lo CD25hi-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- cells were isolated by magnetic bead sorting technique.The CD4- T cells 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 HBVcorel8-27 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 immunospot assay (Elispot).Results The proportion of CD4+ CDl27lo CD25hi-int Treg in the CD4+ T cells 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 difierence 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 CD4+ CD25+ cells in circulation were not depleted (0.17％±0.08％,t=4.75,P＜0.01).The frequency of IFN-γ secreting spots of HBVcorel8-27-specific CD8+ T cells of the CHB patients with their CD4+ CD25+ cells depleted was (112±33),significantly higher than that of the CHB patients whose CD4+ 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 HBVcore18-27 peptide significantly improves the antivirus CTL responses in CHB patients.