慢性乙型肝炎患者抗病毒治疗过程中程序性死亡分子1及其配体表达的变化

目的 探讨慢性乙型肝炎患者在抗病毒治疗过程中程序性死亡分子1(PD-1)及其主要配体PD-L1表达的变化情况及其与HBeAg/抗-Hbe血清学转换的关系.方法 对10例人类白细胞抗原(HLA)-A2阳性的HBeAg阳性慢性乙型肝炎患者予以替比夫定抗病毒治疗24 周,分别于治疗的0、12周和24周随访,检测HBV DNA水平、外周血单个核细胞(PBMC)表面PD-1和PD-L1表达水平、HBV特异性T淋巴细胞数量与其表达PD 1水平,及PBMC体外培养上清液中干扰素(IFN)γ水平.其中HBV DNA定量采用荧光定量PCR法检测;PBMC自新鲜血分离,部分PBMC加入重组HBcAg体外培养7d,加入HBV抗原表位肽五聚体复合体;PD-1和PD-L1阳性细胞、CD8'T淋巴细胞以及CD8和PD-1双阳性细胞用流式细胞仪检测;IFN γ水平用酶联免疫吸附试验检测.比较不同时间点PBMC表面PD-1和PD L1表达水平、HBV特异性CD8'T淋巴细胞数量及其PD-1表达水平、PBMC培养上清液中IFN γ水平.对抗病毒治疗前后对应指标数值的变化采用配对t检验分析.结果 治疗前HBV DNA水平为5.16～8.77 log10拷贝/ml,治疗24周时,7例HBV DNA水平低于检测下限,3例仍可检测到,但明显低于基线水平.2例HBeAg/抗-Hbe血清学转换,2例HBeAg阴转,6例仍维持HBeAg阳性.0、12周和24周PBMC表面PD 1表达水平分别为52.1%±17.0%、39.1%±18.2%和23.4%±16.3%(24周和0周比较,P＜0.01);PD-L1分别为45.6%±15.4%、34.6%±16.2%和20.9%±9.5%(24周和0周,P＜0.01;24周和12周比较P＜0.05);HBV特异性T淋巴细胞上PD-1表达水平分别为76.2%±10.4%、66.5%±15.4%和49.5%±25.3%(24周和0周比较,P＜0.01;12周和0周比较,P＜0.05;24周和12周比较,P＜0.05);HBV特异性T淋巴细胞分别为1.3%±0.5%、1.5%±1.0%和2.2%±1.5%; IFN γ水平(pg/ml)分别为91.7±82.1、99.4+93.5和109.0+86.6.24周HBeAg/抗-Hbe血清学转换者上述指标变化明显大于无HBeAg/抗-Hbe血清学转换者.结论 直接抑制HBV复制能降低PD-1、PD-L1表达水平,并增加HBV特异性CD8'T淋巴细胞的数量和功能.早期PD-1快速下降和HBV特异性T淋巴细胞数量及功能的恢复与早期HBeAg/抗-Hbe血清学转换的关系有待于进一步研究.

Programmed death-1 (PD-1) and PD-L1 expression during antiviral treatment of chronic hepatitis B

Objective To study PD-1 and PD-L1 expressions during 24 weeks telbivudine antiviral treatment in patients with chronic hepatitis B (CHB) and to explore the relationship between PD-1 expression and HBeAg/HBeAb seroconversion. Methods Ten CHB cases with HLA-A2 and HBeAg positive were treated with telbivudine 600 mg/d orally for 24 weeks. Fresh blood samples were collected at week 0, 12 and 24 after treatment. HBV-specific CD8+T cells were expanded in vitro. Cell culture medium were collected for interferon gamma (IFN γ ) detection. Flow cytometry was used to detect the HLA-A type, PD-1, PD-L1 and HBV specific CD8+ T cells. The expressions of PD- 1 and PD-L1, the counts of HBV-specific CD8+ T cells in circulating CD8+ lymphocytes, and IFN γ concentration in culture medium were evaluated during antiviral treatment. Results At week 0, 12 and 24 after telbivudine treatment, 7 of 10 patients were HBV DNA undetectable, 2 were HBeAg seroconvertion and 2 were HBeAg lose but anti-HBe negative. The frequency of PD-1-positive PBMCs were 52.1% ± 17.0%, 39.1% ± 18.2% and 23.4% ± 16.3% (week 24 vs week 0, P ＜0.01) respectively; PD-L1 positive PBMCs were 45.6% ± 15.4%, 34.6% ± 16.2% and 20.9% ± 9.5%respectively(week 24 vs week 0, P ＜ 0.01; week 24 vs week 12, P ＜ 0.05). The frequency of PD-1-positive CD8+ T cells were 76.2% ± 10.4%, 66.5% ± 15.4% and 49.5% ± 25.3% respectively (week 24 vs week 0,P ＜ 0.01; week 12 vs week 0, P ＜ 0.05; week 24 vs week 12, P ＜ 0.05); HBV-specific CD8 cells were 1.3% ±0.5%, 1.5% ± 1.0% and 2.2% ± 1.5%; IFN γ levels in cell culture medium were (91.7 ± 82.1) pg/ml,(99.4 ± 93.5) pg/ml and (109.5 ± 86.6) pg/ml. A remarkable decrease of PD-1 and PD-L1 expressions and increase of HBV-specific CD8+T cells were observed in patients who had HBeAg/HBeAb seroconversion at week 24. Conclusions Direct suppression of HBV replication by telbivudine in CHB patients can decrease PD-1 and PD-L1 expressions and restore HBV-specific CD8+T cells. The relationship between the changes of PD- 1 expression and HBeAg/HBeAb seroconversion during antiviral therapy in HBeAg-positive patients need to confirm by future study.