乙型肝炎患者外周血病毒特异性CD8阳性细胞毒性T淋巴细胞的数量和功能分析

目的 分析急性乙型肝炎(AHB)和慢性乙型肝炎(CHB)患者外周血中乙型肝炎病毒(HBV)特异性细胞毒性T淋巴细胞( CTL)的数量及功能.方法 36例HLA-A2阳性的乙型肝炎患者,其中AHB 12例,CHB 24例.分别用含HBV抗原C、S和P区的c18-27、s183-191和p575-583三个肽段的四聚体Tetramer (Tc 18-27、Ts183-191、Tp 575-583)]检测患者外周血单核淋巴细胞(PBMCs)中HBV特异性CD8阳性CTL细胞的数量,同时用酶联免疫斑点法(EHSPOT)检测其分泌IFN-γ的功能.用SPSS 13.0进行统计学分析.结果 AHB和CHB患者外周血中Tc18-27特异性的CTL数量无明显不同；而Ts 183-191特异性CTL的平均值分别为0.24％±0.39％和0.03％±0.02％,阳性率分别为75％和33.3％；Tp575-583特异性CTL平均值分别为0.08％±0.09％和0.02％±0.01％,阳性率分别为50％和16.7％,AHB较CHB显著升高(P＜0.05).此外,AHB患者平均Tetramer阳性的个数为1.58个,而CHB患者平均为0.67个,AHB较CHB显著增多(P＜0.01).在9例AHB患者中,其外周血Ts183-191特异性的CTL细胞的数量为139～21 735个/106 PBMCs,用ELISPOT方法检测相对应的分泌IFN-γ的斑点形成细胞(SFC)为0～252个/106 PBMCs,AHB患者Tetramer细胞数和ELISPOT检测的IFN-γ斑点数有明显相关性(P＜0.01),而CHB患者则无此相关性.结论 AHB与CHB患者外周血中HBV特异性CTL的数量和分泌IFN-γ的差异提示CTL可能在清除病毒方面发挥着至关重要的作用,是今后慢性乙型肝炎免疫治疗的重要研究方向之一.

Analysis the frequency and function of HBV-specific CD8 positive cytocytoxic T lymphocytes from PBMCs in patients with hepatitis B

Objective To analyze the frequency and the function of HBV-specific CTLs in peripheral blood from 36 cases of HLA-A2+ HBV-infected patients using Tetramer and IFN-γ-ELISPOT assays. Methods The patients enrolled were categorized into two groups named after acute hepatitis B (AHB, 12 cases)and chronic hepatitis B(CHB,24 cases).Peripheral blood mononuclear cells (PBMCs) were isolated via ingredient centrifugation and used for CTL analysis. Three commercial tetramer reagents specifically conjugated with epitopes of HBcAg 18-27(Tc18-27), HBsAg 183-191(Ts183-191) and polymerase 575-583(Tp575-583),respectively, were purchased for analysis the frequencies of HBV-specific CTLs. Furthermore, the assay of ELISPOT was used for analyzing the secreting functions of IFN-γ for CTLs. Results Though no significant difference in Tc18-27-reactive CTLs was between the patients with AHB and CHB, the mean and the positive rate of Ts183-191-specific CTL in AHB were significantly higher than that in the CHB groups(0.24%±0.39% vs 0.03%±0.02% and 75% vs 33.3%,P<0.05),and so were the Tp575-583-specific CTLs(0.08%±0.09% vs 0.02%±0.01% and 50% vs 16.7%). Moreover, averaged 1.58 of 3 tetramers was positive in the patients with AHB, which was higher than that in CHB with a mean value of 0.67. Furthermore, the absolute amounts of Tetramer-positive CTL were positively correlated with IFN-γ-producing cell numbers measured by ELISPOT in AHB patients under the stimulation of HBV correspondingly peptide such as s183-191(P<0.01). Conclusion Our findings suggest that HBV-specific CTLs may play an important role in controlling the HBV replication through producing IFN-γ, which can be one of the main contents for immunotherapy of CHB.