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(Tc 18-27) , HBsAg 183-191 (Ts 183-191) 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-y 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 sl83-191 (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.%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.
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