He X S, Rehermann B, López-Labrador F X, Boisvert J, Cheung R, Mumm J, Wedemeyer H, Berenguer M, Wright T L, Davis M M, Greenberg H B
Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 1999 May 11;96(10):5692-7. doi: 10.1073/pnas.96.10.5692.
It is believed that the hepatitis C virus (HCV)-specific CD8(+) cytotoxic T lymphocytes (CTLs) play a role in the development of liver cell injury and in the clearance of the virus. To develop a direct binding assay for HCV-specific CTLs, we generated two peptide-MHC tetramers by using the recombinant HLA A2.1 molecule and A2-restricted T cell epitopes of the HCV NS3 protein. With these reagents we are able to detect specific CD8(+) cells in the blood of 15 of 20 HLA-A2(+), HCV-infected patients, at a frequency ranging from 0.01% to 1.2% of peripheral CD8(+) T cells. Phenotypic analysis of these specific cells indicated that there is a significant variation in the expression of the CD45 isoforms and CD27 in different patients. A 6-hour incubation of one patient's blood with NS3 peptides resulted in the activation of the epitope-specific CD8(+) cells, as indicated by their expression of CD69 and IFN-gamma. We also detected NS3-specific CD8(+) T cells in the intrahepatic lymphocyte population isolated from liver biopsies of two HCV-infected patients. The frequency of these specific CD8(+) cells in the liver was 1-2%, at least 30-fold higher than in the peripheral blood. All of the intrahepatic NS3-specific CD8(+) T cells were CD69(+), suggesting that they were activated CTLs. Direct quantitation and characterization of HCV-specific CTLs should extend our understanding of the immunopathogenesis and the mechanism of clearance or persistence of HCV.
据信，丙型肝炎病毒（HCV）特异性CD8(+)细胞毒性T淋巴细胞（CTL）在肝细胞损伤的发展以及病毒清除过程中发挥作用。为了开发一种针对HCV特异性CTL的直接结合检测方法，我们利用重组HLA A2.1分子和HCV NS3蛋白的A2限制性T细胞表位生成了两种肽-MHC四聚体。使用这些试剂，我们能够在20名HLA-A2(+)、HCV感染患者中的15名患者的血液中检测到特异性CD8(+)细胞，其频率在外周血CD8(+) T细胞的0.01%至1.2%之间。对这些特异性细胞的表型分析表明，不同患者中CD45异构体和CD27的表达存在显著差异。一名患者的血液与NS3肽孵育6小时后，表位特异性CD8(+)细胞被激活，这通过它们CD69和IFN-γ的表达得以表明。我们还在从两名HCV感染患者的肝活检中分离出的肝内淋巴细胞群体中检测到了NS3特异性CD8(+) T细胞。这些特异性CD8(+)细胞在肝脏中的频率为1%-2%，比外周血中至少高30倍。所有肝内NS3特异性CD8(+) T细胞均为CD69(+)，表明它们是活化的CTL。对HCV特异性CTL的直接定量和表征应能扩展我们对HCV免疫发病机制以及清除或持续存在机制的理解。
