Inflammation and Infection Research Centre, School of Medical Sciences, University of New South Wales, Sydney NSW, 2052, Australia.
J Immunol Methods. 2012 Jan 31;375(1-2):148-58. doi: 10.1016/j.jim.2011.10.004. Epub 2011 Oct 12.
Hepatitis C virus (HCV)-specific CD4(+) effector T cell responses are likely to play a key role in the immunopathogenesis of HCV infection by promoting viral clearance and maintaining control of viraemia. As the precursor frequency of HCV-specific CD4(+) T cells in peripheral blood is low, favoured assay systems such as intracellular cytokine (ICC) or tetramer staining have limited utility for ex vivo analyses. Accordingly, the traditional lymphocyte proliferation assay (LPA) remains the gold standard, despite detecting responses in only a minority of infected subjects. Recently, we reported development and validation of a novel whole blood CD4(+) effector T cell assay based on ex vivo antigen stimulation followed by co-expression of CD25 and CD134 on CD4(+) T cells. Here we report adaptation of this assay to assessment of HCV-specific responses in cryopreserved peripheral blood mononuclear cells using standardised antigens, including peptide pools, viral supernatants and recombinant viral proteins. The assay allowed detection of HCV-specific CD4 responses in donors with both resolved and chronic infection. Responses were highly correlated with those revealed by LPA. Application of this assay will further define the role of CD4(+) T cells in the immunopathogenesis of HCV infection.
丙型肝炎病毒（HCV）特异性 CD4(+)效应 T 细胞反应可能通过促进病毒清除和维持病毒血症控制在 HCV 感染的免疫发病机制中发挥关键作用。由于外周血中 HCV 特异性 CD4(+)T 细胞的前体频率较低，因此细胞内细胞因子（ICC）或四聚体染色等有利的检测系统对于体外分析的应用有限。因此，传统的淋巴细胞增殖试验（LPA）仍然是金标准，尽管仅在少数感染受试者中检测到反应。最近，我们报道了一种基于体外抗原刺激，随后在 CD4(+)T 细胞上共同表达 CD25 和 CD134 的新型全血 CD4(+)效应 T 细胞检测方法的开发和验证。在这里，我们报告了该检测方法在使用标准抗原（包括肽库、病毒上清液和重组病毒蛋白）对冷冻保存的外周血单核细胞中的 HCV 特异性反应进行评估的适应性。该检测方法能够检测到已解决和慢性感染供体中的 HCV 特异性 CD4 反应。反应与 LPA 揭示的反应高度相关。该检测方法的应用将进一步确定 CD4(+)T 细胞在 HCV 感染免疫发病机制中的作用。
