A novel assay for detection of hepatitis C virus-specific effector CD4+ T cells via co-expression of CD25 and CD134

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.