Methods: HCV-specific T lymphocytes s from vaccinated HHD mice were fused with hybridoma partner and screened for CD3 expression by flow cytometry analysis. Capacity to recognize the HLA-A2/HCV peptide complex was tested on human target cells by measuring cytokine production. Beside wild type HCV peptide, mutant peptide analogues were also tested. Minimum peptide concentration required for T cell activation was determined. TCR gene usage was analyzed by PCR V family typing and nucleotide sequencing.
Results: We have identified a large number of murine T cell clones that are able to specifically recognize HCV presented by human HLA molecule. Out of 108 hybridomas 95 were CD3+ and 21 of those were also IL-2 producers of which interferon gamma was detected in majority. TCR gene analysis demonstrated unique Valpha and Vbeta gene usage in all clones. Expression constructs with EGFP tag in polyA- and retroviral vectors for studies of TCR gene transfer in human effector cells are being assembled.
Conclusion: We have generated functional T cell hybridomas with HCV specificities selected on human cells. This allows rapid production and preservation of effector cells for TCR gene transfer. These new reagents will allow simple detection of cells actively presenting HCV and enable studies of HCV related immunopathology.