CD127/PD-1 PHENOTYPE DEFINES THE HCV-SPECIFIC CYTOTOXIC T CELL REACTIVITY AFTER ANTIGEN ENCOUNTER DURING CHRONIC HEPATITIS C VIRUS INFECTION

Methods: 20 HCV-seronegative HLA-A2+ individuals without risk for HCV-acquisition were studied. HCV-NS3(1073)-specific cell lines were generated by short culturing of CD8 T-cells with APCs pulsed with HCV-NS3(1073) peptide. After one-week in-vitro stimulation proliferation by CFSE, after three weeks cytokine responses and tetramer binding were analyzed. Individuals were categorized into non-responder and responder based on proliferation and tetramer frequency. TCR repertoire-analysis of tetramer-sorted cells was performed by RT-PCR with Vb-specific primer and subcloning and sequencing of the CDR3-region. TCR repertoire of HCV-seronegative responder was compared with the TCR repertoire of patients with acute HCV-infection. Results: In 8/20 individuals, HCV-NS3(1073)-specific cells proliferated and variable frequencies (0.4–76%) of tetramer positive cells were documented. TCR reper-analysis of the highly responding individual showed usage of only TCR repertore-Vb6.2. Subcloning of Vb6.2 revealed an oligoclonal TCR repertoire. Interestingly, the CDR3-region of the dominant clone (Vb6.2-QEAGAP-Jb2.2) revealed the presence of similar amino acids found in patients with acute HCV-infection (Acute HCV motifs: Vb6.2-LxGxP-Jb 2.7 and Vb6.2ExAxG-Jb 2.3). Conclusions: Our study confirmed that HCV-NS3(1073)-specific CD8 T-cells exist in HCV-seronegative individuals. The variability between individuals is consistent with the concept of private specificity. The identified TCR repertoire was oligoclonal reminiscent to previously identified cross-reactive responses in mouse models and similar to clones in patients with acute HCVinfection. We suggest that this clone may cross-react during acute HCV-infection leading either to protection, immune pathology or immune-escape due to its oligoclonality. The polyspecificity of memory T-cells needs to be considered when developing peptide based vaccines.