Utilizing a comprehensive approach for the analysis of HCV-specific CD8+ T lymphocytes we showed that these responses can be detected directly ex vivo in the majority of anti-HCV-positive individuals without requiring predictive algorithms or prior knowledge of HLA type. Using 301 overlapping peptides spanning all HCV proteins, we found circulating HCV-specific CD8+ T cells in 9 of 14 subjects tested, whereas only 4 of these HLA A2-positive persons had detectable HLA A2-restricted responses without prior in vitro stimulation. Importantly, half of the responses detected were directed against previously uncharacterized epitopes, and the vast majority of previously reported A2 epitopes were either not targeted or were below the limits of detection in persons with clear CD8+-T-cell responses to other epitopes. These data indicate that HLA type does not predict dominant HCV responses in seropositive individuals, that targeted epitopes are scattered throughout all gene products, and that no epitopes or regions are preferentially targeted by the majority of infected persons.
These studies extend previous studies analyzing the HCV-specific CTL response in PBMC that have usually relied on testing a limited number of previously described or predicted class I epitopes (2
). By relying on HLA A2-restricted epitopes, many individuals are excluded from study, especially those from non-Caucasian populations in whom HLA A2 is less frequently expressed (9
). Our study demonstrates that the analysis of responses restricted by a single HLA allele cannot be used as a surrogate marker for the overall HCV-specific CTL response. A response to any of the 19 previously described HLA A2-restricted HCV epitopes was seen in only 4 of 14 subjects, yet 9 of 14 had responses when the comprehensive approach with overlapping peptides was applied. Moreover, in HLA A2-positive persons with HLA A2-restricted responses, these were not always the dominant responses. In vitro simulation increased the detection of responses to some A2-restricted epitopes, indicating that weak responses were present but underscoring that these responses were minor compared to those directed at non-HLA A2 epitopes in a number of persons. This is consistent with studies in other chronic viral infections such as HIV, in which the expressed HLA alleles are poorly predictive of the targeted epitopes (3
). These findings will be particularly important to consider in studies designed to evaluate correlation of CD8 T-cell responses to other parameters such as liver histology and/or viral load, as assessment of these relationships will require comprehensive analyses such as those described here.
These studies also allowed us to assess the relative predictive ability of algorithms designed to predict CTL epitopes by using the sensitive techniques of Elispot and tetramer analysis. Our results show that a minority of predicted HLA A2-restricted epitopes are detected directly in PBMC. At the time of our experiments, 19 HLA A2-restricted HCV-specific epitopes had been described; only 3 of 19 (NS3 1073, NS3 1406, and NS5B 2594) were recognized by Elispot analysis in any of the patients, and these 3 were repeatedly detected in more than one person. We confirmed these findings by tetramer analysis to exclude the possibility of specific cells being present but undetected due to a defects in functional properties (13
). While frequencies of HCV-specific cells were indeed higher when analyzed by tetramer, the results obtained by Elispot assay correlated significantly in their relative frequency. More importantly, tetramer analysis did not reveal any response that was not also detected by the Elispot assay.
A review of the literature confirmed that the three HLA A2-restricted epitopes detected in our study are the HLA A2-restricted HCV-specific epitopes most commonly detected by all different assays available, be they direct ex vivo assays such as Elispot or tetramer or assays requiring previous specific or nonspecific expansion of cells in vitro (5
). For the six epitopes tested by tetramer, all had similar scores (between 23 and 30; mean, 24.4) in the algorithm described by Rammensee et al. (32
). These scores, ranging from 6 to 31 (mean, 24.4), were also not significantly different from the scores for the other epitopes tested in the Elispot assay. Furthermore, the three epitopes recognized have variable sequences, whereas the epitope in the core is highly conserved through all genotypes but was never detected directly ex vivo. In this context it is noteworthy that we detected responses in individuals infected with various genotypes, despite our peptides being derived from a genotype 1 sequence.
For one of the epitopes (NS3 1073) cross-reactivity with a flu epitope has been proposed as the mechanism leading to preferred recognition (40
). Further studies will be needed to establish whether this is a general phenomenon and also whether there is a difference in function and pathogenicity and in their possible role in vaccines between the epitopes which are more readily detectable and those requiring in vitro expansion.
Using the peptide matrix approach, we were able to detect circulating HCV-specific CTL responses in 6 of 10 individuals with chronic HCV infection, and this is comparable to results obtained by cloning from liver-derived lymphocytes where such a response was detected in 45% of subjects (41
). This suggests that, while the frequencies of HCV-specific cells have been shown to be higher in the liver, this assay is able to detect HCV-specific CTLs with similar sensitivity in the periphery without the need of in vitro expansion. This will facilitate future studies of HCV immunopathogenesis, since liver-derived cells are only occasionally available and the number of extracted cells is usually very low. However, more information is needed regarding how the cells in the two compartments compare in terms of phenotype and functional properties.
In conclusion, we show that the detailed analysis of the HCV-specific CD8+-T-cell response requires a comprehensive screening that cannot be substituted by testing a limited number of epitopes as indicators of the T-cell response. The study of HCV-specific CTLs directly ex vivo without the restriction to certain HLA alleles will be important in determining the role of CD8+ T cells in the control and immunopathology of HCV infection. Already the data indicate that the response is more limited than expected. This finding provides a rationale for future studies designed to measure the impact of augmented HCV-specific immunity on immune control and disease progression.