Comprehensive analysis of CD8(+)-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities

Abstract

The hepatitis C virus (HCV)-specific CD8(+)-T-cell response is thought to play a critical role in HCV infection. Studies of these responses have largely relied on the analysis of a small number of previously described or predicted HCV epitopes, mostly restricted by HLA A2. In order to determine the actual breadth and magnitude of CD8(+)-T-cell responses in the context of diverse HLA class I alleles, we performed a comprehensive analysis of responses to all expressed HCV proteins. By using a panel of 301 overlapping peptides, we analyzed peripheral blood mononuclear cells (PBMC) from a cohort of 14 anti-HCV-positive, HLA A2-positive individuals in an enzyme-linked immunospot assay. Only four subjects had detectable HLA A2-restricted responses in PBMC, and only 3 of 19 predicted A2 epitopes were targeted, all of which were confirmed by tetramer analysis. In contrast, 9 of 14 persons showed responses with more comprehensive analyses, with many responses directed against previously unreported epitopes. These results indicate that circulating HCV-specific CD8(+)-T-cell responses can be detected in PBMC in the majority of infected persons and that these responses are heterogeneous with no immunodominant epitopes consistently recognized. Since responses to epitopes restricted by single HLA alleles such as HLA A2 do not predict the overall response in an individual, more comprehensive approaches, as shown here, should facilitate definition of the role of the CD8(+)-T-cell response in HCV infection. Moreover, the low level or absence of responses to many predicted epitopes provides a rationale for immunotherapeutic interventions to broaden cytotoxic-T-lymphocyte recognition.

FIG.1.

Analysis of the HCV-specific CD8⁺ response against HLA A2 epitopes. (A) Magnitude and breadth of the HCV-specific CD8⁺-T-cell responses as detected with 19 different previously described HLA A2-restricted peptides in an Elispot assay. Individual epitopes tested are indicated by the numbering of the first amino acids in the HCV 1a sequence, and magnitudes are shown as IFN-γ SFC/10⁶ PBMC. Responses were detected only against three of the 19 peptides tested. (B and C) These results were confirmed by tetramer analysis as shown for subject P6 (B), and the results from the Elispot and tetramer assays correlated significantly (C).

FIG.1.

Analysis of the HCV-specific CD8⁺ response against HLA A2 epitopes. (A) Magnitude and breadth of the HCV-specific CD8⁺-T-cell responses as detected with 19 different previously described HLA A2-restricted peptides in an Elispot assay. Individual epitopes tested are indicated by the numbering of the first amino acids in the HCV 1a sequence, and magnitudes are shown as IFN-γ SFC/10⁶ PBMC. Responses were detected only against three of the 19 peptides tested. (B and C) These results were confirmed by tetramer analysis as shown for subject P6 (B), and the results from the Elispot and tetramer assays correlated significantly (C).

FIG.1.

Analysis of the HCV-specific CD8⁺ response against HLA A2 epitopes. (A) Magnitude and breadth of the HCV-specific CD8⁺-T-cell responses as detected with 19 different previously described HLA A2-restricted peptides in an Elispot assay. Individual epitopes tested are indicated by the numbering of the first amino acids in the HCV 1a sequence, and magnitudes are shown as IFN-γ SFC/10⁶ PBMC. Responses were detected only against three of the 19 peptides tested. (B and C) These results were confirmed by tetramer analysis as shown for subject P6 (B), and the results from the Elispot and tetramer assays correlated significantly (C).

FIG. 2.

A screening Elispot with overlapping 20mer peptides can detect HCV-specific epitopes. We first tested the screening Elispot assay in an individual who had been studied previously by limiting dilution cloning. During acute infection eight different HCV-specific epitopes had been defined from that patient (29). Responses to seven of the eight epitopes were still detectable 2 years after the individual had cleared HCV infection (black bars). When compared to responses to the corresponding 20mer peptides used in the screening Elispot assay (gray bars), all seven responses were also detected. However, some of the 20mer peptides elicited responses that were almost as strong as the optimal epitope, whereas other responses were less vigorous. In addition to the previously described epitopes, a response to an additional peptide was identified (p951-970, striped bar). At the time point tested, this peptide elicited the strongest response of all recognized peptides.

FIG. 3.

Peptides detected in the screening Elispot represent CTL epitopes. (A) That stimulation with peptide 951-970 was inducing IFN-γ secretion by CD8⁺ T cells was shown by ICS with PBMC. (B) Use of a peptide-specific T-cell line and partially HLA-matched B-cell lines in the ICS demonstrated that the peptide was presented by HLA B37. (C) The same T-cell line was used to define the minimal epitope in an Elispot with serial dilutions of peptide truncations of the original peptide. (D) The T-cell line specific for peptide 951-970 was also capable of mediating specific cytotoxic activity against autologous peptide pulsed B cells.

FIG. 3.

Peptides detected in the screening Elispot represent CTL epitopes. (A) That stimulation with peptide 951-970 was inducing IFN-γ secretion by CD8⁺ T cells was shown by ICS with PBMC. (B) Use of a peptide-specific T-cell line and partially HLA-matched B-cell lines in the ICS demonstrated that the peptide was presented by HLA B37. (C) The same T-cell line was used to define the minimal epitope in an Elispot with serial dilutions of peptide truncations of the original peptide. (D) The T-cell line specific for peptide 951-970 was also capable of mediating specific cytotoxic activity against autologous peptide pulsed B cells.

FIG. 3.

Peptides detected in the screening Elispot represent CTL epitopes. (A) That stimulation with peptide 951-970 was inducing IFN-γ secretion by CD8⁺ T cells was shown by ICS with PBMC. (B) Use of a peptide-specific T-cell line and partially HLA-matched B-cell lines in the ICS demonstrated that the peptide was presented by HLA B37. (C) The same T-cell line was used to define the minimal epitope in an Elispot with serial dilutions of peptide truncations of the original peptide. (D) The T-cell line specific for peptide 951-970 was also capable of mediating specific cytotoxic activity against autologous peptide pulsed B cells.

FIG. 3.

Peptides detected in the screening Elispot represent CTL epitopes. (A) That stimulation with peptide 951-970 was inducing IFN-γ secretion by CD8⁺ T cells was shown by ICS with PBMC. (B) Use of a peptide-specific T-cell line and partially HLA-matched B-cell lines in the ICS demonstrated that the peptide was presented by HLA B37. (C) The same T-cell line was used to define the minimal epitope in an Elispot with serial dilutions of peptide truncations of the original peptide. (D) The T-cell line specific for peptide 951-970 was also capable of mediating specific cytotoxic activity against autologous peptide pulsed B cells.

FIG. 4.

Comprehensive analysis of the HCV-specific CD8⁺-T-cell response. Elispot responses to previously described HLA A2-restricted epitopes are shown as white bars; Elispot responses to previously described non-HLA A2-restricted epitopes are shown as black bars, and novel responses are indicated as gray bars.

FIG. 5.

Tetramer analysis before and after stimulation of PBMC with peptides representing HCV epitopes. In subject P14, only one of four tetramers (NS3 1073) tested positive directly ex vivo. After 9 days of stimulation with the four respective peptides, three of the four tetramers tested positive, with frequencies of up to 30% of the CD8⁺ T cells.