(A) Short-term T cell line from donor 1144 was generated after restimulation with the HCV-NS3 peptide or the Flu–NA peptide. The lines were tested in an IFN-γ ELISPOT assay (A) and intracellular cytokine staining assay (B). Responses were similar in a line from donor 554.

Representative density plots of ex vivo pMHC staining (left), ex vivo MHC Class I pMHC and magnetic bead enrichment (middle), and in vitro peptide restimulation of PBMCs followed by pMHC staining (right). Top row shows an individual with positive Flu matrix responses, and the bottom row shows the equivalent result with Flu-NA. The frequency of the pMHC-positive cells in the CD8⁺ population is shown in the top right portion of each plot.

(A) A short-term line was established from donor 1144 and stained with CD8^hi pMHCs refolded with HCV-NS3 peptide (fluorochrome label APC) or Flu-NA (fluorochrome label PE) together with anti-CD8. Dual staining profile, gating on live CD8⁺ T cells, after costaining with both HCV and Flu-NA pMHCs. The Flu-NA pMHC-positive cells shown were all positive for HCV-NS3 tetramer staining (circled). (B) Ex vivo analyses. An identical staining protocol as for B was performed ex vivo on PBMCs from 4 donors. Only minimal Flu-NA staining is seen in each case (plots gated on CD8⁺ T cells).

(A) A short-term T cell line was stimulated from HCV⁺ donor 01-07 using the HCV-NS3 peptide (as described in Methods). The line was then stained with conventional or CD8^hi pMHCs refolded with HCV-NS3 peptide (top) or Flu-NA peptide (bottom) as indicated. The percentage of CD8⁺ T cells stained with the pMHCs is indicated in each case. (B) A short-term T cell line was stimulated from donor 1144 using the Flu-NA peptide. The line was then stained with conventional or CD8^hi pMHCs refolded with HCV-NS3 peptide (top) or Flu-NA peptide (bottom) as in A. The percentage of CD8⁺ T cells stained with the pMHCs is indicated in each case. The mean fluorescence intensities for the HCV tetramers are 1173 and 4464, and those for the Flu-NA tetramer are 500 and 1171. (C) Similar results for Flu-NA pMHC staining are shown for 2 of 4 donors tested (donor 949: top; donor 111: bottom) in lines restimulated initially with HCV-NS3.

(A) Examples of staining using a conventional pMHC (left) and a CD8^hi pMHC (right) ex vivo in the same individuals. Top: staining using pMHCs containing a CMV-derived peptide; bottom: pMHCs containing an EBV-derived peptide as described in Methods. (B) Example of staining of cells from a control subject (CMV^–) using the CMV CD8^hi pMHC. Similar results were obtained using the EBV pMHC. (C) Group data comparing the frequency of cells stained with the CMV and EBV tetramers (n = 40 and 24 individuals, respectively). The median values were not significantly different using a Wilcoxon paired test.

Long-term T cell lines were established from HCV⁺ donors (as indicated in Table 2). Staining for these lines was performed using normal pMHCs refolded around the HCV-NS3 peptide or the Flu-NA peptide. (A) Representative staining of lymphocytes showing CD8⁺ tetramer–positive populations. A population staining weakly with the Flu-NA pMHC is observed. (B) Gating on CD8⁺ lymphocytes, the Flu-NA pMHC-positive population lies among the HCV pMHC-positive cells, particularly evident among those cells with the highest level of staining for the HCV-NS3 pMHC. (C) Combined data from T cell lines from the cohort. Control lines were generated using the Flu matrix peptide and then tested with the identical peptide. Test lines were either stimulated with the HCV-NS3 peptide or the Flu-NA peptide and then analyzed with either pMHC. The percentage of CD8⁺ cells for each pMHC is indicated. Analysis was by Wilcoxon paired test.

(A) A short-term cell line was generated from donor 1144, as in Figure 5, by restimulation with HCV-NS3 and then stained using 3 different pMHC constructs (CD8^hi, normal, and CD8^lo) refolded around the Flu-NA peptide (top), HCV-NS3 peptide genotype 1 variant (middle), or genotype 4 variant (lower). (B) ELISPOT assay results from a T cell line derived from donor 1144 after incubation with HCV-NS3 genotype 1 peptide, genotype 4 peptide, or Flu-NA peptide at the concentrations shown. As previously observed, little response was seen against the Flu-NA peptide, but an enhanced response with the genotype 4 variant was observed, consistent with the enhanced binding to the CD8^lo construct. (C) pMHC staining of T cell lines from HCV⁺ donors specific for 2 separate epitopes. CD8^hi, normal, and CD8^lo pMHC constructs were refolded using HCV-NS3 as done previously and HCV A2-2594-2602 ALYDVVTKL derived from NS5b. Preserved staining of the HCV-NS5b line was observed using CD8^lo pMHCs, and staining was not enhanced with the CD8^hi construct, consistent with a population of largely high-affinity, CD8-independent T cells. These data indicate that the failure of the T cells specific to the HCV-NS3 genotype 1 epitope CINGSCWTV to bind CD8^lo pMHCs (as also seen in Figure 6A) is not universal in HCV and that the response to Flu-NA, which is only clearly seen with the CD8^hi construct, is atypical. We previously reported that the HCV-NS5b peptide has a 50% maximal stimulation concentration (SD₅₀) 2 logs lower than that of the NS3 epitope (21). This is consistent with peptide titration ELISPOT data from these lines, as illustrated.