Volume 115, Issue 4, Pages 954-966 (October 1998) Hepatitis C virus variants circumventing cytotoxic T lymphocyte activity as a mechanism of chronicity  Sun-Lung Tsai, Young-Mao Chen, Ming-Huei Chen, Chao-Yuan Huang, I-Shyan Sheen, Chau-Ting Yeh, Jyh-Hsiung Huang, George C. Kuo, Yun-Fan Liaw  Gastroenterology  Volume 115, Issue 4, Pages 954-966 (October 1998) DOI: 10.1016/S0016-5085(98)70268-9 Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 1 Clinical courses and timings for PBMC collection of 5 HLA-A2–positive patients and 3 A2-negative control subjects with acute hepatitis C. The patients and controls had been monitored since peak alanine aminotransferase (ALT) levels were detected. ■, Serum HCV positive; 2, serum HCV RNA negative; ●, seropositive for anti-HCV; ↓, PBMCs used to generate HVR1-specific CTLs; *, liver biopsy. Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 2 Amino acid (398-407) sequences of HVR1 deduced from complementary DNA sequences of virus isolates obtained at different time points of acute-phase serum samples. A comparison of HVR1 sequences with Taiwanese (T), Japanese (J), HCV-1, and RH/H virus isolates is shown at the bottom of figure. *Percentage of 10 independent clones of each serum sample. Single-letter codes for amino acid residues are used. Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 3 Induction of HVR1-specific CTL lines by PBMC stimulation with synthetic decapeptides. CTL activities of PBMCs of HCV1 stimulated by peptide mixture 2 and expanded with IL-2 for 2 weeks were detected against autologous cold-target cells pulsed with decapeptides. The amino acid sequences of these decapeptides are listed in Table 2, and the stimulation method is described in the text. Data are expressed as means of triplicate determinations. ▨, A2-positive B-LCL+ peptide. Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 4 Limiting dilution analysis of HVR1 epitope-specific CTLp. Quantitative analysis of HVR1 epitope (amino acids 398-407)-specific CTLp was assayed in acute-phase PBMCs in patients HCV1 to HCV4 and 1 healthy individual. Higher HVR1 epitope-specific CTLp in patients in recovery was found. Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 5 CTL lines induced by HVR1 decapeptide efficiently lyse target cells expressing endogenously synthesized antigens. Representative CTL lines HCV1-3L1, HCV2-7B7, HCV3-5D2, HCV4-2A1, and HCV1-6E6 generated from PBMCs or liver biopsy samples from patients with acute hepatitis C were stimulated in vitro by HVR1-derived decapeptides. These CTL lines can induce lysis of target cells expressing HCV antigens. They can also lyse autologous cold-target cells preincubated with decapeptides (HVR1 398-407) of the first HCV isolate from each patient. Target cells: ○, HCV-transfected B-LCL cells; ●, pCMV-transfected B-LCL cells; ▵, DP (HVR1 389-407) + autologous B-LCL cells (cold targets); ▴, medium + autologous B-LCL cells (cold targets). Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 6 A shared usage of TCR AV and BV genes in HVR1-specific, HLA-A2–restricted CTL clones. (A) TCR AV usage; (B) TCR BV usage. The junctional region (Nα-Jα or N1-BD-N2) sequences (= CDR3 region) are also shown. V, variable; N, nontemplated; D, diversity; J, joining. The single-letter code for amino acid residues is used. *Vα6.1 by previous designation; likewise, Vβ7.1 for BV17S1A1T. ‡AJ and BJ gene usage were assigned according to those previously described.32,33 Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 7 Map of MHC restriction elements. Autologous and allogeneic B-LCL cells (H1-H6) sharing class I MHC alleles were used as target cells in the cytotoxicity assay. The percentage of specific lysis was assayed at an E/T ratio of 20 and a decapeptide concentration of 1 μg/mL. Rat anti–HLA-I (IgG2a) and anti–HLA-II (IgG2a) monoclonal antibodies (both from Serotec, Oxford, England) were used for the blocking assay at a final concentration of 10 μg/mL in the culture. Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 8 Naturally occurring HVR1 variant-derived decapeptides antagonize CTL activity. The cytotoxic activity of prototype decapeptide epitope-specific CTL clones HCV1-3L1, HCV1-6E6, and HCV3-5D2 could be inhibited by mutant decapeptides. Mutant decapeptide 1 is derived from the second HCV isolate of HCV1, and mutant decapeptide 3 is derived from the second HCV isolate of HCV3. The irrelevant HLA-A2 competitor peptide hepatitis B virus core antigen 18-27 is a well-defined decapeptide recognized by HLA-A2–restricted CTLs in HBV-infected patients.39 No significant inhibition was induced by this A2-competitor. The assay was performed at an E/T ratio of 10:1. Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions

Fig. 9 Titration of naturally occurring HVR1 variant-derived decapeptides. The cytotoxic activity of prototype decapeptide epitope (HVR1 398-407)-specific CTL clones could be inhibited by mutant decapeptides. (A and C) Inhibition of lysis of decapeptide-pulsed target cells by mutant decapeptides at an antagonist/agonist ratio of 1:1. (B and D) Significant inhibition of lysis of decapeptide-pulsed target cells by mutant decapeptides can be detected at antagonist/agonist ratios ranging from 0.1:1 to 10:1. The E/T ratios for these assays are 5:1 for A and C and 10:1 for B and D. Gastroenterology 1998 115, 954-966DOI: (10.1016/S0016-5085(98)70268-9) Copyright © 1998 American Gastroenterological Association Terms and Conditions