Volume 141, Issue 2, Pages 686-695.e1 (August 2011) Delayed Induction, Not Impaired Recruitment, of Specific CD8+ T Cells Causes the Late Onset of Acute Hepatitis C  Eui–Cheol Shin, Su–Hyung Park, Mary DeMino, Michelina Nascimbeni, Kathleen Mihalik, Marian Major, Naga S. Veerapu, Theo Heller, Stephen M. Feinstone, Charles M. Rice, Barbara Rehermann  Gastroenterology  Volume 141, Issue 2, Pages 686-695.e1 (August 2011) DOI: 10.1053/j.gastro.2011.05.006 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Specific chemokines increase in the circulation in the early stage of acute HCV infection. (A) Serum ALT levels (open squares) and serum HCV RNA titers (black diamonds) during the first 6 months after HCV infection have previously been reported for all chimpanzees except ChA3A0258,14,29 and are presented for ease of reference. Plasma (B) CXCL10, (C) CXCL11, (D) CCL4, and (E) CCL5 levels increase during acute HCV infection but (F) CXCL16 levels do not. NT, not tested. The number next to the asterisk specifies a value off scale. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Induction of chemokine mRNA in the liver correlates to the intrahepatic type I IFN response. (A–E) Serial liver biopsy specimens were analyzed for (A) CXCL10, (B) CXCL11, (C) CCL4, (D) CCL5, and (E) CXCL16. (F) 2,5-OAS-1 mRNA levels have previously been reported for all chimpanzees except ChA3A02514 and are presented for ease of reference. mRNA levels were normalized to endogenous references (glyceraldehyde-3-phosphate dehydrogenase and β-actin) and expressed as fold increase over preinfection levels. The number next to the asterisk specifies a value off scale. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Intrahepatic CXCL11, ICAM-1, and VCAM-1 expression during acute hepatitis C. Liver biopsy specimens taken at weeks 0 and 4 of HCV infection from chimpanzee A03A025 were stained for CXCL11, VCAM-1, and ICAM-1 (original magnification 200×). Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 HCV RNA induces selected chemokines in a type I IFN–dependent manner. Huh-7 hepatoma cells were H77 HCV RNA (black squares) or mock transfected (open triangles). (A) IFN-β mRNA was determined by real-time PCR. Secreted IFN-β was detectable by EIA in supernatants of HCV RNA–transfected but not mock-transfected Huh-7 cells (not shown). (B) The concentration of chemokines secreted by H77 HCV RNA (black squares) or mock-transfected (open triangles) Huh-7 hepatoma cells was determined by cytometric bead array or EIA. (C) Blocking of IFN-β with neutralizing antibodies decreased the level of secreted chemokines. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Intrahepatic increase in CXCR3 and CCR5 mRNA levels coincides with accumulation of lymphocytes in the liver and an increase in CD8β mRNA levels. Serial liver biopsy specimens were analyzed for (A) the number of LILs per centimeter biopsy specimen, (B) CD8β (black squares) and CD4 (open triangles), (C) CXCR3, and (D) CCR5 mRNA levels. CD8β mRNA levels have previously been reported for all chimpanzees except ChA3A02514 and are presented for ease of reference. NT, not tested. The number next to the asterisk specifies a value off scale. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 HCV-specific CD8 T cells are recruited to the liver as soon as they appear in the circulation. (A) Representative fluorescence-activated cell sorter dot plots show the percentage of tetramer-positive CD8 T cells. (B) Black bars represent percentage of tetramer-positive T cells in the CD8 T-cell population (mean and SD from 3 independent experiments). The gray line represents intrahepatic CD8β mRNA levels. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Figure 7 Model of the sequence of events in liver and blood culminating in the acute phase of hepatitis C. See text for details. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Representative fluorescence-activated cell sorter dot plot of tetramer-positive CD8 T cells of ChA03025. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 2 HCV-specific CD8 T cells express chemokine receptors after they appear in the circulation. (A) CXCR3 and CCR5 were preferentially expressed by tetramer-positive CD8 T cells compared with the total CD8 T-cell population. Representative results are shown for Ch6461 peripheral blood mononuclear cells at week 8 and Ch6411 peripheral blood mononuclear cells at week 13. Numbers represent the percentage of events in each quadrant. (B) The percentage of CXCR3+ and/or CCR5+ cells within the tetramer-positive T-cell population in the blood decreased after the initial peak. Gastroenterology 2011 141, 686-695.e1DOI: (10.1053/j.gastro.2011.05.006) Copyright © 2011 AGA Institute Terms and Conditions