Volume 134, Issue 7, Pages 1927-1937.e2 (June 2008) Functional Restoration of HCV-Specific CD8 T Cells by PD-1 Blockade Is Defined by PD-1 Expression and Compartmentalization  Nobuhiro Nakamoto, David E. Kaplan, Jennifer Coleclough, Yun Li, Mary E. Valiga, Mary Kaminski, Abraham Shaked, Kim Olthoff, Emma Gostick, David A. Price, Gordon J. Freeman, E. John Wherry, Kyong–Mi Chang  Gastroenterology  Volume 134, Issue 7, Pages 1927-1937.e2 (June 2008) DOI: 10.1053/j.gastro.2008.02.033 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Increased PD-1 expression in circulating HCV-specific CD8 T cells from viremic patients with acute and chronic but not resolved hepatitis C. (A) Percent PD-1 expression in CD8 and CD4 T cells in 10 acute (A), 27 chronic (C), and 8 resolved (R) patients with HCV infection and 12 healthy HCV-seronegative (H) controls. Median percent PD-1–positive/CD8 T cells: A 15.4% vs C 6.8% vs R 7.5% vs H 6.6% (P = .006). Median percent PD-1–positive/CD4 T cells: A 8.5% vs C 5.8% vs R 5.7% vs H 6.4% (P = .47). (B) Percent tetramer-positive CD8 T cells specific for HCV (circle), EBV (triangle), and Flu (diamond) in 7 acute, 19 chronic, 8 recovered, and 3 healthy patients. Median percent HCV-specific: A 0.00% vs C 0.00% vs R 0.08% (P = .018); median percent EBV or Flu-specific: A 0.11% vs C 0.10% vs R 0.04% vs H 0.08 (P = .68). (C) Representative PD-1 stainings for peripheral HCV- and Flu-specific tetramer-positive CD8 T cells from acute, chronic, and recovered patients. The top panel shows the PD-1 cutoff strategy with isotype control (dotted red line). (D) Percent PD-1–positive per tetramer-positive CD8 T cells (circle, NS3 1073; diamond, NS3 1406; triangle, NS5B 2594), EBV (filled triangle), and Flu (filled diamond) in 7 acute, 19 chronic, 8 resolved, and 3 healthy individuals. Median percent PD-1–positive HCV-specific CD8 T cells: A 87.4% vs C 26.7% vs R 5.1% (P < .0001); median percent PD-1–positive non–HCV-specific CD8 T cells: A 11.1% vs C 6.5% vs R 11.8% vs H 11.8% (P = .50). Red horizontal bars indicate the medians. P values were determined by the Kruskal–Wallis test. Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 HCV-specific CD8 T cells in patients with chronic HCV infection display impaired antigen-specific expansion and effector function in vitro. (A) HCV- and Flu-specific tetramer-positive CD8 T-cell frequency and expression of perforin and granzyme B on day 0 (open circle) and day 7 of culture (filled circle) with peptides (10 μg/mL) and 100 IU/mL rIL-2 for 15 chronic (C) and 6 recovered (R) patients. Percent Tet positive CD8 positive: median C 0.69% vs R 4.16% on day 7 (P = .003). Percent perforin positive/Tet positive CD8 positive: median C 49.1% vs R 92.3% on day 7 (P = .0006). Percent Granzyme B positive/Tet positive CD8 positive: median C 55.2% vs R 96.2% on day 7 (P = .007). Red horizontal bars indicate the median. (B) Percent CD107a positive and percent IFN-γ in HCV- and Flu-specific CD8 T cells on day 7. HCV-specific CD8 T cells, median percent CD107a positive (C 40.7% vs R 89.8%; P = .01); median percent IFN-γ (C 14.7% vs R 74.7%; P = .01). (C) Representative FACS plots comparing HCV-specific and Flu-specific tetramer-positive CD8 T-cell expansion and effector function in chronic (C75) and resolved (R23) patients on day 0 and after 7 days of antigenic stimulation. Events are gated on CD8-positive cells except for the far right intracellular staining gating on tetramer-positive CD8 T cells. (D) Inverse correlation between percent PD-1 expression ex vivo and antigen-specific IFN-γ, CD107a, and perforin expression on day 7 by HCV-specific CD8 T cells. P values were determined by Mann–Whitney U test or Spearman rank correlation test. Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 PD-1 expression is increased in HCV-specific CD8 T cells in the liver compared with peripheral blood. (A) Representative FACS plots for PBLs and LILs from HLA-A2–positive HCV-infected liver transplant recipients (T51 and T9) gating on CD8 T cells. The top histogram shows the PD-1 cutoff strategy with isotype control (dotted red line). (B) Frequency of tetramer-positive CD8 T cells specific for HCV (NS3 1073, NS3 1406, NS5B 2594), EBV, and Flu epitopes in 8 HLA-A2–positive patients. Median values are indicated by a red horizontal line. Median percent HCV-specific CD8 T cells: PBL 0.00% vs LIL 0.21%; P = .001. Median percent Flu- or EBV-specific CD8 T cells: PBL 0.02% vs LIL 0.16%; P = .06. (C) Percent PD-1 expression in CD8 and CD4 T cells in 16 HCV-infected patients: median percent PD-1 positive/CD8, PBL 6.1% vs LIL 17.1% (P = .0004); median percent PD-1 positive/CD4, PBL 5.4% vs LIL 21.9% (P = .001). (D) Percent PD-1 expression and (E) PD-1 MFI on CD8 T cells specific for HCV and non-HCV epitopes (EBV, triangle; Flu, diamond) from 8 HLA-A2–positive HCV-infected patients. HCV-specific CD8 T cells: median percent PD-1 positive, PBL 23.0% vs LIL 83.3% (P < .0001); median PD-1 MFI, PBL 281 vs LIL 1375 (P = .0001). Non–HCV-specific CD8 T cells: median percent PD-1 positive, PBL 16.1% vs LIL 22.5% (P = .28); median PD-1 MFI, PBL 356 vs LIL 406 (P = .16). P values were determined by paired t test or Mann–Whitney U test. Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Phenotypic characteristics of PD-1–positive CD8 T cells in peripheral blood and liver of patients with chronic HCV infection. (A) Representative FACS density plots comparing phenotypic markers for PD-1–negative and PD-1–positive CD8 T cells in PBLs and LILs from an HCV-infected patient. Numbers in each quadrant reflect percentage of gated PD-1–negative and PD-1–positive CD8 T cells. (B) Phenotype of PD-1–positive and PD-1–negative CD8 T cells in PBL (n = 24) and LIL (n = 14) shown as median percentages. P values were calculated by Mann–Whitney U test. Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Impaired expansion and effector function unresponsive to PD-1/PD-L blockade in highly PD-1–positive HCV-specific CD8 T cells in the liver of HCV-infected patients. (A) The frequency, fold expansion, and perforin expression of HCV-specific and Flu-specific tetramer-positive CD8 T cells on day 0 (white bars) and on day 7 following antigenic stimulation without (gray bars) or with anti–PD-L1 (black bars). *Concurrent blockade not done. All subjects were studied with NS3 1073 tetramer except T47 and T51, which displayed a dominant NS3 1406 response. (B) FACS plots showing HCV-specific and Flu-specific expansion and effector function in PBLs and LILs from patient T9. (C) HCV-specific T-cell IFN-γ response by IFN-γ ELISPOT in 6 HLA-A2–negative HCV-infected patients following stimulation with overlapping NS3 peptides with or without PD-1/PD-L blockade. Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 PD-1/PD-L blockade does not enhance expansion of highly PD-1–positive HCV-specific CD8 T cells in acute HCV infection. Expansion of HCV- and Flu-specific CD8 T cells in an acute hepatitis C patient during (A) the acute (week 1) and (B) resolved phase (week 80). Carboxyfluorescein succinimidyl ester–labeled peripheral blood mononuclear cells were stimulated with peptides (10 μg/mL) and rIL-2 (100 IU/mL) with/without anti–PD-L1 and/or anti–PD-L2 for 7 days. Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Inverse relationship between PD-1 expression and HCV-specific CD8 T-cell expansion with anti–PD-L1 blockade. (A) Comparison of HCV tetramer-positive CD8 T-cell expansion in vitro after 7 days of antigenic stimulation with anti–PD-L1 and ex vivo PD-1 expression directly (left) and in subgroups by percent PD-1 cutoff of 50% (right). (B) Correlation between the frequency and MFI for PD-1 expression on HCV-specific CD8 T cells with an exponential trend line (32 PBLs, unfilled triangles; 10 LILs, red filled triangles). Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 αPD-1/FITC (clone M1H4) defines a highly PD-1–positive CD8 T-cell subset in comparison to αPD-1/PE (clone EH12). (A) Upper histograms show staining characteristics for CD8 T cells stained with anti–PD-1/FITC (clone M1H4; green line) used in this study; relative isotype control stained lymphocytes (gray shaded area) in 4 HCV-seropositive subjects (patients 1–4). The cutoff for PD-1 positivity was determined based on isotype controls (>99.5%) for anti–PD-1/FITC. The lower histograms showing an overlay of gated PD-1/FITC-positive CD8 T cells onto CD8 T cells costained with anti–PD-1/PE (clone EH12; red shaded area) indicate that they correspond to PD-1 high cells. (B) Anti–PD-1 PE staining characteristics of gated HCV tetramer-positive CD8 T cells positive for anti–PD-1/FITC (red dots) are shown as an overlay (left graphs) using samples costained with anti–PD-1 FITC (M1H4), anti–PD-1 PE (EH12), CD8 PerCP, and tetramer allophycocyanin. The gated PD-1 FITC-positive tetramer-positive CD8 T cells (red dots the overlay) indicate that they are highly positive for PD-1 expression per anti–PD-1 PE (EH12 clone). The middle graph for each subject shows the anti–PD-1 FITC and tetramer staining for CD8 T cells with the gating strategy for PD-1–positive tetramer-positive cells (red square) in the overlay. The right graph for each subject shows the anti–PD-1 PE and tetramer staining for gated CD8 T cells. The bottom graphs shows the isotype staining and gating strategy to define PD-1 positivity (99.5% negative for isotype). Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 2 Additional allophycocyanin could not enhance the effect of PD-L1 blockade on intrahepatic HCV-specific T cells. A total of 20 million PBLs from an HLA-A2–positive transplant recipient (T65) were depleted of CD3-positive cells by Dynabeads (Dynal Inc, Carlsbad, CA). The resulting CD3-depleted antigen-presenting cells were added to PBLs or LILs being stimulated for 7 days in vitro with HCV peptides and rIL-2 with or without anti–PD-L1 as described in Subjects and Methods. For each stimulation condition (2 million PBLs or LILs per condition), 1 million CD3-depleted PBLs were added. On day 7, the frequency of HCV NS3 1073-specific CD8 T cells with perforin expression as well as the frequency of IFN-γ–positive CD8 T cells were examined by intracellular staining. Gastroenterology 2008 134, 1927-1937.e2DOI: (10.1053/j.gastro.2008.02.033) Copyright © 2008 AGA Institute Terms and Conditions