Volume 143, Issue 1, Pages 78-87.e3 (July 2012) Increased Levels of Arginase in Patients With Acute Hepatitis B Suppress Antiviral T Cells  Elena Sandalova, Diletta Laccabue, Carolina Boni, Tsunamasa Watanabe, Anthony Tan, Ho Zi Zong, Carlo Ferrari, Antonio Bertoletti  Gastroenterology  Volume 143, Issue 1, Pages 78-87.e3 (July 2012) DOI: 10.1053/j.gastro.2012.03.041 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Dysfunction of HBV-specific CD8+ T cells in AHB infection. (A) Discrepancy between activated/proliferating and functional CD8+ T cells. Bars represent the frequency of TNF-α/IL-2/IFN-γ–producing, degranulating (CD107a+), and CD38/HLA-DR+ CD8+ T cells in 6 patients with AHB ex vivo. (B) Direct ex vivo visualization of activation (CD38/HLA-DR+ and CD38/Ki-67+) and IFN-γ production of HBV pentamer–positive CD8+ T cells in representative patients at the onset and after resolution of acute HBV infection. PBMCs were stained with HBV core-18-27 pentamer and CD38, HLA-DR, and Ki67 or stimulated with c18-27 peptide (100 nmol/L) for 4 hours before ICS. Graphs show the frequencies of HBV pentamer–positive CD8+ T cells producing IFN-γ, TNF-α, or IL-2 or degranulating (CD107a+) after peptide stimulation in the indicated patients with AHB at the onset and resolution. (C) PBMCs of AHB were stimulated with pools of peptides covering the whole HBV proteome ex vivo or expanded in vitro for 10 days. The upper graph compares production of IFN-γ ex vivo and after in vitro expansion. The lower graph shows the number of responses that were found ex vivo and after in vitro expansion. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Analysis of functional recovery of HBV-specific CD8+ T cells. (A) Patients' PBMCs were stained with CD38 and HLA-DR or stimulated with known specific peptides in different conditions: (1) in medium alone; (2) with IL-15 alone or IL-15 and IL-2; (3) increasing the concentration of specific peptides; (4) with apoptosis inhibitors, anti-Bax peptide, and QVD alone or with IL-15; and (5) blocking PD-L1, CTLA-4, or both. The cells were incubated for 12 hours and then stimulated with specific peptides overnight in the presence of brefeldin A, and ICS was performed. The experiment was performed in 3 individual patients. (B and C) PBMCs of patients with AHB were stimulated with peptide ex vivo or after 2 days of in vitro culture in medium alone or with IL-2, IL-7, and IL-15 or in 50% patients' serum with IL-2, IL-7, and IL-15. Fluorescence-activated cell sorter plots show the IFN-γ production by HBV pentamer–positive CD8+ T cells ex vivo and after 2 days of culture. Graphs show IFN-γ production by HBV pentamer–positive cells (black bars) and the frequency of pentamer-positive cells (gray bars). Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Functional inhibition of heterologous CD8+ T cells during AHB. (A) Production of IFN-γ by EBV pentamer–positive CD8+ T cells during AHB. PBMCs were stained with EBV BZLF1190–197 and EBNA193–201 pentamers and stimulated with the relevant peptides. After 4 hours of incubation with brefeldin A, ICS was performed as described in Materials and Methods. The cells were gated on CD3+, CD8+, and EBV pentamer positive. Data of a representative patient at the onset, 10 days after onset, and at resolution of acute HBV infection are shown. (B) Altered IFN-γ production and CD107a+ expression on EBV, HCMV, and influenza pentamer–positive CD8+ T cells in 5 patients with AHB over time. (C) Frequencies of CMV (n = 4) or influenza (n = 3) pentamer-positive CD8+ T cells and IFN-γ–positive pentamer-positive cells in patients with AHB compared with healthy controls (n = 3) ex vivo and after 2 days in culture with IL-2, IL-7, and IL-15. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Sera of patients with acute hepatitis inhibit CD8+ T cell function. (A) IL-10 and (B) arginase were measured in patients' serum (n = 9; n = 12) at the onset of acute hepatitis and at the resolution. (C) Correlation of serum levels of arginase I and IL-10 with ALT. Linear regression analysis of serum arginase I levels (left panel) and IL-10 levels (right panel) against ALT from patients with acute HBV (n = 12) was performed. A P value of <.05 indicates a gradient with significant deviation from 0. (D) Effect of the serum of AHB on T-cell responses to HCMV. PBMCs of healthy individuals with a defined CMV response were cultured for 48 hours in presence of IL-15 (10 ng/mL) and 50% of the serum of HBV-positive patients or patients with drug-induced hepatitis at onset or resolution (n = 4); alternatively, healthy AB serum or no serum was used. (Acute onset sera ALT levels ranged from 3500 to 2600 U/L, arginase from 220 to 160 pg/mL, and IL-10 from 8 to 6 pg/mL.) After incubation, ICS was performed for IFN-γ after 5 hours of stimulation with specific peptide. Percent inhibition by serum was calculated based on the percent of IFN-γ–positive CMV-specific CD8+ T cells. The number of patients' sera tested is indicated. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Arginase, but not IL-10, inhibits CD8+ T cell function. (A) HBV core18-27 specific CD8+ T cell clone was incubated with arginase or IL-10 at indicated concentration for 2 days, and killing assay and intracellular cytokine staining for IFN-γ was performed with GFP-expressing HepG2 cells as targets. The plots show percent inhibition of cytotoxicity or IFN-γ production (measured as mean fluorescent intensity) compared with that of untreated T cells. (B) Healthy PBMCs were incubated either with IL-10 (10 pg/mL) or arginase (250 pg/mL) with or without IL-15 for 48 hours and specific IFN-γ production was evaluated. The cells were gated on an HCMV pentamer–positive population (B, left plot). The level of activation of HCMV-specific T cells is shown based on HLA-DR/CD38+, the histogram plots show the levels of IFN-γ production by HCMV-specific T cells, and the bar charts indicate the percent inhibition of IFN-γ production. Experiments were performed in 2 individuals 3 times. (C) Arginase inhibitor reverted the suppressive effect of the sera from patients with AHB. HBV pentamer–positive CD8+ T cells present in patients with AHB were stimulated with HBV peptide after 2 days of culture with IL-2, IL-7, and IL-15 with or without patients' serum (ALT 3100 U/L, arginase 210 pg/mL, IL-10 7 pg/mL), arginase inhibitor (nor-NOHA), or anti–IL-10 antibody. The percent inhibition of IFN-γ production by HBV-specific CD8+ T cells compared with culture without serum is shown. (D) Addition of l-arginine reverted the suppressive effect of the sera from patients with AHB. A total of 0.3 μmol/L arginine was added to PBMCs cultured in the presence of sera from patients with AHB (ALT 3100 U/L, arginase 210 pg/mL). Here, the arginine level was adjusted according to the arginine concentration in culture medium (AIM-V), used to restore T-cell responses. HCMV-specific IFN-γ response was evaluated after 5 hours of activation. Percent inhibition is shown. Experiments were performed in 2 individuals 3 times. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Regulation of immune T-cell response during AHB infection. Schematic representation of different mechanisms of T-cell functional modulation potentially active during AHB. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 (A) Dysfunction of HBV-specific CD8+ T cells in AHB infection. PBMCs of patients with AHB were stimulated with pools of peptides covering the whole HBV proteome, or with staphylococcal enterotoxin B, and the cells were incubated for 12 hours for enzyme-linked immunospot assay. Results of an enzyme-linked immunospot assay for 3 representative acute patients are displayed. (B) The frequencies of IFN-γ pentamer–positive CD8+ T cells are plotted together with total HBV core 18-27 pentamer–positive cells in 2 representative patients over time. Corresponding ALT values are shown below each graph. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Healthy PBMCs were incubated with either IL-10 (10 pg/mL) or arginase (250 ng/mL) with or without IL-15 for 48 hours, and specific CD107a up-regulation was evaluated. The cells were gated on an EBV or influenza pentamer–positive population. The bar charts indicate the percent inhibition of degranulation (CD107a up-regulation). Experiments were performed in 2 individuals 3 times. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Arginase, but not IL-10, inhibits EBV- and influenza-specific responses. Healthy PBMCs were incubated with either IL-10 (10 pg/mL) or arginase (250 ng/mL) with or without IL-15 for 48 hours, and specific IFN-γ production was evaluated. The cells were gated on an EBV or influenza pentamer–positive population. The bar charts indicate the percent inhibition of IFN-γ production. Experiments were performed in 2 individuals 3 times. Gastroenterology 2012 143, 78-87.e3DOI: (10.1053/j.gastro.2012.03.041) Copyright © 2012 AGA Institute Terms and Conditions