Volume 153, Issue 5, Pages 1392-1403.e2 (November 2017) Intra-Hepatic Depletion of Mucosal-Associated Invariant T Cells in Hepatitis C Virus- Induced Liver Inflammation  Fabian J. Bolte, Ashley C. O’Keefe, Lauren M. Webb, Elisavet Serti, Elenita Rivera, T. Jake Liang, Marc Ghany, Barbara Rehermann  Gastroenterology  Volume 153, Issue 5, Pages 1392-1403.e2 (November 2017) DOI: 10.1053/j.gastro.2017.07.043 Copyright © 2017 AGA Institute Terms and Conditions

Figure 1 MAIT cells are depleted in HCV-induced liver inflammation. (A) Representative flow cytometry plots for identification of MAIT cells (time gate and singlet gate are not shown). FSC-A, forward scatter area; SSC-A, side scatter area. (B) Frequency of MAIT cells in the blood of 37 HCV patients and 57 uninfected controls (left panel). Frequency of MAIT cells in the liver of 37 HCV patients and 10 uninfected controls (right panel). (C) Linear regression analysis (non-parametric Spearman correlation) of intrahepatic MAIT cell frequency and liver fibrosis (ISHAK) in chronic HCV infection. (D) Linear regression analysis (non-parametric Spearman correlation) of intrahepatic MAIT cell frequency and liver inflammation (histologic activity index) in chronic HCV infection. (E) Serum HCV RNA levels (n = 37) and liver biopsy HAI score (n = 28) of HCV-infected patients prior to and at week 4 of antiviral therapy. Liver biopsies of 11 patients were of insufficient size for staging and grading. (F) Frequency of MAIT cells in the liver of HCV-infected patients prior to and at week 4 of antiviral therapy compared with uninfected controls (n = 10). Frequency of MAIT cells in the blood of HCV-infected patients prior to, at week 4 of antiviral therapy, and at week 24 after the end of antiviral therapy (sustained virological response) compared with uninfected controls (n = 57). DAA, direct acting antivirals. Median and IQR are shown. Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions

Figure 2 MAIT cell activation and cytotoxicity decrease in blood and liver within 4 weeks of antiviral therapy for HCV infection. Frequency of CD69+ MAIT cells (A) and CD69 expression of MAIT cells (B) in blood and liver of HCV-infected patients (left panel). Effect of antiviral therapy on the frequency of CD69+ MAIT cells in blood (middle panel) and liver (right panel). Mean and SD (A, right panel) or median and IQR (all other panels) are shown. Frequency of HLA-DR+ MAIT cells (C) and HLA-DR expression of MAIT cells (D) in blood and liver of HCV-infected patients (left panel). Effect of antiviral therapy on the frequency of HLA-DR+ MAIT cells in blood (middle panel) and liver (right panel). Mean and SD (right panels) or median and IQR (left and middle panels) are shown. (E) Frequency of degranulated (CD107a+) MAIT cells in blood and liver of HCV-infected patients (left graph). Effect of antiviral therapy on CD107a MFI of MAIT cells in blood (middle graph) and liver (right panel). Median and IQR (left and middle panels) are shown. Outliers in panels A, B, and D do not affect statistical significance. MFI, mean fluorescence. Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions

Figure 3 The frequency of intermediate/pro-inflammatory monocytes correlates with the activation of MAIT cells. (A) Representative flow cytometry plots for identification of monocytes and their subsets: a, CD14++CD16- ‘classical’ monocytes; b, CD14++CD16+ ‘intermediate/pro-inflammatory’ monocytes; c, CD14+CD16++ ‘non-classical’ monocytes. SSC-A, side scatter area. (B) HLA-DR expression on blood and liver monocytes of HCV-infected patients. Median and IQR are shown. (C) Linear regression analysis (non-parametric Spearman correlation) of the frequency of CD14++CD16+ intermediate/pro-inflammatory monocytes and MAIT cell activation in the blood of HCV-infected patients. (D) Plasma IL-18 levels of HCV-infected patients prior to and at week 4 of antiviral therapy compared with uninfected controls. Mean and SD are shown. HLA-DR expression of total monocytes and monocyte subsets in blood (E) and liver (F) of HCV-infected patients prior to and at week 4 of antiviral therapy. Median and IQR (E) and mean and SD (F) are shown. Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions

Figure 4 Changes in MAIT cell and monocyte phenotype during antiviral therapy are greater in the liver than in the blood. Change in the frequency of HLA-DR+ MAIT cells (A) and the HLA-DR MFI (B) and CD107a MFI (C) of MAIT cells by week 4 of antiviral therapy in blood and liver. Change in HLA-DR MFI of total monocytes (D) and their CD14++CD16- ‘classical’ (E) and CD14++CD16+ ‘intermediate/pro-inflammatory’ subsets (F). Median and IQR are shown. Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions

Figure 5 Stimulation with IL-12/IL-18 or E coli activates CD8+ rather than CD4+ MAIT cells and results in a loss of CD8+ MAIT cells. (A) Representative flow cytometry plot for identification of CD8+ and CD4+ MAIT cells. (B) Frequency of CD8+, CD4+, double positive (DP), and double negative (DN) MAIT cells in the peripheral blood of 20 chronic HCV patients and 23 uninfected controls. Median and IQR are shown. *P ≤ .05 and **P < .01. Frequency of IFN-γ+ cells and IFN-γ MFI within the CD8+ and CD4+ MAIT cell populations after 18-hour stimulation of PBMC from chronic HCV patients with either IL-12/IL-18 (C) or E coli/IL-15 (D). Mean and SD (left panel in C and D) or median and IQR (right panel in C) are shown. (E) Change in the frequency of CD8+ and CD4+ MAIT cells after IL-12/IL-18 (left panel) or E coli/IL-15 (right panel) stimulation of PBMC from chronic HCV patients. (F) Comparison of the ex vivo frequency of CD8+ (left panel) and CD4+ MAIT cells (right panel) of 20 HCV patients before (Pre) and at week 24 after the end of antiviral therapy (Wk 24 post). Median and IQR are shown. DAA, direct acting antivirals; W/o, without. Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions

Figure 6 Stimulation with IL-12/IL-18 or E coli induces different cytokine profiles of MAIT cells. (A) Frequency of IFN-γ+ (left graph), TNF-α+ (middle graph) or IL-17+ (right graph) CD8+ MAIT cells after stimulation of PBMC from chronic HCV patients with either IL-12/IL-18 (18 hour), E coli/IL-15 (6 hour) or E coli/IL-15 (18 hour). (B) IL-18 concentration in supernatants of PBMC from chronic HCV patients after stimulation with E coli/IL-15 for 6 or 18 hours. (C) Frequency of IFN-γ+ (left graph) and IFN-γ+ TNF-α+ MAIT cells (right graph) and IFN-γ MFI of MAIT cells (middle graph) after 18-hour stimulation of PBMC with E coli/IL-15 in the presence or absence of IL-18 neutralizing antibodies. (D) IL-18 concentration in supernatants after 18-hour stimulation of PBMC from chronic HCV patients and uninfected controls with E coli/IL-15. Median and IQR are shown. Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions

Figure 7 IFN-γ production of MAIT cells in response to antigen but not cytokine stimulation is impaired in chronic HCV infection and does not change with antiviral therapy. (A) Frequency of IFN-γ+ and TNF-α+ CD8+ MAIT cells (left graph) of PBMC from HCV patients before (Pre) and at the end (Wk 12) of antiviral therapy and PBMC from uninfected controls after stimulation with IL-12/IL-18 (18 hour). (B) Frequency of IFN-γ+ and TNF-α+ CD8+ MAIT cells (left graph) of PBMC from HCV patients before (Pre) and at the end (Wk 12) of antiviral therapy and PBMC from uninfected controls after stimulation with E coli/IL-15 (6 hour). (C) IFN-γ and TNF-α MFI of MAIT cells in PBMC from HCV patients and PBMC from uninfected controls after stimulation with E coli/IL-15 (6 hour). (D) Linear regression analysis (non-parametric Spearman correlation) of FIB-4 index and the percentage of IFN-γ+ and TNF-α+ MAIT cells in response to stimulation with E coli/IL-15 (6 hour). Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions

Supplementary Figure 1 The decreased MAIT cell frequency is not accompanied by an increase of CD161-TCR-Vα7.2+ cells in blood and liver. (A) Frequency of CD161-TCR-Vα7.2+ cells in 37 paired blood and liver biopsies of HCV patients. (B) Linear regression analysis (non-parametric Spearman correlation) of the frequency of intrahepatic CD161-TCR-Vα7.2+ cells and liver inflammation (HAI) in chronic HCV infection. (C) Frequency of CD161-TCR-Vα7.2+ cells in blood and liver prior to and at week 4 of antiviral therapy. (D) HLA-DR MFI of CD161-TCR-Vα7.2+ cells in blood and liver of HCV-infected patients. (E) HLA-DR MFI of CD161-TCR-Vα7.2+ cells and MAIT cells in the liver of HCV-infected patients. (F) HLA-DR MFI of CD161-TCR-Vα7.2+ cells in blood and liver of HCV-infected patients prior to and at week 4 of antiviral therapy. Samples were excluded if the number of events in the CD161-TCR-Vα7.2+ gate were less than 41 events. Wk, week. Mean and SD (C, left graph) or median and IQR (all other graphs) are shown. Gastroenterology 2017 153, 1392-1403.e2DOI: (10.1053/j.gastro.2017.07.043) Copyright © 2017 AGA Institute Terms and Conditions