Volume 135, Issue 4, Pages 1333-1343 (October 2008) Fatal Autoimmune Hepatitis Induced by Concurrent Loss of Naturally Arising Regulatory T Cells and PD-1-Mediated Signaling  Masahiro Kido, Norihiko Watanabe, Taku Okazaki, Takuji Akamatsu, Junya Tanaka, Kazuyuki Saga, Akiyoshi Nishio, Tasuku Honjo, Tsutomu Chiba  Gastroenterology  Volume 135, Issue 4, Pages 1333-1343 (October 2008) DOI: 10.1053/j.gastro.2008.06.042 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 CD4+ T-cell phenotypes in normal BALB/c or PD-1-deficient mice with or without neonatal thymectomy (NTx). (A, upper panels) The cells were isolated from spleen of 3-week-old indicated mice, stimulated with anti-CD3 for 48 hours, and stained with FITC-anti-CD4 and PE-anti-PD-1. Shaded histograms represent staining of anti-PD-1 in CD4+ T cells; open histograms represent the isotype control. Numbers indicate the mean fluorescence intensity of PD-1. (A, lower panels) The cells were isolated from spleen and stained with FITC-anti-CD4 and biotinylated-anti-Foxp3 followed by PE-streptavidin. Percentages of CD4+Foxp3+ cells and CD4+Foxp3− cells in a viable lymphocyte gate are shown. (B and C) Numbers of Treg or non-Treg cells were calculated by (percentage of the cells in viable cells) × (number of viable cells). Error bars represent SD. (n = 4, *P < .05). (D) Dot plot and mean intensity of forward scatter (FSC) and side scatter (SSC) of CD4+ T cells. Error bars represent SD (n = 3, *P < .05). Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Histologic analysis of various organs from normal BALB/c or PD-1-deficient mice with or without neonatal thymectomy. The sections of various tissues from 3-week-old indicated mice were fixed in formalin and stained with H&E. All scale bars, 100 μm. Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Fatal autoimmune hepatitis in NTx-PD-1−/− mice. (A) Survival of PD-1-deficient mice that received neonatal thymectomy (NTx; n = 24) or sham operation (sham, n = 10). (B and C) Macroscopic view and histologic findings of the liver and spleen from 3-week-old NTx-PD-1−/− mice. (C, i and ii) Whole liver shows massive degeneration of hepatocytes and severe mononuclear cell infiltration. (C, iii and iv) Mononuclear cell infiltration is found in the portal area and parenchyma without bile duct destruction (white arrowheads). (C, v) Although some clusters of hepatocytes (black arrowheads) remain normal, most of the hepatocytes exhibit vacuolar and vesicular cytoplasmic changes; the remaining hepatocytes show reduction of cytoplasm and nuclear condensation. (C, vi) TUNEL staining for these cells is positive. All scale bars, 100 μm. (D) Serum levels of the liver transaminase, aspartate aminotransferase (AST) and alanine aminotransferase (ALT), and total bilirubin. Sera from indicated mice are measured at the indicated time points. Data are shown as mean of at least 3 mice. Error bars represent SD. Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Progression of organ-specific autoimmunity and autoAb production. (A) Histologic findings of the liver and the stomach in NTx-PD-1−/− mice at indicated age in weeks. Arrowheads show a few foci of remaining hematopoietic cells because of residual hematopoiesis in the embryonic stage. All scale bars, 100 μm. (B) AutoAbs detected by fluorescence immunohistology. Indicated tissues of normal BALB/c mice were stained with ×100 diluted sera from indicated mice at 3 weeks of age, followed by FITC-anti-mouse IgG. All scale bars, 100 μm. The inset shows nuclei of hepatocytes with a higher magnification. (C) Serum titers of ANAs in the indicated mice at 3 weeks of age. Open circles indicate the maximum dilution of sera from individual mouse detected by fluorescence immunohistology. (D) Serum titer of ANAs in a panel of normal mouse indicated tissues. Results from different tissues in individual mice are connected by lines. Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Characterization of mononuclear cell infiltration in fatal AIH of NTx-PD-1−/− mice. (A) Cell numbers of each subset of liver mononuclear cells in the indicated mice at 3 weeks of age. Data represent the cell number of CD11b+CD11c− macrophages, CD11c+ dendritic cells (DCs), CD11b+Gr-1+ myeloid cells, CD3−DX5+ natural killer cells (NK cells), CD3+DX5+ natural killer T cells (NKT cells), CD3+DX5− T cells, and B220+ B cells in 1 of 3 separate experiments. (B) CD4+ and CD8+ T cells in CD3-gated liver mononuclear cells of the indicated mice. Numbers in quadrants in upper panels indicate percent cells in that gate. Open and solid bars in the lower panel indicate cell numbers of CD4+ and CD8+ T cells, respectively. Data shown are 1 of 3 separate experiments. (C) Fluorescence immunohistology of the hepatitis in NTx-PD-1−/− mice. Serial sections of the liver of NTx-PD-1−/− mice were stained with FITC-anti-CD4 and -anti-CD8. All scale bars, 100 μm. Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 Characterization of infiltrating T cells and cytokine production in fatal AIH. (A) The cells were isolated from the spleen and liver of the indicated mice at 3 weeks of age and stained with Abs against indicated cell surface markers and Ki-67, a nuclear antigen associated with cell proliferation. Phenotypes of CD4+ and CD8+ T cells were determined by flow cytometry. Shaded histograms represent staining of CD4+ and CD8+ T cells; open histograms represent the isotype controls. Numbers indicate percent cells in the gates. Data represent 1 of 3 separate experiments. (B and C) The percentages of CD62L or Ki-67 positive cells in CD4+ and CD8+ T cells (*P < .05, **P < .01). (D) CCR5 and CCR6 expression of CD4+ and CD8+ T cells in the spleen and liver from NTx-PD-1−/− mice. Data represent 1 of 3 separate experiments. (E) Intracellular cytokine staining of CD3+CD8− T cells and CD3+CD8+ T cells in the spleen and liver of the indicated mice at 3 weeks of age. Numbers in quadrants indicate percent cells in that gate. Data represent 1 of 3 separate experiments. (F) Serum IFN-γ and TNF-α levels of indicated mice at 3 weeks were measured by ELISA. Data are shown as mean of at least 6 mice. Error bars represent SD. Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Transfer of splenocytes of NTx-PD-1−/− mice into RAG2-deficient recipient mice. (A) Total, CD4+ cell-depleted, or CD8+ cell-depleted splenocytes (3 × 107 cells) of 3-week-old NTx-PD-1−/− mice were transferred into RAG2-deficient recipient mice. Data shown are anti-CD4, anti-CD8, TUNEL, and H & E (HE) staining. All scale bars, 100 μm. (B) Serum levels of the liver ALT, (C) the numbers of TUNEL-positive hepatocytes, and (D) gastritis score of recipient mice transferred with indicated donor cells. Data are shown as mean of 3 mice. Error bars represent SD. Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Suppression of AIH by transfer of Treg cells from normal BALB/c or PD-1-deficient mice. (A) Treg cells (1 × 106) isolated from splenocytes of either adult normal BALB/c or PD-1-deficient mice were transferred into NTx-PD-1−/− mice at 4 days after thymectomy. (B) Survival of NTx-PD-1−/− mice transferred with Treg cells (1 × 106) isolated from splenocytes of adult normal BALB/c mice at 0 (T = 0), 1 (T = 1), 2 (T = 2), and 3 (T = 3) weeks after NTx (each group, n = 5 or 6). (C) Total splenocytes (1 × 106) of 3-week-old NTx-PD-1−/− mice were transferred into RAG2-deficient mice with or without the same number of Treg cells from either adult normal BALB/c or PD-1-deficient mice. Two (A) and 3 weeks (C) after the transfer, mice were killed, and sections of the liver were fixed in formalin and stained with H&E. All scale bars, 100 μm. Gastroenterology 2008 135, 1333-1343DOI: (10.1053/j.gastro.2008.06.042) Copyright © 2008 AGA Institute Terms and Conditions