1. Effects and Regulation of Autoreactive CD8+ T Cells in a Transgenic Mouse Model of Autoimmune Hepatitis 
Mario Zierden, Elisabeth Kühnen, Margarete Odenthal, Hans-Peter Dienes 
Gastroenterology 
Volume 139, Issue 3, Pages e3 (September 2010)
DOI: /j.gastro
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2. Figure 1
Liver-specific hemagglutinin expression in Alb-HA transgenic mice leads to priming of adoptively transferred autoreactive CD8+ T cells eliciting transient hepatitis. (A) Transgenic Alb-HA expression vector. (B) HA and β-actin messenger RNA (mRNA) expression analysis by RT-PCR of different organs from Alb-HA and WT mice. Negative controls were performed by omitting mRNA (−). Eight × 106 CFSE-labeled CL4-TCR cells were injected into Alb-HA or WT mice either (C) 3 days before analysis of their proliferation and activation marker expression or (D) at day 0, and ALT levels were determined at indicated days. Data represent mean values (± standard deviation) of 3 experiments (n ≥ 6 per group). (E) At day 5 after transfer of 8 × 106 CL4-TCR cells, liver sections were obtained from Alb-HA or WT recipients and stained by H&E or for CD8+ T cells (brown).
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3. Figure 2
Efficient thymic generation of HA-specific CD8+ T cells is a prerequisite for the development of spontaneous chronic hepatitis in male Alb-HA/CL4-TCR mice. (A) Thymic T-cell development in Alb-HA/CL4-TCR and CL4-TCR mice. Gated CD4−CD8+ thymocytes were analyzed for transgenic TCRβ chain (Vβ8) expression and HA specificity using an H-2Kd:HA512–520 pentamer (H-2Kd:HA). (B) Vβ8 expression and affinity for the H-2Kd:HA512–520 pentamer of gated HA-specific CD4−CD8+ thymocytes from indicated mice. A and B represent mean values of 3 experiments (n = 9 per group). (C) ALT levels (mean ± standard deviation) of male and female Alb-HA/CL4-TCR and CL4-TCR mice (n = 6–27 per group) determined weekly at indicated age. Values depicted for CL4-TCR mice represent both genders.
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4. Figure 3
Male Alb-HA/CL4-TCR mice suffer from chronic liver inflammation. Liver sections obtained from 8-week-old male Alb-HA/CL4-TCR and CL4-TCR mice were stained with H&E, for CD8+ T cells (brown), or for collagen fibers (red) by van Gieson staining.
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5. Figure 4
Phenotype of splenic and liver-infiltrating CD8+ T cells from male Alb-HA/CL4-TCR mice. (A) CD8+ T cells from indicated organs of Alb-HA/CL4-TCR and CL4-TCR mice were analyzed for transgenic TCRβ chain (Vβ8) expression and HA specificity (H-2Kd:HA). Histograms show Vβ8 expression on gated HA-specific CD8+ T cells and activation marker expression on gated CD8+Vβ8+ T cells. (B) Forward scatter profiles of gated HA-specific and HA-nonspecific CD8+Vβ8+ T cells from different organs of indicated mice. A and B represent average values of 3 experiments (n = 9 per group). (C) Total numbers of CD8+Vβ8+ and CD4+CD3+ T cells (mean ± standard deviation) in different organs of indicated mice calculated from 5 experiments (n = 15 per group) (*P < .05; ***P < .0001).
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6. Figure 5
Liver-infiltrating HA-specific CD8+ T cells of male Alb-HA/CL4-TCR mice possess impaired effector functions. (A) Identical numbers of HA-specific CFSE-labeled CD8+ T cells from indicated organs of Alb-HA/CL4-TCR mice and of splenic CL4-TCR cells were cultured with APCs in the presence of HA512–520 peptide for 4 days. Histograms show average proliferation of CD8+CFSE+ T cells from 2 experiments performed in triplicate. (B) CD8+ T cells from indicated organs of Alb-HA/CL4-TCR and CL4-TCR mice were analyzed for intracellular IFN-γ expression. Gated CD8+CD90.1+ T cells from Ad-HA-infected Alb-HA recipients at day 4 after transfer of CL4-TCR cells were used as positive controls for IFN-γ expression. (C) In vivo CTL assay in Alb-HA/CL4-TCR mice. Equal numbers of CFSEhigh HA512–520 peptide-pulsed and CFSElow unpulsed splenocytes from BALB/c mice were injected into indicated mice. After 6 hours, CFSE+propidium iodide− cells from different organs of mice were analyzed for the ratio (r) of unspecific CFSElow vs specific CFSEhigh target cells. Ad-HA-infected Alb-HA recipients served as positive controls for cytotoxicity at day 5 after transfer of CL4-TCR cells. HA-specific cytolysis was calculated as described in Supplementary Materials and Methods and depicted as mean ± standard deviation (right panel). (D) Frequency of apoptotic CD8+Vβ8+ T cells in indicated organs of Alb-HA/CL4-TCR and CL4-TCR mice determined by cell surface Annexin V and intracellular active caspase-3 staining of gated CD8+ T cells. Alb-HA thymocytes cultured for 19 hours served as positive controls for apoptosis (right panel). Gates were set considering positive and enhanced Alb-HA/CL4-TCR autofluorescence controls (see Supplementary Figure 2). B, C, and D represent average values of 2 experiments (n = 6 per group).
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7. Figure 6
Spontaneous encounter with a liver-specific autoantigen induces CD8+ T-cell tolerance. CFSE-labeled or unlabeled CL4-TCR cells were injected into Alb-HA and WT mice or Ad-HA-infected Alb-HA recipients. At day 4 after transfer, gated CD8+CD90.1+ T cells from different organs of indicated mice were analyzed either for (A) CFSE intensity and intracellular IFN-γ expression or (B) cell surface Annexin V and intracellular active caspase-3 staining. WT thymocytes cultured for 24 hours served as positive controls for apoptosis (right panel). Data represent mean values of 2 experiments (n = 5 per group).
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8. Figure 7
Accumulation of functional Tregs in the liver of male Alb-HA/CL4-TCR mice. (A) Gated CD4+ T cells from indicated organs of Alb-HA/CL4-TCR and control mice were analyzed for CD25 and intracellular Foxp3 expression. (B) Total numbers of Tregs in different organs of indicated mice. (*P < .05; **P < .001; NS, not significant). A and B represent mean values (± standard deviation) of 2 experiments (n = 6 per group). (C) CFSE-labeled CL4-TCR cells cultured with equal numbers of intrahepatic CD4+CD25+ Tregs from Alb-HA/CL4-TCR mice or with splenic CD4+CD25− control T cells from WT mice in the presence of APCs and anti-CD3 antibody for 88 hours. Histograms show average proliferation of CD8+CFSE+ T cells from 2 experiments performed in duplicate.
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9. Supplementary Figure 1
Purity of isolated CL4-TCR cells. Naïve splenic HA-specific CD8+ T cells were isolated from CL4-TCR mice (CL4-TCR cells) and analyzed for purity and expression of the indicated activation markers. Data represent average values of 3 experiments.
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10. Supplementary Figure 2
Enhanced autofluorescence of paraformaldehyde-fixed intrahepatic CD8+ T cells from male Alb-HA/CL4-TCR mice. Paraformaldehyde-fixed gated CD8+ T cells from indicated organs of Alb-HA/CL4-TCR and CL4-TCR mice were analyzed for Vβ8 expression and either for intracellular active caspase-3 staining (see Figure 5D) or, because an appropriate isotype-control antibody is not available, for the respective autofluorescence intensity (FL-2). Note the enhanced autofluorescence of fixed intrahepatic CD8+Vβ8+ T cells from Alb-HA/CL4-TCR mice compared with the spleen and CL4-TCR controls considered in setting the gate. Data represent average values of 2 experiments (n = 6 per group).
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11. Supplementary Figure 3
Ad-HA adenovirus infection of Alb-HA mice elicits severe but transient hepatitis. Alb-HA and WT mice were infected with 109 infectious units of Ad-HA adenovirus on day −3 and were then either adoptively transferred with 8 × 106 CL4-TCR cells on day 0 or remained without further treatment. Alanine aminotransferase levels (mean ± standard deviation) were determined at indicated days (n = 3–5 per group). Note that alanine aminotransferase declined to normal, preinfection levels by day 28 and that histologic analysis at days 28 and 42 revealed no significant signs of liver inflammation in any groups of mice (not shown) that would have suggested the induction of a chronic autoimmune hepatitis.
Gastroenterology  , e3DOI: ( /j.gastro )
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