1
MATERIALS AND METHODS
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Histopathology and Immunohistochemistry.
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Mice were sacrificed, and their livers were dehydrated, embedded in paraffin, sectioned,
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and stained with hematoxylin-phloxine-safran. Scoring of liver inflammation was done
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blindly using the modified Ishak histological activity index1.
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Lymphocyte isolation from liver and spleen.
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Liver from xenoimmunized mice were perfused via the portal vein with RPMI 1640 and
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removed. Liver sections and spleen were finely minced in RPMI 1640, passed through a
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100-gauge steel-mesh and centrifuged at 400 g for 5 min at 4oC. Cells contained in the
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supernatant were washed 3 times with RPMI 1640/5% foetal calf serum before being
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centrifuged on a Percoll gradient to purify lymphocytes2.
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Enzyme-Linked Immunosorbent Assay.
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Detection of autoantibodies against FTCD assay was performed as previously described3.
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Briefly, the fusion protein produced by the pEt-30C-FTCD (murine FTCD) was purified
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and used as antigen in the enzyme-linked immunosorbent assay (0.2 μg/well). IL-10
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levels from proliferation supernatant were measured using the Mouse IL-10 ELISA
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Ready-SET-Go! Kit (eBioscience, San Diego, California).
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CD4+CD25+ and CD8+ T cell isolation.
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CD4+CD25+ T cells were purified from isolated lymphocytes from the spleen or liver
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using the CD4+CD25+ mouse regulatory T cell isolation Kit (Miltenyi Biotec, Bergisch
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Gladbach, Germany) and the isolation of untouched CD8+ T cell was performed using the
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CD8a+ T Cell Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany).
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Proliferation assay
1
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Isolated lymphocytes and/or purified CD8+ T cells (Miltenyi Biotech, Bergisch Gladbach,
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Germany) from the spleen and liver of vaccinated animals were labeled with CFSE
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(5μM) (Vybrant™ CFDA SE Cell Tracer Kit, Molecular Probes, Eugene, Oregon) and
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incubated with syngenic purified antigen presenting cells (APC) from spleen4 previously
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incubated with 100ug/1x106 cells of purified mouse FTCD in RPMI1640 supplemented
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with 5% foetal calf serum and 20 U/ml of murine rIL-2 (Invitrogen, Carlsbad,
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California). In proliferation suppression assays, purified CD4+CD25+ T cells (Miltenyi
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Biotech, Bergisch Gladbach, Germany) were co-incubated with CFSE-labelled CD8+ T
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cells at different ratios. After 3 days, cells were labeled with APC coupled anti-CD8+
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(eBioscience, San Diego, California) and 7-AAD (Invitrogen, Carlsbad, California). Cells
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were analyzed by flow cytometry (BD biosciences, Mississauga, Canada) and the
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precursor frequency was determined using FlowJo (Treestar, Ashland, Oregon).
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Combined CD107a mobilisation assay and intracellular Granzyme B and IFN-γ
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staining.
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1x106 lymphocytes isolated from 7 month-old xenoimmunized C57BL/6 mice were
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incubated for 5 hours at 37°C with 100ug of purified mouse FTCD in RPMI1640
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supplemented with 5% foetal calf serum and in presence of FITC-labelled CD107a
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antibody, brefeldin A and monensin (eBioscience, San Diego, California). Surface
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staining with PE-Cy7-CD8a and Alexa700-CD62L and viability staining with 7-AAD
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was then performed according to the manufacturer guideline (eBioscience, San Diego,
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California).
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(eBioscience, San Diego, California) was performed according to the manufacturer
Intracellular
staining
with
PE-IFN-γ
and
eFluor450-granzyme
B
2
46
guideline. Cells were analyzed by flow cytometry (BD biosciences, Mississauga, Canada)
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and the FlowJo analysis software (Treestar, Ashland, Oregon).
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CYP2D9, FTCD, AIRE, CXCL9 and CXCL10 expression Level.
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For the measurement of CYP2D9, FTCD and AIRE thymic expression, RNA was
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isolated from thymuses of newborn mice (1-2 days old) using the RNeasy Micro kit
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(QIAGEN, Santa Clarita, California). Expression levels of FTCD and CYP2D9 in the
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liver were performed using RNA isolated from liver of 8 week-old mice. Expression of
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FTCD, CYP2D9 and AIRE in these organs was quantified using specific primers (5’-
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TGCTGCCTGTTTGGAGGCAA-3’,
5’-AAGCAAGGCTTGGGCCACTT-3’;
5’-
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GAGCAGAGGCGATTCTCTGT-3’,
5’-CCCAGGTGGTCCTATTCTCA-3’;
5’-
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GGGGCGGCCAGGTCCAGCAC-3’ and 5’-GGTGTGCCTGGCGTGGGAGTCG-3’,
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respectively). For the expression level of CXCL9 (MIG) and CXCL10 (IP10), RNA was
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isolated from livers of xenoimmunized animals and quantification was performed using
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specific
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CCGGATTCAGACATCTCTGC-3’; CXCL9: 5’-GACATTCTCGGACTTCACTC-3’,
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5’-GATTCAGGGTGCTTGTTGGT-3’). PCR was performed using one-step RT-PCR
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(QIAGEN, Santa Clarita, California) and murine β-actin expression level was used as
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internal reference.
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Flow Cytometry Analysis.
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The frequency of CD4+ regulatory T cells in blood, spleen, lymph nodes or amongst
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liver-infiltrating lymphocytes was assessed using the mouse regulatory T cell staining kit
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(eBioscience, San Diego, California). Fluorochrome-labeled antibodies against CXCR3,
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CD8, CD4, CD25 were obtained from eBioscience (San Diego, California).
primers
(CXCL10:
5’-GTGCTGCCGTCATTTTCTGC-3’,
5’-
3
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Statistical Analysis.
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Differences between groups were tested using one-way analysis of variance (ANOVA)
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with Tukey's post-hoc test. Correlation coefficients were computed using Pearson's test.
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In all graphs, error bars represent standard error of means. All statistical analyses were
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performed using GraphPad Prism version 5 (GraphPad Software, CA).
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4
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SUPPORTING FIGURE LEGEND.
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Figure 1. CD8+ T cells from xenoimmunized C57BL/6 mice are cytotoxic. A. Isolated
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T cells from xenoimmunized C57BL/6 mice were incubated with murine FTCD and
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degranulation was assayed using CD107a staining combined with Granzyme B and
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Interferon-γ intracellular staining. B. 15.9 +/- 5.5% of activated CD8+ T cells
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(CD8+CD62L-) degranulated in presence of FTCD of which, 33.1 +/- 2.3% expressed
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Granzyme B, 26.4 +/- 5.4% expressed IFN-gamma and 17.2 +/- 3.5% expressed both.
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REFERENCES
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1.
2.
3.
4.
Ishak, K., et al. Histological grading and staging of chronic hepatitis. J Hepatol
22, 696-699 (1995).
Lapierre, P., Beland, K., Martin, C., Alvarez, F., Jr. & Alvarez, F. Forkhead box
p3+ regulatory T cell underlies male resistance to experimental type 2
autoimmune hepatitis. Hepatology 51, 1789-1798.
Lapierre, P., Beland, K., Martin, C., Alvarez, F., Jr. & Alvarez, F. Forkhead box
p3+ regulatory T cell underlies male resistance to experimental type 2
autoimmune hepatitis. Hepatology 51, 1789-1798 (2010).
Inaba, K., et al. Isolation of dendritic cells. Curr Protoc Immunol Chapter 3, Unit
3 7 (2009).
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