Cutting Edge: Chronic Inflammatory Liver
Disease in Mice Expressing a CD28-Specific
Ligand
This information is current as
of June 17, 2017.
Emily Corse, Rachel A. Gottschalk, Joon Seok Park, Manuel
A. Sepulveda, P'ng Loke, Timothy J. Sullivan, Linda K.
Johnson and James P. Allison
J Immunol published online 17 December 2012
http://www.jimmunol.org/content/early/2012/12/16/jimmun
ol.1202621
http://www.jimmunol.org/content/suppl/2012/12/17/jimmunol.120262
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Supplementary
Material
Published December 17, 2012, doi:10.4049/jimmunol.1202621
Cutting Edge: Chronic Inflammatory Liver Disease in Mice
Expressing a CD28-Specific Ligand
Emily Corse,* Rachel A. Gottschalk,*,†,1,2 Joon Seok Park,*,†,1 Manuel A. Sepulveda,*,3
P’ng Loke,‡,4 Timothy J. Sullivan,‡,5 Linda K. Johnson,x,6 and James P. Allison*,7
C
D28 and CTLA-4 are related T cell transmembrane
receptors that share the ligands B7-1 and B7-2 but
have opposing effects upon T cell responses (1–3).
Although stimulation of CD28 results in augmentation of
TCR-signaling pathways and increased IL-2 production,
CTLA-4 binding inhibits T cell responses, potentially by
multiple mechanisms (4). Mechanistic insight into these opposing pathways of T cell regulation has been obscured by the
inability to specifically ligate the CD28 and CTLA-4 receptors in cell culture systems. The development of membranebound single chain V region Ab reagents specific for CD28
and CTLA-4, known as single chain Fragment variable (scFv),
represents a reductionist solution to this problem (5). Indeed,
transgenic mice expressing anti–CTLA-4 scFv in B cells are
resistant to autoimmune diabetes, underscoring the role of
CTLA-4 in immune self-tolerance (6).
The liver is subject to a greater degree of immune tolerance
than other organs, because of the unique APC environment
(7) and its consequences for potentially reactive T cells (8).
Disruption of this tolerogenic microenvironment occurs
during chronic liver disease, which can result from persistent
viral infection, drug toxicity, and autoimmune reactivity
toward the liver. Although several mouse models of immunemediated liver injury exist, models that reflect the chronic and
complex pathologies of autoimmune liver diseases, such as
autoimmune hepatitis (AIH), have been elusive (9, 10). Existing
chronic mouse models of liver disease involve virus infection
(11) and overexpression of inflammatory mediators (12).
According to the National Institutes of Health, development
of new models that reflect features of autoimmune liver diseases, such as AIH, including spontaneous development of
chronic lymphocytic inflammation and fibrosis, is important
for understanding the pathogenesis of this group of diseases
(13). Activated CD4+ T cells have long been known to be in
the liver and peripheral blood of AIH patients, and cytochrome P450 2D6 is an important autoantigen in the type
2 form of AIH (14). CD8+ T cells are also likely to be important in pathogenesis given the correlation between disease
severity and IFN-g secretion by cytochrome P450 2D6–reactive
CD8+ T cells in AIH type 2 patients (15).
*Program in Immunology, Howard Hughes Medical Institute, and Ludwig Center for
Cancer Immunotherapy, Memorial Sloan-Kettering Cancer Center, New York, NY
10021; †Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate
School of Medical Sciences, New York, NY 10021; ‡Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA
94720; and xLaboratory of Comparative Pathology, Memorial Sloan-Kettering Cancer
Center, New York, NY 10021
7
Current address: Department of Immunology, The University of Texas MD Anderson
Cancer Center, Houston, TX.
1
2
Address correspondence and reprint requests to Dr. Emily Corse at the current address:
The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard,
Unit 901, Houston, TX 77030. E-mail address: [email protected]
3
The online version of this article contains supplemental material.
R.A.G. and J.S.P. contributed equally to this work.
Current address: Laboratory of Systems Biology, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, MD.
Current address: Janssen Research and Development, Spring House, PA.
4
Current address: Department of Microbiology, New York University School of Medicine, New York, NY.
5
Received for publication September 19, 2012. Accepted for publication November 16,
2012.
This work was supported by the Howard Hughes Medical Institute and the National
Institutes of Health.
Abbreviations used in this article: AIH, autoimmune hepatitis; ALT, alanine aminotransferase; AST, aspartate aminotransferase; DKO, double knockout; MSKCC, Memorial Sloan-Kettering Cancer Center; scFv, single chain fragment variable.
Current address: ChemoCentryx Inc., Mountain View, CA.
6
Current address: School of Veterinary and Biomedical Sciences, James Cook University,
Townsville, Queensland, Australia.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202621
Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00
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Inflammation of the normally tolerant liver microenvironment precedes the development of chronic liver
disease. Study of the pathogenesis of autoimmune liver
diseases, such as autoimmune hepatitis (AIH), has been
hampered by a lack of autochthonous chronic animal
models. Through our studies of T cell costimulation,
we generated transgenic mice expressing a ligand specific
for the CD28 receptor, which normally shares ligands
with the related inhibitory receptor CTLA-4. The mice
spontaneously develop chronic inflammatory liver disease with several pathologies found in AIH, including
elevated serum aminotransferases in the context of normal alkaline phosphatase and bilirubin levels, lymphocytic inflammation, focal necrosis, oval cell hyperplasia,
and fibrosis. The prevalence of IFN-g–producing CD8+
T cells in the livers of transgenic mice suggests a role for
autoimmune cytotoxicity in the chronic disease state.
The CD28 ligand–specific transgenic mice will facilitate
evaluation of CD8+ T cell function in liver disease pathologies found in AIH. The Journal of Immunology,
2013, 190: 000–000.
2
CUTTING EDGE: MURINE MODEL OF CHRONIC LIVER INFLAMMATION
In the course of studies on T cell costimulation, we generated
anti-CD28 scFv transgenic mice that allow selective ligation of
the T cell transmembrane receptor CD28, which normally
shares the ligands B7-1 and B7-2 with the T cell inhibitory
receptor CTLA-4. The CD28 scFv mice, when maintained
on a B7-1, B7-2 double-deficient background (16), spontaneously develop chronic inflammatory liver disease characterized
by infiltration of IFN-g–secreting CD8+ T cells, necrosis, and
fibrosis. Engagement of CD28 in the absence of CTLA-4 may
cause inflammatory liver disease by lowering the threshold for
T cell reactivity in the normally tolerant liver microenvironment, a notion supported by the association between polymorphisms in the human CTLA-4 gene and susceptibility to
the autoimmune liver diseases AIH and primary biliary cirrhosis (17). The CD28 scFv mice are ideal for the study of
CD8+ T cell contributions to pathologies found in autoimmune liver diseases, such as AIH.
Mice
The anti-CD28 scFv ligand was generated by fusing 37N.51 variable regions
to B7-2 in place of its membrane distal Ig domain (5) and subcloned into the
pD0I6 vector containing the invariant chain promoter, a gift of D. Mathis,
Department of Microbiology and Immunology, Harvard Medical School,
Boston, MA (18). Linearized plasmid was injected into C57BL/6 embryos
by the Memorial Sloan-Kettering Cancer Center (MSKCC) Mouse Genetics Facility. Founder mice were identified by PCR. B7-1, B7-2 double
knockout (DKO) mice were purchased from The Jackson Laboratory and
bred to anti-CD28 scFv mice. Male anti-CD28 scFv mice were analyzed.
OTII RAG22/2 mice (19) were purchased from The Jackson Laboratory
and bred to B7-1, B7-2 DKO mice. All mice were maintained in microisolator cages and treated in accordance with National Institutes of Health
and American Association of Laboratory Animal Care regulations. Experiments in this study were approved by the MSKCC Institutional Animal
Care and Use Committee.
In vitro T cell proliferation
CD11c+ APCs were positively selected (Miltenyi Biotec), pulsed with OVA
(323–339) peptide, and used to stimulate OT-II B7-1, B7-2 DKO T cells.
Proliferation was monitored by the addition of [3H]thymidine. Cells were
cultured at 37˚C/5% CO2 in RPMI 1640 supplemented with 10% FCS, 2
mM glutamine, 100 U/ml penicillin/streptomycin, and 2 mM 2-ME. Cells
were harvested onto glass-fiber filters (Tomtec), and filters were counted with
a MicroBeta counter (Perkin-Elmer).
CD28-Fc staining, Abs, and flow cytometry
Splenocytes treated with 0.3 mg Liberase TL and 0.2 mg DNase I (both from
Roche) were incubated with 20 mg CD28-Fc (R&D Systems). The human
Fc region of CD28-Fc was detected with biotinylated goat anti-human
secondary Ab (Jackson ImmunoResearch) and PE-streptavidin (eBioscience).
All other Abs were from BD Pharmingen, eBioscience, or BioLegend. Liver and
spleen T cells were positively selected for CD90.2 (Miltenyi Biotec) prior to
staining with TCR Vb Abs (BD Pharmingen). Flow cytometry was performed on a BD LSRII and data analyzed with FlowJo software (TreeStar).
Histology, immunohistochemistry, and liver enzyme measurements
Preparation of liver tissue, sectioning, H&E and Masson’s trichrome staining,
anti-CD3 immunohistochemistry, and liver chemistry panels were done by
the Laboratory of Comparative Pathology, MSKCC.
Isolation, quantitation, and stimulation of leukocytes from liver
Livers were digested with Liberase TL (1.2 mg) and DNase I (0.8 mg) and passed
through a 100-mm filter. Leukocytes were isolated by centrifugation in 33%
isotonic Percoll. For analysis of IFN-g secretion, cells stimulated with PMA/
ionomycin were stained for intracellular IFN-g with BD Biosciences reagents.
Statistical analysis
Data were analyzed for significance by one-way ANOVA with Bonferroni
correction using Prism 5 software (GraphPad).
Anti-CD28 scFv mice express a functional ligand for CD28 on APCs
The anti-CD28 scFv mice express anti-CD28 Ab fragments
fused to the CD28 ligand B7-2 (5) (Fig. 1A) in MHC
II–expressing APCs (18). Expression was visualized with
recombinant soluble CD28-Fc and flow cytometry (Fig. 1B).
Like natural CD28 ligands, the anti-CD28 scFv fusion protein is most highly expressed on APCs bearing the dendritic
cell marker CD11c+ (Fig. 1B). To specifically activate CD28
with scFv ligand, anti-CD28 scFv–transgenic mice were
crossed to mice deficient in CD28 and CTLA-4 ligands B7-1
and B7-2 (16). These mice, referred to hereafter as “B7-1, B7-2
DKO,” serve as littermate controls lacking CD28-mediated
costimulation.
To test the function of the anti-CD28 scFv ligand, CD11c+
leukocytes from transgenic mice were used as APCs to
stimulate Ag-specific T cells (Fig. 1C). The anti-CD28 scFv
APCs induce increased proliferation compared with B7-1,
B7-2 DKO APCs, consistent with costimulatory function of
CD28 (Fig. 1C). Thus, the anti-CD28 scFv fusion protein is
a functional CD28 ligand that enhances T cell stimulation.
Anti-CD28 scFv mice have chronic inflammatory liver disease
Upon observing enlarged livers from 3 mo of age (Fig. 1D,
Supplemental Table I), we were prompted to evaluate liver
chemistry in the anti-CD28 scFv mice. Elevated levels of
serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were observed in all mice examined between
4 and 8 mo of age (Fig. 1E), whereas alkaline phosphatase,
g-glutamyl transpeptidase, bilirubin, and other liver chemistry
measurements were normal (data not shown). Elevated
transaminases in the context of normal levels of alkaline
phosphatase and bilirubin is often seen in AIH patients (20).
H&E staining of liver sections from anti-CD28 scFv mice
showed periportal lymphocytic inflammation (Fig. 1F, right
panel). Microscopic signs of liver damage, such as single cell
necroses and oval cell hyperplasia, were often observed in the
inflamed anti-CD28 scFv livers (Fig. 2A, right panel, arrow
and arrowhead, respectively). In addition, Masson’s trichrome
staining highlighted collagen deposition, which is indicative
of fibrosis, or formation of scar tissue, extending from the
portal tract in anti-CD28 livers (Fig. 2B, right panel). A summary of liver pathology in mice of various ages can be found in
Supplemental Table I.
The anti-CD28 scFv mice also exhibit splenomegaly
(Supplemental Fig. 1A, 1B). Although this could result from
constitutive costimulation in the anti-CD28 scFv mice,
splenomegaly is also often found in patients with chronic liver
disease, including that caused by AIH (21, 22), due to shunting
of blood from liver to spleen as a result of fibrosis and portal
hypertension. Examination of other organs revealed occasional
mild inflammation of the lung, kidney, pancreas, and skin
(data not shown). Although present in only some anti-CD28
scFv mice, these phenotypes are reminiscent of additional
autoimmune disorders in patients with AIH (21–23). We did
not find evidence of circulating autoantibodies (data not
shown), which is likely precluded by the defect in germinal
center formation and diminished serum IgG levels in B7-1,
B7-2 DKO mice (16). Although autoantibodies are commonly seen in autoimmune liver disease, evidence for their
involvement in pathogenesis is lacking (21, 24). In summary,
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Materials and Methods
Results and Discussion
The Journal of Immunology
3
anti-CD28 scFv mice spontaneously develop Ab-independent
chronic liver disease that recapitulates several features of
human AIH, including persistently elevated ALT and AST,
periportal lymphocytic inflammation, and portal-based fibrosis.
IFN-g–secreting CD8+ T cells are prevalent in livers of anti-CD28
scFv mice
FIGURE 1. (A) Anti-CD28 scFv ligand (5). (B) Anti-CD28 scFv detected
with CD28-Fc. Graphs are gated on indicated populations (unshaded, antiCD28 scFv; shaded, B7-1, B7-2 DKO control). (C) OTII B7-1, B7-2 DKO
T cells stimulated with OVA-pulsed CD11c+ APCs from anti-CD28 scFv
mice (DKO aCD28 scFv) or B7-1, B7-2 DKO mice. Data in (B) and (C) are
representative of three independent experiments. (D) Livers from 4-mo-old
anti-CD28 scFv and control mice (left panel); scale bars, 1 cm. Liver weights
from 3–5-mo-old anti-CD28 scFv, B7-1, B7-2 DKO, and B6 control mice
(right panel). ***p = 0.0002. (E) Serum ALT (left panel) and AST (right panel)
were measured in 4–8-mo-old mice. ALT, ***p = 0.0006; AST, ***p =
0.0004. Each symbol in (D) and (E) represents an individual mouse. (F) H&E
staining of livers from 4-mo-old B7-1, B7-2 DKO mouse (left panel) and
anti-CD28 scFv mouse (right panel). These photomicrographs are representative of the 11 mice evaluated per group. Scale bars, 100 mm.
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We examined liver infiltrates for the presence of T cells using
anti-CD3 immunohistochemistry (Fig. 2C), and this revealed
substantial numbers of CD3+ lymphocytes in anti-CD28 scFv
liver tissue (brown-labeled cells, Fig. 2C, right panel) compared with very few in B7-1, B7-2 DKO tissue (Fig. 2C, left
panel). To further characterize liver infiltrates, we analyzed
isolated leukocytes by flow cytometry. Significantly more
CD8+ T cells exist in livers of anti-CD28 scFv mice (31% of
total leukocytes) compared with control livers (11% of leukocytes) (Fig. 2D). This corresponded to a 5-fold average
increase in the absolute number of CD8+ T cells in livers of
3–5-mo-old anti-CD28 scFv mice compared with controls
(Fig. 2D). We did not observe increases in CD4+ or NK
T cells (data not shown), making it likely that a significant
portion of the CD3+ staining in Fig. 2C represents CD8+
T cells. Enrichment of CD8+ T cells in liver of CD28 scFv
mice is in contrast to increases in CD4+ and CD8+ T cells
and B cells in spleen (data not shown).
Trapping of T cells in liver was described as a mechanism of
apoptotic removal (25); to directly examine the functional
activity of CD8+ T cells in livers of anti-CD28 scFv mice,
we analyzed IFN-g production upon ex vivo restimulation.
Compared with B7-1, B7-2 DKO controls, nearly twice the
percentage of CD8+ T cells in the livers of anti-CD28 scFv
mice secrete the inflammatory cytokine IFN-g (Fig. 2E).
Thus, in addition to increased numbers of CD8+ T cells in
livers of anti-CD28 scFv mice, more of these cells produce
IFN-g, corresponding to a .8-fold increase in the number of
IFN-g–secreting CD8+ T cells, suggesting that liver disease
in anti-CD28 scFv mice could be IFN-g mediated. IFN-g
production by CD8+ T cells correlates with disease severity in
AIH type 2 patients (15). Initial experiments show that CD8+
T cell depletion minimizes lymphocytic infiltration and other
pathological features, such as fibrosis, in anti-CD28 scFv liver
tissue (Supplemental Fig. 1C–F).
To examine the diversity and potential specificity of liver
CD8+ T cells in anti-CD28 scFv mice, we stained T cells
from liver and spleen of anti-CD28 scFv and B7-1, B7-2
DKO mice with Abs against specific TCR Vb subunits
(Supplemental Fig. 1G–I). The frequency of Vb8.1/8.2+
4
CUTTING EDGE: MURINE MODEL OF CHRONIC LIVER INFLAMMATION
Acknowledgments
FIGURE 2. (A) H&E-stained anti-CD28 scFv liver tissue from 4-mo-old
mouse (right panel), with B7-1, B7-2 DKO tissue as control (left panel). Arrow
indicates single cell necrosis, and arrowhead indicates oval cell hyperplasia.
Photomicrographs are representative of 11 mice/group. Scale bars, 50 mm. (B)
Trichrome staining (blue) of anti-CD28 scFv liver tissue from 4-mo-old mouse
(right panel) compared with B7-1, B7-2 DKO control tissue (left panel). Photomicrographs are representative of three mice/group. Scale bars, 100 mm. (C)
Anti-CD3 immunohistochemistry on B7-1, B7-2 DKO liver tissue (left panel)
and anti-CD28 scFv liver tissue (right panel) from 4-mo-old mice. Photomicrographs are representative of two mice/group. Scale bars, 50 mm. (D) Flow
cytometric analysis of CD45+ leukocytes from anti-CD28 scFv (right panel) and
B7-1, B7-2 DKO (left panel) liver tissue. Representative plots show the percentage of CD8+ T cells of total liver leukocytes (outside gates) and the percentage of IFN-g+ CD8+ T cells (inside gates). Graph shows absolute number of
liver CD8+ T cells (right panel). (E) Representative plots and graph show percentage of IFN-g+ CD8+ T cells upon PMA/ionomycin stimulation. Control,
unstimulated leukocyte samples. Each symbol in (D) and (E) represents an individual mouse; mice were 3–5 mo of age at the time of analysis. ***p , 0.0001.
We thank Diane Mathis for the invariant chain promoter vector. We also
thank Jacqueline Candelier (Laboratory of Comparative Pathology, MSKCC),
Elisa Cardenas, and Gaurav Singh for technical assistance and Robert
Anders, Danielle Bello, Katharina Kreymborg, and Dmitriy Zamarin for critical
comments on the manuscript.
Disclosures
The authors have no financial conflicts of interest.
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