Chronic Inflammatory Liver Disease in Mice Expressing a CD28-specific Ligand

Abstract

Inflammation of the normally tolerant liver microenvironment precedes 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-γ-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.

Introduction

CD28 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). Whereas 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 membrane-bound single chain variable region antibody 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, due to the unique antigen presenting cell 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 towards the liver. Although several mouse models of immune-mediated 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 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 (CYP2D6) 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-γ secretion by CYP2D6-reactive CD8+ T cells in AIH type 2 patients (15).

In the course of studies on T cell costimulation, we generated anti-CD28 scFv (αCD28 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 cytotoxic T lymphocyte antigen (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-γ-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 study of CD8+ T cell contributions to pathologies found in autoimmune liver diseases such as AIH.

Materials and Methods

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 (18). Linearized plasmid was injected into C57BL/6 embryos by the MSKCC Mouse Genetics Facility. Founder mice were identified by PCR. B7-1, B7-2 DKO mice were purchased from Jackson Labs and bred to αCD28 scFv mice. Male αCD28 scFv mice were analyzed. OTII RAG2−/− mice (19) were purchased from Jackson Labs and bred to B7-1, B7-2 DKO mice. All mice were maintained in microisolator cages, and treated in accordance with NIH and AALAC regulations. Experiments in this study were approved by the MSKCC IACUC.

In vitro T cell proliferation

CD11c+ APC were positively selected (Miltenyi), pulsed with OVA (323–339) peptide, and used to stimulate OT-II B7-1, B7-2 DKO T cells. Proliferation was monitored by addition of ³H-thymidine. Cells were cultured at 37°C/5% CO₂ in RPMI1640 supplemented with 10% FCS, 2 mM glutamine, 100 U/mL pen/strep, and 2 mM 2-mercaptoethanol. Cells were harvested onto glass-fiber filters (Tomtec), and filters counted with a MicroBeta counter (Perkin-Elmer).

CD28-Fc staining, antibodies, and flow cytometry

Splenocytes treated with 0.3 mg Liberase TL and 0.2 mg DNase I (both from Roche) were incubated with 20 μg CD28-Fc (R&D Systems). The human Fc region of CD28-Fc was detected with biotinylated goat anti-human secondary antibody (Jackson ImmunoResearch) and PE-streptavidin (eBioscience). All other antibodies were from BD Pharmingen, eBioscience, or BioLegend. Liver and spleen T cells were positively selected for CD90.2 (Miltenyi) prior to staining with TCR Vβ antibodies (BD Pharmingen). Flow cytometry was on a BD LSRII and data analyzed with FlowJo (Treestar, Inc.).

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 μm filter. Leukocytes were isolated by centrifugation in 33% isotonic Percoll. For analysis of IFN-γ secretion, cells stimulated with PMA/ionomycin were stained for intracellular IFN-γ with BD Biosciences reagents.

Statistical analysis

Data were analyzed for significance by one-way ANOVA with Bonferroni correction using Prism 5 software (GraphPad).

Results and Discussion

αCD28 scFv mice express a functional ligand for CD28 on APCs

The αCD28 scFv mice express αCD28 antibody fragments fused to the CD28 ligand B7-2 (5); Figure 1A) in major histocompatibility class (MHC) II-expressing antigen presenting cells (APC; reference (18). Expression was visualized with recombinant soluble CD28-Fc and flow cytometry (Figure 1B). Like natural CD28 ligands, the αCD28 scFv fusion protein is most highly expressed on APCs bearing the dendritic cell marker CD11c+ (Figure 1B). To specifically activate CD28 with scFv ligand, αCD28 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.

Figure 1.

A) αCD28 scFv ligand (5). B) αCD28 scFv detected with CD28-Fc. Histograms gated on indicated populations (unshaded, αCD28 scFv; shaded, B7-1, B7-2 DKO control). C) OTII B7-1, B7-2 DKO T cells stimulated with OVA-pulsed CD11c+ APCs from αCD28 scFv mice (DKO αCD28 scFv; open squares) or B7-1, B7-2 DKO mice (closed squares). Data in B) and C) are representative of three independent experiments. D) Left, livers from 4 month old αCD28 scFv and control mice. Right, liver weights from 3–5 month oldαCD28 scFv, B7-1, B7-2 DKO, and B6 control mice. Scale bars=1 cm. ***P=.0002. E) Serum ALT (left) and AST (right) were measured in 4–8 month old mice. ***P=.0006. Each symbol in D) and E) represents an individual mouse. F) H&E staining of livers from 4 month old B7-1, B7-2 DKO and αCD28 scFv mice, left and right, respectively, is representative of the 11 mice evaluated per group. Scale bars=100 μm.

To test the function of theαCD28 scFv ligand, CD11c+ leukocytes from transgenic mice were used as APC to stimulate antigen-specific T cells (Figure 1C). TheαCD28 scFv APC induce increased proliferation compared to B7-1, B7-2 DKO APC, consistent with costimulatory function of CD28 (Figure 1C). Thus, the αCD28 scFv fusion protein is a functional CD28 ligand that enhances T cell stimulation.

αCD28 scFv mice have chronic inflammatory liver disease

Upon observing enlarged livers from three months of age (Figure 1D, Supplemental Table S1), we were prompted to evaluate liver chemistry in the αCD28 scFv mice. Elevated levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were observed in all mice examined between 4 and 8 months of age (Figure 1E), while alkaline phosphatase, γ-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 αCD28 scFv mice showed periportal lymphocytic inflammation (Figure 1F, right panel). Microscopic signs of liver damage such as single cell necroses and oval cell hyperplasia were often observed in the inflamed αCD28 scFv livers (Figure 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, that extends from the portal tract in αCD28 livers (Figure 2B, right panel). A summary of liver pathology in mice of various ages can be found in Supplemental Table 1.

Figure 2.

A) H&E-stained αCD28 scFv liver tissue from 4 month old mouse (right panel), with B7-1, B7-2 DKO tissue as control (left panel). Scale bars=50 μm. Arrow indicates single cell necrosis, and arrowhead indicates oval cell hyperplasia. Micrographs are representative of 11 mice per group. B) Trichrome staining (blue) of αCD28 scFv liver tissue from 4 month old mouse (right panel) compared to B7-1, B7-2 DKO control tissue (left panel). Scale bars=100 μm. Micrographs are representative of 3 mice per group. C) αCD3 IHC on B7-1, B7-2 DKO liver tissue (left panel) and αCD28 scFv liver tissue (right panel) from 4 month old mice. Scale bars=50 μm. Micrographs are representative of 2 mice per group. D) Flow cytometric analysis of CD45+ leukocytes from αCD28 scFv (right) and B7-1, B7-2 DKO liver tissue (left). Representative plots show percent of CD8+ T cells of total liver leukocytes (outside gates), and percent of IFN-γ+ CD8+ T cells (inside gates). Graph shows absolute number of liver CD8+ T cells, ***P<.0001. E) Representative plots and graph show percent of IFN-γ+ CD8+ T cells upon PMA/ionomycin stimulation, ***P<.0001. ‘Control’-unstimulated leukocyte samples. Each symbol in D) and E) represents an individual mouse; mice were 3–5 months of age at time of analysis.

The αCD28 scFv mice also exhibit splenomegaly (Supplemental Figure 1A, B). Although this could result from constitutive costimulation in the αCD28 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 αCD28 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). While autoantibodies are commonly seen in autoimmune liver disease, evidence for their involvement in pathogenesis is lacking (21, 24). In summary, αCD28 scFv mice spontaneously develop antibody-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-γ-secreting CD8+ T cells are prevalent in livers of αCD28 scFv mice

We examined liver infiltrates for the presence of T cells using αCD3 immunohistochemistry (Figure 2C), and this revealed substantial numbers of CD3+ lymphocytes in αCD28 scFv liver tissue (brown-labeled cells, Figure 2C, right panel) compared to very few in B7-1, B7-2 DKO tissue (Figure 2C, left panel). To further characterize liver infiltrates, we analyzed isolated leukocytes by flow cytometry. Significantly more CD8+ T cells exist in livers of αCD28 scFv mice (31% percent of total leukocytes), compared to control livers (11% of leukocytes) (Figure 2D). This corresponded to a 5-fold average increase in the absolute number of CD8+ T cells in livers of 3–5 month old αCD28 scFv mice compared to controls (Figure 2D). We did not observe increases in CD4+ or natural killer T cells (data not shown), making it likely that a significant portion of the CD3+ staining in Figure 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 has been described as a mechanism of apoptotic removal (25); to directly examine functional activity of CD8+ T cells in livers of αCD28 scFv mice, we analyzed IFN-γ production upon ex vivo restimulation. Compared to B7-1, B7-2 DKO controls, nearly twice the percent of CD8+ T cells in the livers of αCD28 scFv mice secrete the inflammatory cytokine IFN-γ (Figure 2E). Thus, in addition to increased numbers of CD8+ T cells in livers of αCD28 scFv mice, more of these cells produce IFN-γ, corresponding to a >8-fold increase in the number of IFN-γ-secreting CD8+ T cells, suggesting that liver disease in αCD28 scFv mice could be IFN-γ mediated. IFN-γ 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 αCD28 scFv liver tissue (Supplemental Figure 1C–F).

To examine the diversity and potential specificity of liver CD8+ T cells in αCD28 scFv mice, we stained T cells from liver and spleen of αCD28 scFv and B7-1, B7-2 DKO mice with antibodies against specific TCR Vβ subunits (Supplemental Figure 1G–I). The frequency of Vβ8.1/8.2+ CD8+ T cells is significantly increased in livers of αCD28 scFv mice compared to controls (29.1% and 15.8% of total CD8+ T cells, respectively)(Supplemental Figure 1G). Vβ8.1+/8.2+ CD8+ T cells are not overrepresented in spleens of αCD28 scFv mice (Supplemental Figure 1H), and the splenic CD8+ TCR Vβ repertoire is normal (Supplemental Figure 1I). These data suggest that a liver-specific T cell response could contribute to the inflammatory liver disease in αCD28 scFv mice.

Here we report a new spontaneous mouse model of chronic liver disease characterized by infiltration of IFN-γ-secreting CD8+ T cells and the development of fibrosis. Engagement of the T cell costimulatory receptor CD28 with scFv ligand in the absence of inhibition by the T cell inhibitory receptor CTLA-4 may lower the threshold for T cell-mediated immune attack in the normally tolerant liver microenvironment. Interestingly, polymorphisms in human CTLA-4 are associated with AIH and another autoimmune liver disease, primary biliary cirrhosis (17). Of the three types of autoimmune liver disease, AIH, primary biliary cirrhosis, and primary sclerosing cholangitis, the pathologies found in the αCD28 scFv mice most closely resemble those seen in AIH, including lymphocytic inflammation, development of portal-based fibrosis, elevated transaminases, and occasional mild inflammation of other organs.

Although early studies of AIH emphasized the involvement of CD4+ T cells, it is now appreciated that CD8+ T cell reactivity is relevant (14), and the αCD28 scFv mice will facilitate study of CD8+ T cell contributions to AIH-like pathologies. Transgenic mice expressing liver-specific IFN-γ were previously shown to have inflammatory liver disease, although these mice did not develop fibrosis (26). Deletion of the immunomodulatory cytokine TGF-β also results in inflammatory liver disease and death of Balb/c mice at around two weeks of age (27). While genetic manipulation of IFN-γ and other cytokines demonstrates their ability to mediate liver disease (9), the αCD28 scFv mice are unique due to the spontaneous CD8+ T cell response initiated in the absence of virus infection, immunization, or other manipulation, and this makes them an advantageous experimental model.