Medullary thymic epithelial cell depletion leads to autoimmune hepatitis

TRAF6, an E3 ubiquitin protein ligase, plays a critical role in T cell tolerance by regulating medullary thymic epithelial cell (mTEC) development. mTECs regulate T cell tolerance by ectopically expressing self-antigens and eliminating autoreactive T cells in the thymus. Here we show that mice with mTEC depletion due to conditional deletion of Traf6 expression in murine thymic epithelial cells (Traf6ΔTEC mice) showed a surprisingly narrow spectrum of autoimmunity affecting the liver. The liver inflammation in Traf6ΔTEC mice exhibited all the histological and immunological characteristics of human autoimmune hepatitis (AIH). The role of T cells in AIH establishment was supported by intrahepatic T cell population changes and AIH development after transfer of liver T cells into immunodeficient mice. Despite a 50% reduction in natural Treg thymic output, peripheral tolerance in Traf6ΔTEC mice was normal, whereas compensatory T regulatory mechanisms were evident in the liver of these animals. These data indicate that mTECs exert a cell-autonomous role in central T cell tolerance and organ-specific autoimmunity, but play a redundant role in peripheral tolerance. These findings also demonstrate that Traf6ΔTEC mice are a relevant model with which to study the pathophysiology of AIH, as well as autoantigen-specific T cell responses and regulatory mechanisms underlying this disease.

T cell development and establishment of tolerance to self-antigens occurs in the thymus and depends on cross-talk between thymocytes and the thymic epithelium (1). This cellular compartment is composed of 2 major populations: cortical and medullary thymic epithelial cells (cTECs and mTECs, respectively). Whereas cTECs regulate positive selection of thymocytes and the diversity of the T cell repertoire, mTECs ectopically express tissue-restricted antigens (TRAs) for the purpose of deleting autoreactive T cells that recognize these antigens with high affinity (2–6). In addition, mTECs also affect peripheral tolerance through the production of natural Tregs (nTregs), which suppress aberrant T cell activation in peripheral tissues (7–9). The importance of mTECs in preventing autoimmunity has been revealed through the analysis of genetic mutations that control mTEC development and function in mice and humans (5, 10). In mutant mice, the absence or disruption of the 3-dimensional medullary architecture and defective TRA expression correlates with development of autoimmunity of varied severity, manifested as lymphocyte infiltrates within and autoantibody production against peripheral tissues (11–15). In humans, mutations in the AIRE protein (which controls TRA expression in mTECs) result in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; also known as autoimmune polyglandular syndrome type 1) (16, 17). This syndrome is characterized by chronic mucocutaneous candidiasis, hypoparathyroidism, and adrenal insufficiency as well as by a higher incidence of diabetes, inflammatory bowel disease, and autoimmune hepatitis (AIH). Thus, proper development of — and antigen presentation by — mTECs plays a key role in the establishment of central tolerance and prevention of autoimmunity.

Existing animal models with mutations that disrupt mTEC development and T cell selection have advanced our understanding of the role of mTECs in induction of central tolerance. However, as most of these mutations also affect the hematopoietic compartment, it is difficult to ascertain the individual contributions of the epithelial versus the hematopoietic compartments to abnormal T cell selection and development of autoimmunity. For example, while members of the TNF receptor superfamily and their corresponding signaling partners have been shown to inhibit TEC development (11, 12, 14, 15), these molecules are also expressed in hematopoietic cells, where they exert their effects in mediating immune responses. In this report, we describe the generation and characterization of conditional knockout mice with deletion of Traf6 specifically in TECs (Traf6ΔTEC mice). These mice developed a T cell–mediated inflammatory response against the liver that exhibited histopathological features of human AIH, representing a novel animal model for the study and treatment of this disease.

TEC-specific deletion of Traf6 inhibits mTEC development. Conditional deletion of Traf6 in TECs was achieved by crossing floxed Traf6 mice (18) to animals expressing the Cre recombinase under the control of the foxn1 promoter (ref. 19 and Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI65414DS1). This gene encodes the transcription factor FOXN1, which regulates thymic epithelial cell development (20). Deletion of Traf6 expression was confirmed by staining thymic sections with an anti-TRAF6 antibody and the mTEC-specific marker lectin ulex europeous agglutinin–1 (UEA) (21), showing a drastic reduction of TRAF6⁺UEA⁺ mTECs in Traf6ΔTEC mice (Supplemental Figure 1B). H&E staining of thymic sections revealed diminished medullary areas in Traf6ΔTEC mice compared with WT controls (Supplemental Figure 1C). Further, staining of thymic sections with the cortical and medullary markers keratin-8 (K8) and K5, respectively, revealed a marked reduction in K5⁺ mTECs in Traf6ΔTEC mice (Supplemental Figure 2A). The diminished medulla and lack of K5⁺ mTECs also correlated with markedly reduced UEA⁺AIRE⁺ mature mTECs (Supplemental Figure 2A). Quantitative analysis of mTEC subpopulations by flow cytometry showed drastically decreased production of immature and mature mTECs (Supplemental Figure 2B), previously described as UEA⁺MHCII^int and UEA⁺MHCII^hi, or intermediate and mature MHCII^hiAIRE^– and MHCII^hiAIRE⁺ cells (22–24). In contrast, cTECs identified as UEA^–MHCII⁺ cells were unaffected by Traf6 deficiency (Supplemental Figure 2B), consistent with a specific role for TRAF6 in mTEC development. Depletion of mTECs in Traf6ΔTEC mice had no apparent effect on thymocyte development, as the total numbers of the different thymocyte populations were comparable to those in WT controls (Supplemental Figure 3A). In addition, mature T cells in the spleen and lymph nodes were unaffected in Traf6ΔTEC mice compared with controls (Supplemental Figure 3, B and C). These results showed that TRAF6 is required for, and plays an intrinsic role in, mTEC differentiation, whereas Traf6 deletion and mTEC depletion has no apparent effect on cTEC and T cell development.

Traf6ΔTEC mice develop AIH. Defects in mTEC development correlate with the presence of peripheral autoimmune manifestations, such as autoantibody production against and presence of inflammatory infiltrates in multiple organs (5, 10). While young Traf6ΔTEC mice (6–12 weeks old) appeared healthy, older Traf6ΔTEC mice (∼6 months old) were smaller compared with WT controls and appeared hunched with ruffled fur (Figure 1A). Despite looking healthy, approximately 80% of young Traf6ΔTEC mice developed autoantibodies with reactivity against the liver, lung, kidney, and small and large intestines (Figure 1B and Table 1). Inflammatory infiltrates were also present, but exhibited a more narrow distribution, found predominantly in the liver (100%), and to a lesser extent in the lung (50%) and kidney (33%), of Traf6ΔTEC mice (Figure 1C and Table 1). The small intestine and colon appeared normal, despite the presence of autoantibodies against these tissues in the majority of Traf6ΔTEC mice (Figure 1C and Table 1). In older Traf6ΔTEC animals (6–9 months), the penetrance and distribution of the infiltrates and autoantibodies was similar to that of younger mice, although infiltrates and autoantibodies were also found in some WT mice analyzed (Table 1). Together, these results suggest that the incidence and distribution of autoimmune manifestations in Traf6ΔTEC mice appears relatively constant over time, but the phenotype may be compounded as the mice age, which is due to age rather than mTEC depletion–related effects.

Figure 1

Traf6ΔTEC mice develop peripheral autoantibodies and inflammatory infiltrates. (A) Images of WT and Traf6ΔTEC conditional knockout (cKO) mice. Mouse weight at approximately 6 months of age is also shown. n = 10 per genotype. Red bars represent mean. (B) Frozen tissue sections from Rag1^–/– animals (used to eliminate mouse Ig background) were incubated sequentially with sera from 6- to 12-week-old WT or Traf6ΔTEC mice, and anti–mouse IgG–FITC and visualized with fluorescence microscopy. (C) Paraffin-embedded sections of organs of WT and Traf6ΔTEC mice as in B were stained with H&E and evaluated for the presence of infiltrates (arrows) by light microscopy. n ≥ 8 per genotype in B and C. Scale bars: 200 μm. ***P < 0.001.

Table 1

Penetrance of autoantibody production and presence of inflammatory infiltrates in young and old WT and Traf6ΔTEC mice

Because 100% of the Traf6ΔTEC animals examined exhibited liver infiltrates (Table 1), we targeted this organ for further analysis. The hepatic inflammation in older Traf6ΔTEC animals was diagnosed as AIH based on several histopathological features typically observed in humans with AIH, including portal inflammation (defined by the presence of inflammatory infiltrates surrounding hepatic portal tracts), interface hepatitis (characterized by infiltrates crossing the limiting plate of inflammation and invading the liver parenchyma), and lobular inflammation (consisting of infiltrates within the liver parenchyma) (Figure 2, A–C). The consequences of chronic hepatic inflammation and tissue damage in Traf6ΔTEC mice were reflected by increased collagen deposition and fibrosis surrounding the hepatic vessels and elevated serum alanine aminotransferase (ALT) levels, respectively (Figure 2D). Collagen deposition and fibrosis was quantified by morphometry of picrosirius red–stained, paraffin-embedded liver sections (see Methods and Figure 2D). Analysis of young mice also showed significant differences in AIH histopathology between Traf6ΔTEC and WT mice; however, the scores were lower than those of older animals, in which AIH histopathological features peaked at 6 months of age (Supplemental Figure 4).

Figure 2

Liver inflammation in Traf6ΔTEC mice exhibits pathognomonic features of AIH. (A–C) Paraffin-embedded liver sections of approximately 6-month-old WT and Traf6ΔTEC mice were stained with H&E. Portal inflammation, interface hepatitis, and lobular inflammation (arrows) was scored by a pathologist as described in Methods. (D) Picrosirius red staining was used for quantification of liver fibrosis as described in Methods. Serum ALT levels were determined by reflectance spectrophotometry. n = 8 per genotype in A–D. Red bars represent mean. Scale bars: 100 μm (A–C); 200 μm (D). *P < 0.05; **P < 0.01.

Traf6ΔTEC mice exhibit classic hallmarks of AIH, such as the presence of plasma cells and anti-nuclear autoantibodies (ANAs). In addition to the histological manifestations discussed above, development of AIH in humans correlates with increased presence of plasma cells in intrahepatic inflammatory lesions as well as elevated total serum Ig levels and organ-specific and non–organ-specific autoantibodies (25–27). To further characterize the AIH phenotype of Traf6ΔTEC mice, we assayed for the presence of plasma cells in the liver and the nature of autoantibodies in the serum of these mice. Staining of paraffin-embedded liver sections from Traf6ΔTEC and WT mice with the plasma cell–specific dye methyl green-pyronin revealed an increased presence of plasma cells in the portal infiltrates of Traf6ΔTEC livers (Figure 3A). A marked increase in the number of CD138⁺ plasma cells in Traf6ΔTEC mice was also found when intrahepatic lymphocyte populations were analyzed by flow cytometry (Figure 3B). This increase in plasma cells, in turn, correlated with ANA presence in Traf6ΔTEC mice, as assayed by staining HEp-2 cells with sera from Traf6ΔTEC or control animals and determining fluorescence intensity based on arbitrary scores of 1–5 (Figure 3C). The majority of Traf6ΔTEC mice (75%) stained positive for ANAs, whereas 20% had autoantibodies against perinuclear proteins and 5% had autoantibodies against both nuclear and cytoplasmic antigens (Figure 3D).

Figure 3

Traf6ΔTEC mice exhibit increased numbers of liver plasma cells and elevated serum ANAs. (A) Paraffin-embedded liver sections from Traf6ΔTEC mice were stained with methyl green-pyronin, and the presence of plasma cells (arrows) was scored as described in Methods. n = 8 per genotype. (B) Liver cell suspensions from 6- to 8-week-old WT and Traf6ΔTEC mice were stained with anti-CD19–eFluor 450 and anti-CD138–biotin/streptavidin PE-Cy7 and analyzed by flow cytometry. n = 9 per genotype. Data are pooled results from 3 independent experiments. Bar graph represents mean + SEM. (C) Sera of 8- to 10-week-old mice were used to stain HEp-2 cells, and the presence of ANAs was revealed by anti–mouse IgG–FITC and fluorescence microscopy. Data are presented on an arbitrary scale of 1–5, based on HEp-2 cell staining and ANA fluorescence intensity. n = 25 per genotype. Data are pooled results from at least 3 independent experiments. Red bars represent mean. (D) Representative patterns of autoantibody staining and frequency within the sample population shown in C. Scale bars: 20 μm (A); 40 μm (C and D). *P < 0.05; ***P < 0.001.

The presence of autoantibodies in Traf6ΔTEC mice correlated with a significant increase in total serum Ig and, more specifically, IgG1 and IgM isotype antibody levels (Figure 4, A and B). Western blot analysis of liver extracts showed that autoantibodies in the sera of different Traf6ΔTEC animals exhibited similar binding specificities against several liver proteins, whereas sera from WT mice showed limited reactivity (Figure 4C). Both non–organ-specific and organ-specific autoantibodies were present in the sera of Traf6ΔTEC mice: some protein bands were present in all tissue extracts, whereas others were unique to the liver (Figure 4D). Together, the presence of plasma cells in the hepatic infiltrates, the increased level of total serum Ig, the presence of ANA, and the organ-specific and non–organ-specific autoantibodies in Traf6ΔTEC animals are consistent with the classic features of AIH described in human patients (25–27).

Figure 4

Traf6ΔTEC mice produce autoantibodies that bind to liver-specific proteins. (A and B) Concentrations of different antibody isotypes (A) and total Ig (B) in the sera of 6- to 12-week-old WT and Traf6ΔTEC mice were determined by ELISA. n ≥ 8 per genotype. Data are pooled results from 3 independent experiments run in triplicates. Red bars represent mean. Bar graph represents mean + SEM. (C and D) Western blots of normal tissue lysates (100 μg) from Rag1^–/– mice were incubated with sera (1:100) from WT and Traf6ΔTEC mice as shown, followed by horseradish peroxidase–conjugated anti–mouse IgG secondary antibody. Protein bands in C and D were visualized by chemiluminescence as described in Methods. Blots were stripped and reprobed with anti-Erk antibody (loading control). In C, blots were cut apart in order to be incubated with different sera, then reassembled (black lines). Arrows in D denote protein bands that are unique to the liver. Representative blots from 1 of at least 2 independent experiments are shown. Li, liver; Lu, lung; Sp, spleen; Ki, kidney. (E) Autoantibody concentrations in the sera of 6- to 12-week-old WT and Traf6ΔTEC mice, determined by ELISA. n ≥ 7 per genotype. Red bars represent mean. **P < 0.01; ***P < 0.001.

Based on the nature of the autoantibodies present, 2 types of AIH have been identified: type 1, characterized by ANA and/or anti-SMA antibodies; and type 2, characterized by anti–liver and kidney microsomal type 1 (anti-LKM) or anti–liver cytosol type I (anti-LC1) antibodies (26, 28, 29). In addition, anti–soluble liver antigen (anti-SLA) antibodies serve as highly specific serological markers for AIH and exhibit 99% specificity for diagnosing this disease (30–33). Anti-SLA antibodies have been found in 11%–22% of white North American and northern European adult patients with type 1 AIH and are common (18%–44% incidence) in children with type 1 and type 2 AIH (30, 34–36). To further characterize the nature of the autoantibodies in Traf6ΔTEC mice, we tested for the presence of anti-SLA, anti-SMA, and anti-LKM autoantibodies. While there was no difference in the levels of anti-SMA and anti-LKM antibodies between Traf6ΔTEC and control mice, levels of anti-SLA autoantibodies were significantly increased in Traf6ΔTEC mice compared with controls (Figure 4E). We therefore concluded that the presence of ANA and anti-SLA antibodies together in the serum of Traf6ΔTEC mice support an AIH phenotype in these animals similar to human type 1 AIH.

Traf6ΔTEC mice exhibit abnormal hepatic T cell populations and T cell function. Since mTECs regulate negative selection of T cells in the thymic medulla (3, 4, 37), and given the observed inflammatory lesions in the liver of Traf6ΔTEC mice, we examined whether mTEC depletion correlates with abnormal T cell presence and function in these animals. Costaining frozen liver sections with anti-CD3 and anti-CD4 or anti-CD8 antibodies revealed that the portal infiltrates primarily consisted of CD3⁺CD4⁺ T cells, whereas CD3⁺CD8⁺ cells were more prevalent within the liver parenchyma (Figure 5A and data not shown). Further analysis of resident liver T cell populations by flow cytometry confirmed an increase in the total number of CD4⁺ and CD8⁺ T cells in Traf6ΔTEC mice compared with WT controls (Figure 5B). In contrast to the liver, we found no significant quantitative or qualitative differences in T cell populations in the spleen and lymph nodes of Traf6ΔTEC and control mice (Supplemental Figure 3, B and C, and Supplemental Figure 5B), suggestive of organ-specific, rather than systemic, autoimmunity in Traf6ΔTEC animals. Furthermore, analysis of T cells subsets in tissues such as the lung and kidney showed that T cell accumulation was restricted to the liver of Traf6ΔTEC mice, as T cell numbers in these tissues were similar to those of controls (Supplemental Figure 5, A, C, and D). In addition to increased numbers of CD4⁺ and CD8⁺ T cell subsets, an aberrant CD4⁺CD8⁺ double-positive population of T cells was markedly increased in the liver of Traf6ΔTEC mice, but not in other tissues (Figure 5B and Supplemental Figure 5). CD4⁺CD8⁺ cells have been identified in peripheral blood of patients with autoimmune diseases and are thought to have escaped thymic selection (38, 39). Therefore, it is possible that the presence of such cells in the liver of Traf6ΔTEC mice corresponds to abnormal thymic selection of T cells due to the observed mTEC depletion. Alternatively, as CD4⁺CD8⁺ T cells have been shown to represent peripherally hyperactivated T cells in animals and humans (40, 41), their presence in the liver may result from the inflammation induced in this organ by mTEC depletion.

Figure 5

Abnormal T cell presence and immune responses in the liver of Traf6ΔTEC mice. (A) Staining of frozen liver sections from Traf6ΔTEC mice with anti-CD3 and anti-CD4 or anti-CD8 antibodies showed increased presence of CD3⁺CD4⁺ T cells in hepatic portal tracts. (B) Total T cell population numbers were quantified by flow cytometry in liver hematopoietic cell suspensions from WT and Traf6ΔTEC mice stained with different antibodies. n = 8 per genotype. (C) Liver T cells purified from WT or Traf6ΔTEC mice were stained with CFSE, then incubated with liver syngeneic (C57BL/6) or allogeneic (Balb/c) CD11c⁺ APCs in vitro for 72 hours. Percent CFSE-labeled CD4⁺ cells was determined by flow cytometry. n = 7 per genotype, run in triplicates. MLR, mixed lymphocyte reaction. (D) CFSE-labeled liver T cells were incubated 1:1 with syngeneic CD11c⁺ APCs purified from the indicated tissues. 72 hours later, percent CD4⁺CFSE⁺ T cells was determined by flow cytometry. n = 6, run in triplicates. Data are pooled results from 2 independent experiments. (E) CFSE-labeled liver T cells were incubated 1:1 with GM-CSF bone marrow–derived DCs unloaded or loaded with the indicated lysates for 5 days. Percent CD4⁺CFSE⁺ T cells was analyzed by flow cytometry. n = 9 per genotype, run in triplicates. Data are pooled from 3 independent experiments. (F) Intracellular staining of purified WT and Traf6ΔTEC liver T cells was performed with anti–IL-4–PE and anti–IFN-γ–allophycocyanin antibodies and flow cytometry. n = 8 per genotype. Bar graphs represent mean + SEM. Scale bar: 50 μm. **P < 0.01; ***P < 0.001.

Further analysis of Traf6ΔTEC and WT liver T cells revealed that CD44 and CD69 expression increased 3-fold in the Traf6ΔTEC CD4⁺ T cell subset and 6-fold in the Traf6ΔTEC CD8⁺ T cell subset compared with controls (Supplemental Figure 6, A and B), suggesting an activated phenotype for these cells. In contrast, and although activated T cells subsets were found in the spleen and, to a lesser extent, the lung and kidney of Traf6ΔTEC mice, the increase was <1-fold compared with WT controls (Supplemental Figure 6, A and B). These results were consistent with the low prevalence and distribution of infiltrates in the lung and kidney of Traf6ΔTEC animals compared with widespread infiltration in the liver. These data also suggest targeted homing of T cells to the liver of Traf6ΔTEC mice, as opposed to sequestration of activated T cells from the periphery. In support of targeted sequestration, perfusion of the liver prior to T cell isolation and the finding that T cell subsets in the blood of Traf6ΔTEC mice were composed entirely of naive (CD62⁺CD44^–) T cells (Supplemental Figure 6C) argue against recruitment of activated T cells into the liver from circulating blood.

Although the trigger for human AIH is currently unknown, existing evidence suggests that disrupted immune homeostasis plays a permissive role in disease development. Liver damage is thought to be mediated by autoreactive CD4⁺ T cells that recognize and respond to liver-specific peptide(s) (26, 42). Given that mTECs regulate elimination of autoreactive T cells in the thymus and the presence of activated T cells in the liver of Traf6ΔTEC mice, we examined whether liver T cells respond to liver APCs in syngeneic mixed lymphocyte reactions. We found significantly increased proliferation of Traf6ΔTEC liver T cells compared with controls in response to syngeneic APC stimulation (Figure 5C); in contrast, these cells responded to the same extent when stimulated with allogeneic APCs or anti-CD3/CD28 antibodies (Figure 5C and data not shown), which suggests that there were no inherent defects in the ability of WT cells to proliferate. In addition, consistent with normal peripheral responses in Traf6ΔTEC animals, WT and Traf6ΔTEC splenic T cells responded similarly to syngeneic or allogeneic stimulation (Supplemental Figure 7A). The specificity of liver Traf6ΔTEC T cells for liver APC/antigen complexes was further assessed by incubating liver T cells from WT and Traf6ΔTEC animals with syngeneic APCs isolated from different organs. Whereas Traf6ΔTEC liver T cells responded robustly to liver APCs compared with controls, these cells failed to proliferate when stimulated with APCs isolated from the lung or kidney of the same animals and responded similarly to WT T cells when stimulated with splenic APCs (Figure 5D). Furthermore, when the specificity of Traf6ΔTEC liver T cells for liver antigens was tested using in vitro–differentiated APCs loaded with cell extracts from different tissues, we found a significant increase in Traf6ΔTEC liver T cell proliferation in response to stimulation with liver extracts, but not lung or kidney extracts (Figure 5E). The proliferation of Traf6ΔTEC liver T cells under these conditions exhibited longer kinetics as well as a lower level of activation than those observed with liver-derived APCs (Figure 5D), likely reflecting the rarity of autoreactive T cells responding to liver antigens. In contrast, similar to the results with splenic APCs (Figure 5D), there was no significant difference in the proliferation of WT versus Traf6ΔTEC liver T cells when these were stimulated with unloaded APCs (Figure 5E), which suggests that both types of cells respond to endogenous antigens to a similar extent. Collectively, these results suggest that mTEC depletion in the thymus results in the production of autoreactive T cell clones that home predominantly to the liver of Traf6ΔTEC mice, where they bind and respond to liver antigens and promote the development of inflammatory lesions and AIH.

As mentioned above, liver damage associating with AIH development is thought to be mediated by autoreactive CD4⁺ T cells that recognize and respond to liver-specific peptides. Once activated, Th0 cells differentiate into Th1 cells secreting IFN-γ, an important intermediary in tissue destruction, and/or into Th2 cells, which produce IL-4, IL-10, and IL-13 cytokines and promote antibody production by plasma cells (26, 42). Consistent with this model, we found significantly increased numbers of IFN-γ– and IL-4–producing cells and, to a lesser extent, IL-17–producing cells in the liver of Traf6ΔTEC versus WT mice (Figure 5F and data not shown), whereas normal numbers of cells producing these cytokines were found in the spleens of the same animals (Supplemental Figure 7B). Similarly, we found no difference in serum Th1, Th2, Th17, or inflammatory cytokine levels between Traf6ΔTEC and control mice (Supplemental Figure 7C), consistent with normal systemic immune responses in Traf6ΔTEC animals. Thus, the T cell–containing infiltrates in the liver of Traf6ΔTEC mice exhibit an effector T cell phenotype that recapitulates that observed in human AIH and may be driving chronic liver damage and fibrosis in these mice.

Transfer of liver T cells from Traf6ΔTEC donors into immunodeficient mice leads to AIH development. One hallmark of autoimmune disease is the ability of immune cells to adoptively transfer the disease to another host. To determine whether the T cells present in the liver of Traf6ΔTEC animals were able to promote AIH development, sorted liver CD3⁺ T cells were adoptively transferred into Rag1^–/– recipient mice. Rag1^–/– recipients of Traf6ΔTEC liver T cells developed AIH of increased severity compared with the donor mice (Supplemental Figure 8) and with the mice analyzed in Figure 2. Portal and lobular inflammation and interface hepatitis were all significantly increased in Rag1^–/– recipients of Traf6ΔTEC versus WT liver T cells and compared with donor mice (Figure 6, A and B, and Supplemental Figure 8, A and B). The hepatic inflammation correlated with significantly increased levels of ALT in recipients of Traf6ΔTEC liver T cells compared with WT controls and donor mice (Figure 6C and Supplemental Figure 8C). Liver fibrosis was also significantly increased in Rag1^–/– recipients of Traf6ΔTEC liver T cells, with evident tissue damage shown as hepatocyte dropout areas around fibrotic vessels (Figure 6, D and E). The fibrosis was more severe than in Traf6ΔTEC mice (compare Figure 6D and Figure 2D), whereas minimal fibrosis was observed in Rag1^–/– mice receiving WT liver T cells (Figure 6, D and E). Some Rag1^–/– recipient animals developed colitis regardless of the nature of the donor cells (data not shown), which suggests that liver T cells exhibit colitogenic properties. In contrast, transfer of splenic T cells or total splenocytes isolated from the same mice failed to promote disease development in Rag1^–/– recipients (data not shown). Together, these data suggest that Traf6 deletion in TECs and mTEC depletion associates with organ-specific rather than systemic autoimmunity and that production of aberrant liver T cell populations is responsible for AIH development in Traf6ΔTEC mice.

Figure 6

Transfer of Traf6ΔTEC liver T cells in immunodeficient recipients results in development of AIH. (A and B) Liver CD3⁺ T cells purified from WT and Traf6ΔTEC mice were injected into Rag1^–/– mice. Paraffin-embedded, H&E-stained liver sections of recipient animals were analyzed 4–6 weeks after transfer for the presence of infiltrates (arrows). For histopathologic parameters used to define AIH, see Methods and Figure 2. (C) ALT levels in the sera of Rag1^–/– recipients of WT or Traf6ΔTEC liver T cells, indicative of tissue damage, were determined as described in Methods. (D and E) Fibrosis (arrows) due to collagen deposition was quantified by picrosirius red staining, as described in Methods and Figure 2. Arrowheads indicate tissue damage around fibrotic vessels, known as hepatocyte dropout areas. Liver CD3⁺ T cells from 8-week-old WT and Traf6ΔTEC mice were transferred into an equal number of recipient Rag1^–/– mice in these experiments. n ≥ 6 per genotype. Data in A–E are pooled results from 3 independent transfer experiments. Red bars represent mean. Scale bars: 200 μm. *P < 0.05; **P < 0.01; ***P < 0.001.

To further determine whether a specific T cell subpopulation is responsible for AIH transfer in Rag1^–/– recipients, we performed adoptive transfers of sorted T cell subsets from WT and Traf6ΔTEC mice. Total CD3⁺ T cell transfers from different mice were included in these experiments as positive controls (Figure 7, A and B). Transfer of Traf6ΔTEC CD4⁺ T cells led to accumulation of infiltrates in hepatic portal tracks in the majority of recipient Rag1^–/– mice (Figure 7A). However, the histopathological scores of AIH in these recipients were not significantly different from those of mice adoptively transferred with WT cells (Figure 7C). On the other hand, transfer of CD8⁺ cells sorted from the same mice failed to promote infiltrate formation or significant differences in AIH scores in the majority of the recipient animals (Figure 7, A and D). Similar to CD4⁺ T cells, transfer of serum from Traf6ΔTEC and WT mice into Rag1^–/– recipients led to some infiltrate formation, but again, no significant differences were observed in the histopathological AIH scores of the different recipients (Figure 7, A and E). In these experiments, we also attempted to transfer CD4⁺CD8⁺ T cells; however, we failed to purify enough cells from several mice, as these cells were exceedingly rare, especially in WT mice. Therefore, the ability of the CD4⁺CD8⁺ cells to transfer the disease could not be assessed. Finally, analysis of ALT levels of the different recipient mice was consistent with their histopathological scores: increased ALT levels were observed only in recipients of total Traf6ΔTEC CD3⁺ T cells, not in recipients of T cell subsets (Figure 7F). These results suggest that although liver T cells are important for disease development in Traf6ΔTEC mice, a complex interplay among different T cell subsets, and likely B cells, is required for disease development. Thus, CD4⁺ T cells may be responsible for initiating the inflammatory response in an antigen-specific manner, whereas other T cell subsets and/or B cells play a secondary/complimentary role in disease development.

Figure 7

Development of AIH requires the contribution of more than 1 lymphocyte cell subset. (A–E) Sorted liver CD3⁺, CD4⁺, and CD8⁺ T cells and sera purified from 8-week-old WT and Traf6ΔTEC mice were injected into an equal number of Rag1^–/– mice. Paraffin-embedded and H&E-stained liver sections of recipient animals were analyzed approximately 6 weeks after transfer for the presence of infiltrates. (F) ALT levels in the sera of Rag1^–/– recipients adoptively transferred with liver WT or Traf6ΔTEC T cells or sera, determined as described in Figure 6 and Methods. n ≥ 6 per genotype in all transferred groups. Data in A–E are pooled results from 3 independent transfer experiments. PI, portal inflammation; IH, interface hepatitis; LI, lobular inflammation. Red bars represent mean. Scale bar: 100 μm. *P < 0.05; ***P < 0.001.

Traf6ΔTEC mice exhibit normal peripheral tolerance, despite reduced thymic output of nTregs. In addition to regulating the negative selection of autoreactive T cells, mTECs have also been shown to regulate production of nTregs in the thymus (7–9). Because a reduction in the numbers and/or functional impairment of Tregs is thought to correlate with AIH development (43, 44) and because Tregs are potent suppressors of inflammation and autoimmunity, we next examined whether Treg production and function is impaired in Traf6ΔTEC mice. Staining of thymocyte cell suspensions with the Treg-specific marker FOXP3 revealed a 50% decrease in the total number of FOXP3⁺ thymocytes (Figure 8A), consistent with a role for mTECs in nTreg production. However, there was no difference in the number of splenic FOXP3⁺ T cells between Traf6ΔTEC and WT mice, and Traf6ΔTEC CD25⁺FOXP3⁺ splenic T cells were equally able to suppress T effector function in vitro compared with WT controls (Supplemental Figure 9, A and B). On the other hand, we found a marked increase in the number of FOXP3⁺ T cells in Traf6ΔTEC versus WT liver (Figure 8B). The increase in cell numbers correlated with increased production of induced CD25⁺FOXP3⁺ Tregs (iTregs) when Traf6ΔTEC CD4⁺CD25^– liver T cells were incubated with TGF-β in vitro (Figure 8C). In contrast, and consistent with the normal numbers of CD25⁺FOXP3⁺ Tregs in the spleen of Traf6ΔTEC mice (Supplemental Figure 9A), we observed no difference in the percentage of iTregs when splenic Traf6ΔTEC or WT CD4⁺CD25^– T cells were similarly stimulated in vitro (Figure 8C). Consistent with the in vitro production of iTregs, we found significantly increased protein and RNA levels of TGF-β and IL-10 in liver extracts from Traf6ΔTEC animals compared with controls (Figure 8, D and E). Thus, despite the decreased thymic output of nTregs, peripheral mechanisms such as homeostatic expansion of nTregs in the spleen and/or inflammation-driven production of iTregs in the liver of Traf6ΔTEC mice may compensate for the reduced nTreg thymic output in these animals.

Figure 8

Increased numbers of iTregs and cytokines in the liver of Traf6ΔTEC mice. (A and B) Thymocytes and liver hematopoietic cell suspensions isolated from approximately 8-week-old WT and Traf6ΔTEC mice were stained with anti-CD45–Alexa Fluor 750, anti-CD3–PerCP-Cy5.5, anti-CD4–allophycocyanin, anti-CD8–Pacific Blue, anti-CD25–FITC, and anti-FOXP3–PE antibodies and analyzed by flow cytometry. n = 9 per genotype. (C) Sorted liver or spleen CD4⁺CD25^– T cells isolated from approximately 8-week-old WT and Traf6ΔTEC mice were stimulated with TGF-β in vitro and stained with the above antibodies, and iTregs were analyzed by flow cytometry. n = 9 per genotype, run in triplicates. Data in A–C are pooled results from 3 independent experiments. (D and E) Liver lysates from WT and Traf6ΔTEC mice were analyzed for the production of TGF-β and IL-10 cytokines. (D) ELISA. n ≥ 7 per genotype. Data are pooled results from 2 independent experiments run in duplicates. Red bars represent mean. (E) RT-PCR. n = 11 per genotype. Data are pooled results from 2 independent experiments run in triplicates. Bars represent mean + SEM. *P < 0.05; ***P < 0.001.

In this report, we examined the role of impaired mTEC development on T cell–mediated autoimmunity within the context of a normal hematopoietic cell compartment, which provided a unique opportunity to decipher the mechanisms of mTEC differentiation and central tolerance. Peripheral T cell subsets and T cell immune responses were normal in Traf6ΔTEC mice, supporting organ-specific (rather than systemic) development of autoimmunity. Consistent with this, and although the majority of Traf6ΔTEC mice developed autoantibodies against most of the tissues tested, the distribution of inflammatory lesions was largely restricted to the liver — and, to a lesser extent, the lung and kidney — of these mice. The reduced prevalence or absence of infiltrates in nonhepatic tissues was not due to different kinetics of autoimmunity development, as the incidence of inflammatory infiltrates in the different organs was similar between young and older animal cohorts. In addition, the reduction or lack of T cell–containing infiltrates in tissues other than the liver was not due to altered (patchy or diffuse) distribution of immune cells, as quantification of total T cells from the spleen, lung, and kidney of several mice revealed similar numbers of T cells between WT and Traf6ΔTEC animals. This was in contrast to the liver, where we found large increases in the total numbers of both CD4⁺ and CD8⁺ T cells. Given the presence of autoantibodies in the serum and inflammatory infiltrates in the lung and kidney of some Traf6ΔTEC mice, we currently cannot exclude the possibility that these effects also contribute to the overall disease phenotype in Traf6ΔTEC animals. Nonetheless, our results showing (a) complete penetrance of liver inflammation in all Traf6ΔTEC animals tested; (b) increased numbers of total and activated/autoreactive T cells in Traf6ΔTEC liver compared with other tissues; (c) stimulation of Traf6ΔTEC liver T cell proliferation by liver, but not by other APCs, and by APCs loaded with liver cell extracts, but not lung and kidney extracts, and (d) the presence of anti-SLA and other liver-specific autoantibodies in the serum of Traf6ΔTEC mice suggest that the liver is targeted in these mice by autoreactive T cells that escape deletion in the thymus due to mTEC depletion.

It is currently unclear why the liver is the organ predominantly affected in Traf6ΔTEC mice, as expression of all peripheral antigens in mTECs should be similarly affected by mTEC depletion. As different TRAs are stochastically expressed in mTECs (45), it is possible that autoimmune manifestations vary in a manner dependent on the genetic background of the animals, or the nature of the genetic mutation resulting in mTEC depletion. In terms of genetic background, whereas inflammation was mostly contained within the liver in C57BL/6 mice, we observed a wider range of phenotypes affecting different tissues, including the skin, eye, joints, and muscle, in a mixed (129/SvJ/C57BL/6) genetic background. It is possible that mTEC depletion by Traf6 deletion in a more permissive, autoimmune-prone background may reveal a wider spectrum of autoimmune manifestations than in the current mouse model. In terms of the genetic mutations used to deplete mTECs, the phenotype of Traf6ΔTEC mice was in sharp contrast to previous experiments with straight Traf6 knockout mice and other models with defective mTEC development. In Traf6ΔTEC mice, TEC-specific deletion of Traf6 correlates with chronic inflammation, and despite looking unhealthy, the mice survive for at least 1 year after birth. In contrast, in straight knockout mouse models with mutations affecting proteins that regulate the noncanonical NF-κB pathway and mTEC development (including TRAF6), mTEC depletion in homozygous mice correlated not only with peripheral autoimmune manifestations, but also early lethality (12). In those animal models, the hematopoietic compartment was also shown to contribute to autoimmunity development and reduced survival, as the affected proteins were expressed in both the epithelial and the hematopoietic compartments. Autoimmune manifestations in these mice were more pronounced and led to early death due to systemic effects and lymphoproliferation, effects that are absent in Traf6ΔTEC mice, in which the hematopoietic compartment is intact. Consistent with this, T cell–specific deletion of Traf6 (Traf6ΔT mice) led to multiorgan inflammatory disease and lymphadenopathy due to defective Treg-mediated suppression of Traf6ΔT T cell responses (46). Despite the differences in survival between existing models and our Traf6ΔTEC mouse, liver inflammation has been found in all animals with mTEC defects; however, a connection to AIH had not previously been established. Therefore, the extended lifespan of the Traf6ΔTEC mice, together with the chronic, organ-specific (instead of systemic) nature of the inflammation, makes these animals an attractive and convenient model with which to study AIH.

The chronic nature of liver inflammation in Traf6ΔTEC mice correlated with increased presence of FOXP3⁺ T cells in the liver of these animals. Therefore, despite the reduced thymic nTreg output in Traf6ΔTEC animals, the normal presence and function of the peripheral Treg compartment, the increased frequency of iTregs, and the increased levels of TGF-β and the immunosuppressive cytokine IL-10 in the liver of Traf6ΔTEC mice may all be responsible for their lack of overt peripheral T cell responses and the chronic nature of their hepatic inflammation. A correlation between reduced Treg numbers and/or functional impairment of Tregs and development of AIH has been observed in human patients (26). However, a recent report showed that the numbers and suppressive function of FOXP3⁺ T cells isolated from AIH patients were similar to those of normal controls. In addition, the intrahepatic frequency of these cells was higher and correlated with the degree of inflammation in the liver of AIH patients (47). Our results support the latter observations and suggest that in Traf6ΔTEC mice, inflammation-driven production of iTregs in the liver may compensate for the reduced nTreg thymic output and drive the chronic nature of the disease. Consistent with this, existing evidence supports a protective role for iTregs in established or induced autoimmunity in several mouse models (48, 49).

The liver inflammation and AIH development in Traf6ΔTEC mice is quite intriguing, given that the liver is considered a privileged organ from an immunological standpoint. It is well known that transplanted livers are less frequently rejected than other solid organ transplants (50–53). In addition, liver autoimmune diseases like AIH develop with lower frequency compared with other autoimmune conditions, for example, type 1 diabetes and multiple sclerosis (54). Although the causes of AIH remain unknown, current models of AIH development propose that hepatitis development and ensuing tissue damage is orchestrated by naive CD4⁺ T lymphocytes recognizing liver self-peptides. Following antigen binding, activated CD4⁺ Th0 cells differentiate into IFN-γ–secreting Th1 cells that activate macrophages and increase MHCI expression on hepatocytes, rendering liver cells susceptible to CD8⁺ T cell–mediated cytotoxicity. Th0 cells can also differentiate into Th2 effector cells that secrete IL-4, IL-10, and IL-13 cytokines, favoring autoantibody production by plasma cells (26, 42). The major components of this model were recapitulated in the Traf6ΔTEC mice, shown by increased total numbers of CD4⁺ and CD8⁺ T cells, increased production of both Th1 and Th2 cytokines, and increased presence of plasma cells and autoantibody production. In addition, our results showing that individual T cell subsets failed to recapitulate AIH development in recipient mice further supported the idea that complex interplay among lymphocyte cell subsets is required for disease development, with self-antigen recognition by autoreactive CD4⁺ T cells playing a key role in disease initiation. Consistent with this, susceptibility of human patients to AIH has been linked to deviant DRB1 alleles within the human leukocyte antigen (HLA) region (55–57). As these molecules present peptide antigens to CD4⁺ T cells, compromised antigen presentation by susceptibility alleles and recognition of liver self-antigens by naive T lymphocytes is thought to contribute to AIH pathogenesis. A similar mechanism may be in effect in Traf6ΔTEC mice, as antigen presentation was indeed compromised by mTEC depletion in these animals. In addition, our observation of anti-SLA autoantibodies present in the sera of Traf6ΔTEC mice supports faulty antigen presentation in these mice, as anti-SLA autoantibody production has been linked to HLA DRB1*0301 alleles (33, 58), which, together with DRB1*0401 alleles, are the principal genetic risk factors for development of type 1 AIH in certain patients (30, 34–36).

Finally, impaired antigen presentation due to mTEC depletion and elimination of autoreactive T cells in Traf6ΔTEC mice may mimic or replicate the conditions that promote pathogenesis of AIH in humans in different disease settings. The observation that 10%–15% of patients with APECED (the result of impaired AIRE function and antigen expression in mTECs) develop AIH (59, 60) provides further support for impaired antigen presentation and mTEC function in the pathogenesis of AIH. Therefore, despite the existence of several mouse models of AIH involving chemically induced or liver-intrinsic perturbations of hepatic immune responses (25, 54, 61), we believe that the Traf6ΔTEC mouse represents a relevant model by which to study the pathogenic mechanisms leading to development of this disease and explore new therapeutic approaches. Further characterization of the Traf6ΔTEC mouse model can not only improve our understanding of the pathophysiology of AIH, but may also lead to a broader understanding of how liver tolerance is established and maintained, with wider impact on other liver autoimmune diseases and liver transplantation.

View Supplemental data

The authors thank Andrea D. Branch and Arielle Klepper for protocols and advice, Scott L. Friedman and Andrea D. Branch for critically reading the manuscript, Nancy R. Manley for providing foxn1-Cre knockin mice, and Marie E. Grace for advice and technical support with serum biochemical analysis. A.J. Bonito was supported by Icahn School of Medicine at Mount Sinai’s Immunology Institute training T32 AI007605-09 grant and by R01 grant AI068963-01. C. Aloman was supported by grant 1K08DK088954. E.G. Weinstein was supported by R01 grants AI088106-01 and AI068963-01. This work was supported by NIAID grants R01 AI49387-01, R56 AI049387-05, and R01 AI068963-01.

Address correspondence to: Konstantina Alexandropoulos, Icahn School of Medicine at Mount Sinai, One Gustave L. Levy Place, Box 1089, New York, New York 10029, USA. Phone: 212.659.8610; Fax: 212.987.5593; E-mail: k.alexandropoulos@mssm.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2013;123(8):3510–3524. doi:10.1172/JCI65414.

1. Nitta T, Murata S, Ueno T, Tanaka K, Takahama Y. Thymic microenvironments for T-cell repertoire formation. Adv Immunol. 2008;99:59–94.
   View this article via: PubMed Google Scholar
2. Klein L, Hinterberger M, Wirnsberger G, Kyewski B. Antigen presentation in the thymus for positive selection and central tolerance induction. Nat Rev Immunol. 2009;9(12):833–844.
   View this article via: PubMed Google Scholar
3. Anderson MS, et al. Projection of an immunological self shadow within the thymus by the aire protein. Science. 2002;298(5597):1395–1401.
   View this article via: PubMed CrossRef Google Scholar
4. Derbinski J, Schulte A, Kyewski B, Klein L. Promiscuous gene expression in medullary thymic epithelial cells mirrors the peripheral self. Nat Immunol. 2001;2(11):1032–1039.
   View this article via: PubMed CrossRef Google Scholar
5. Tykocinski LO, Sinemus A, Kyewski B. The thymus medulla slowly yields its secrets. Ann N Y Acad Sci. 2008;1143:105–122.
   View this article via: PubMed CrossRef Google Scholar
6. Alexandropoulos K, Danzl NM. Thymic epithelial cells: antigen presenting cells that regulate T cell repertoire and tolerance development. Immunol Res. 2012;54(1–3):177–190.
   View this article via: PubMed CrossRef Google Scholar
7. Aschenbrenner K, et al. Selection of Foxp3+ regulatory T cells specific for self antigen expressed and presented by Aire+ medullary thymic epithelial cells. Nat Immunol. 2007;8(4):351–358.
   View this article via: PubMed CrossRef Google Scholar
8. Apostolou I, Sarukhan A, Klein L, von Boehmer H. Origin of regulatory T cells with known specificity for antigen. Nat Immunol. 2002;3(8):756–763.
   View this article via: PubMed Google Scholar
9. Jordan MS, et al. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat Immunol. 2001;2(4):301–306.
   View this article via: PubMed CrossRef Google Scholar
10. Irla M, Hollander G, Reith W. Control of central self-tolerance induction by autoreactive CD4+ thymocytes. Trends Immunol. 2010;31(2):71–79.
    View this article via: PubMed CrossRef Google Scholar
11. Burkly L, et al. Expression of relB is required for the development of thymic medulla and dendritic cells. Nature. 1995;373(6514):531–536.
    View this article via: PubMed CrossRef Google Scholar
12. Akiyama T, et al. Dependence of self-tolerance on TRAF6-directed development of thymic stroma. Science. 2005;308(5719):248–251.
    View this article via: PubMed CrossRef Google Scholar
13. Kajiura F, et al. NF-kappa B-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner. J Immunol. 2004;172(4):2067–2075.
    View this article via: PubMed Google Scholar
14. Boehm T, Scheu S, Pfeffer K, Bleul CC. Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho-epithelial cross talk via LTbetaR. J Exp Med. 2003;198(5):757–769.
    View this article via: PubMed CrossRef Google Scholar
15. Akiyama T, et al. The tumor necrosis factor family receptors RANK and CD40 cooperatively establish the thymic medullary microenvironment and self-tolerance. Immunity. 2008;29(3):423–437.
    View this article via: PubMed CrossRef Google Scholar
16. Mathis D, Benoist C. Aire. Annu Rev Immunol. 2009;27:287–312.
    View this article via: PubMed CrossRef Google Scholar
17. Akirav EM, Ruddle NH, Herold KC. The role of AIRE in human autoimmune disease. Nat Rev Endocrinol. 2011;7(1):25–33.
    View this article via: PubMed CrossRef Google Scholar
18. Kobayashi T, et al. TRAF6 is a critical factor for dendritic cell maturation and development. Immunity. 2003;19(3):353–363.
    View this article via: PubMed CrossRef Google Scholar
19. Gordon J, et al. Specific expression of lacZ and cre recombinase in fetal thymic epithelial cells by multiplex gene targeting at the Foxn1 locus. BMC Dev Biol. 2007;7:69.
    View this article via: PubMed CrossRef Google Scholar
20. Manley NR, Condie BG. Transcriptional regulation of thymus organogenesis and thymic epithelial cell differentiation. Prog Mol Biol Transl Sci. 2010;92:103–120.
    View this article via: PubMed Google Scholar
21. Farr AG, Anderson SK. Epithelial heterogeneity in the murine thymus: fucose-specific lectins bind medullary epithelial cells. J Immunol. 1985;134(5):2971–2977.
    View this article via: PubMed Google Scholar
22. Gray D, Abramson J, Benoist C, Mathis D. Proliferative arrest and rapid turnover of thymic epithelial cells expressing Aire. J Exp Med. 2007;204(11):2521–2528.
    View this article via: PubMed CrossRef Google Scholar
23. Rossi SW, et al. RANK signals from CD4(+)3(–) inducer cells regulate development of Aire-expressing epithelial cells in the thymic medulla. J Exp Med. 2007;204(6):1267–1272.
    View this article via: PubMed CrossRef Google Scholar
24. Danzl NM, Donlin LT, Alexandropoulos K. Regulation of medullary thymic epithelial cell differentiation and function by the signaling protein Sin. J Exp Med. 2010;207(5):999–1013.
    View this article via: PubMed CrossRef Google Scholar
25. Czaja AJ. Animal models of autoimmune hepatitis. Expert Rev Gastroenterol Hepatol. 2010;4(4):429–443.
    View this article via: PubMed CrossRef Google Scholar
26. Mieli-Vergani G, Vergani D. Autoimmune hepatitis. Nat Rev Gastroenterol Hepatol. 2011;8(6):320–329.
    View this article via: PubMed CrossRef Google Scholar
27. Krawitt EL. Autoimmune hepatitis. N Engl J Med. 2006;354(1):54–66.
    View this article via: PubMed CrossRef Google Scholar
28. Homberg JC, et al. Chronic active hepatitis associated with antiliver/kidney microsome antibody type 1: a second type of “autoimmune” hepatitis. Hepatology. 1987;7(6):1333–1339.
    View this article via: PubMed CrossRef Google Scholar
29. Martini E, Abuaf N, Cavalli F, Durand V, Johanet C, Homberg JC. Antibody to liver cytosol (anti-LC1) in patients with autoimmune chronic active hepatitis type 2. Hepatology. 1988;8(6):1662–1666.
    View this article via: PubMed CrossRef Google Scholar
30. Czaja AJ. Autoantibodies as prognostic markers in autoimmune liver disease. Dig Dis Sci. 2010;55(8):2144–2161.
    View this article via: PubMed CrossRef Google Scholar
31. Baeres M, et al. Establishment of standardised SLA/LP immunoassays: specificity for autoimmune hepatitis, worldwide occurrence, and clinical characteristics. Gut. 2002;51(2):259–264.
    View this article via: PubMed CrossRef Google Scholar
32. Czaja AJ, Shums Z, Norman GL. Frequency and significance of antibodies to soluble liver antigen/liver pancreas in variant autoimmune hepatitis. Autoimmunity. 2002;35(8):475–483.
    View this article via: PubMed CrossRef Google Scholar
33. Czaja AJ, Donaldson PT, Lohse AW. Antibodies to soluble liver antigen/liver pancreas and HLA risk factors for type 1 autoimmune hepatitis. Am J Gastroenterol. 2002;97(2):413–419.
    View this article via: PubMed CrossRef Google Scholar
34. Kanzler S, et al. Clinical significance of autoantibodies to soluble liver antigen in autoimmune hepatitis. J Hepatol. 1999;31(4):635–640.
    View this article via: PubMed CrossRef Google Scholar
35. Ballot E, Homberg JC, Johanet C. Antibodies to soluble liver antigen: an additional marker in type 1 auto-immune hepatitis. J Hepatol. 2000;33(2):208–215.
    View this article via: PubMed CrossRef Google Scholar
36. Krawitt EL. Discrimination of autoimmune hepatitis: autoantibody typing and beyond. J Gastroenterol. 2011;1:39–41.
    View this article via: PubMed Google Scholar
37. Anderson MS, Venanzi ES, Chen Z, Berzins SP, Benoist C, Mathis D. The cellular mechanism of Aire control of T cell tolerance. Immunity. 2005;23(2):227–239.
    View this article via: PubMed CrossRef Google Scholar
38. Parel Y, Chizzolini C. CD4+ CD8+ double positive (DP) T cells in health and disease. Autoimmun Rev. 2004;3(3):215–220.
    View this article via: PubMed CrossRef Google Scholar
39. Parel Y, Aurrand-Lions M, Scheja A, Dayer JM, Roosnek E, Chizzolini C. Presence of CD4+CD8+ double-positive T cells with very high interleukin-4 production potential in lesional skin of patients with systemic sclerosis. Arthritis Rheum. 2007;56(10):3459–3467.
    View this article via: PubMed CrossRef Google Scholar
40. Pahar B, Lackner AA, Veazey RS. Intestinal double-positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines. Eur J Immunol. 2006;36(3):583–592.
    View this article via: PubMed CrossRef Google Scholar
41. Xie D, et al. Peripheral CD4+CD8+cells are the activated T cells expressed granzyme B (GrB), Foxp3, interleukin 17 (IL-17), at higher levels in Th1/Th2 cytokines. Cell Immunol. 2009;259(2):157–164.
    View this article via: PubMed CrossRef Google Scholar
42. Czaja AJ, Manns MP. Advances in the diagnosis, pathogenesis, and management of autoimmune hepatitis. Gastroenterology. 2010;139(1):58–72.e54.
    View this article via: PubMed CrossRef Google Scholar
43. Longhi MS, Ma Y, Mieli-Vergani G, Vergani D. Aetiopathogenesis of autoimmune hepatitis. J Autoimmun. 2010;34(1):7–14.
    View this article via: PubMed CrossRef Google Scholar
44. Ichiki Y, Aoki CA, Bowlus CL, Shimoda S, Ishibashi H, Gershwin ME. T cell immunity in autoimmune hepatitis. Autoimmun Rev. 2005;4(5):315–321.
    View this article via: PubMed CrossRef Google Scholar
45. Derbinski J, Pinto S, Rosch S, Hexel K, Kyewski B. Promiscuous gene expression patterns in single medullary thymic epithelial cells argue for a stochastic mechanism. Proc Natl Acad Sci U S A. 2008;105(2):657–662.
    View this article via: PubMed CrossRef Google Scholar
46. King CG, et al. TRAF6 is a T cell-intrinsic negative regulator required for the maintenance of immune homeostasis. Nat Med. 2006;12(9):1088–1092.
    View this article via: PubMed CrossRef Google Scholar
47. Peiseler M, et al. FOXP3+ regulatory T cells in autoimmune hepatitis are fully functional and not reduced in frequency. J Hepatol. 2012;57(1):125–132.
    View this article via: PubMed CrossRef Google Scholar
48. Lan Q, Fan H, Quesniaux V, Ryffel B, Liu Z, Zheng SG. Induced Foxp3(+) regulatory T cells: a potential new weapon to treat autoimmune and inflammatory diseases? J Mol Cell Biol. 2012;4(1):22–28.
    View this article via: PubMed CrossRef Google Scholar
49. Zhou X, et al. Therapeutic potential of TGF-beta-induced CD4(+) Foxp3(+) regulatory T cells in autoimmune diseases. Autoimmunity. 2011;44(1):43–50.
    View this article via: PubMed CrossRef Google Scholar
50. Calne RY, et al. Induction of immunological tolerance by porcine liver allografts. Nature. 1969;223(5205):472–476.
    View this article via: PubMed CrossRef Google Scholar
51. Qian S, Demetris AJ, Murase N, Rao AS, Fung JJ, Starzl TE. Murine liver allograft transplantation: tolerance and donor cell chimerism. Hepatology. 1994;19(4):916–924.
    View this article via: PubMed Google Scholar
52. Kamada N. The immunology of experimental liver transplantation in the rat. Immunology. 1985;55(3):369–389.
    View this article via: PubMed Google Scholar
53. Crispe IN. Hepatic T cells and liver tolerance. Nat Rev Immunol. 2003;3(1):51–62.
    View this article via: PubMed CrossRef Google Scholar
54. Hardtke-Wolenski M, Jaeckel E. Mouse models for experimental autoimmune hepatitis: limits and chances. Dig Dis. 2010;28(1):70–79.
    View this article via: PubMed CrossRef Google Scholar
55. Donaldson PT. Genetics of liver disease: immunogenetics and disease pathogenesis. Gut. 2004;53(4):599–608.
    View this article via: PubMed CrossRef Google Scholar
56. Czaja AJ, Donaldson PT. Genetic susceptibilities for immune expression and liver cell injury in autoimmune hepatitis. Immunol Rev. 2000;174:250–259.
    View this article via: PubMed CrossRef Google Scholar
57. Donaldson PT. Genetics in autoimmune hepatitis. Semin Liver Dis. 2002;22(4):353–364.
    View this article via: PubMed CrossRef Google Scholar
58. Ma Y, et al. Antibodies to conformational epitopes of soluble liver antigen define a severe form of autoimmune liver disease. Hepatology. 2002;35(3):658–664.
    View this article via: PubMed CrossRef Google Scholar
59. Lankisch TO, Jaeckel E, Strassburg CP. The autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy or autoimmune polyglandular syndrome type 1. Semin Liver Dis. 2009;29(3):307–314.
    View this article via: PubMed CrossRef Google Scholar
60. Strassburg CP. Autoimmune hepatitis. Best Pract Res Clin Gastroenterol. 2010;24(5):667–682.
    View this article via: PubMed CrossRef Google Scholar
61. Christen U, Hintermann E, Jaeckel E. New animal models for autoimmune hepatitis. Semin Liver Dis. 2009;29(3):262–272.
    View this article via: PubMed CrossRef Google Scholar
62. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol. 1991;13(3):372–374.
    View this article via: PubMed CrossRef Google Scholar
63. Ward SC, Schiano TD, Thung SN, Fiel MI. Plasma cell hepatitis in hepatitis C virus patients post-liver transplantation: case-control study showing poor outcome and predictive features in the liver explant. Liver Transpl. 2009;15(12):1826–1833.
    View this article via: PubMed CrossRef Google Scholar
64. Safadi R, et al. Immune stimulation of hepatic fibrogenesis by CD8 cells and attenuation by transgenic interleukin-10 from hepatocytes. Gastroenterology. 2004;127(3):870–882.
    View this article via: PubMed CrossRef Google Scholar