A total of 20 patients with new onset were enrolled in this study at Southwest Hospital (Chongqing, China). Of these 20 patients, 5 were carriers of the -1993TC genotype and 15 were carriers of the -1993TT genotype. All the patients fulfilled the criteria for AIH according to the 1999 revised scoring system of the International Autoimmune Hepatitis Group (IAIHG)[25] and were therefore found to be eligible for the study. Only pretreatment scores were analyzed. A definite diagnosis of AIH based on the IAIHG criteria requires a pretreatment score exceeding 15. Patients were excluded from the study if there was histological evidence of cholangitis or non-alcoholic steatohepatitis. In addition, patients who were positive for hepatitis B virus (HBV)-surface antigen (HBsAg) or hepatitis C virus (HCV)-RNA were also excluded. Patients with other causes of liver disease, such as excess alcohol or drug use, or those who had received immune-suppressive therapies or glucocorticoid therapies within the past 6 mo were excluded based on reviews of their appropriate history and investigations. The control population comprised 35 healthy Chinese Han adults, who were volunteer blood donors from Chongqing, China. They were matched with type 1 AIH patients for age, gender, and TBX21 genotyping (10-1993TC and 25-1993TT genotype carriers), and had no history of any chronic inflammatory disease. All subjects provided written consent to participate in the present study. This study was approved by the Ethics Committee of Southwest Hospital, Chongqing. The demographic and clinical characteristics of these subjects are shown in Table 1.

The clinical data of each subject were collected from the hospital records. These data included age, sex and age at diagnosis, time of onset of symptoms or other evidence of liver disease, markers of infection with hepatitis viruses HBV and HCV, alcohol intake, coexisting autoimmune diseases, serum levels of alanine transaminase (ALT), aspartate aminotransferase (AST), alkaline phosphatase and bilirubin, and full blood cell counts. Autoantibodies were determined by indirect immunofluorescence on murine tissue sections (EuroImmun Medizinische Labordiagnostika AG, Lübeck, Germany), and a serum titer of 1:100 or greater was considered positive for all antibodies. All patients underwent liver biopsies to evaluate disease activity at the time of entry. The histological diagnosis of cirrhosis required a loss of the normal lobular architecture, reconstruction of hepatic nodules and presence of regenerative nodules.

Jurkat cells (CD4⁺ human T lymphoblasts) and Raji cells (human B lymphocytes) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin and 50 mg/mL streptomycin as described previously[23]. Human peripheral blood mononuclear cell (PBMC) were obtained by Ficoll-Paque density centrifugation (Amerhsam Pharmacia, Uppsala, Sweden) of whole blood from AIH-1 patients.

The electrophoretic mobility shift assay (EMSA) was performed by the method described previously[22]. The synthesized oligonucleotides (5’-TACGGAGAAATGG(T)GGGTAAGGTGTTG-3’ corresponding to the -2006/-1980 region of the human TBX21 gene with -1993T) and 5’-TACGGAGAAATGG(C)GGGTAAGGTGTTG-3’, -1993C), labeled with biotin at the 5’-end, were annealed to the complementary oligonucleotide labeled with biotin at the 5’-end. Nuclear extracts were prepared from Jurkat and Raji cells as described previously. For the competition assay, non-labeled double stranded DNA (dsDNA) fragments consisting of the following sequences were used: YY1 5’-CGCTCCCCGGCCATCTTGGCGGCTGGT-3’, and the sequences containing --1993T or -1993C. Supershifts were performed with antibodies to YY1, C/EBPβ (N-10) and c-Jun (N) that were purchased from Santa Cruz Biotechnology. All EMSA experiments were repeated more than three times, and a representative result for each experiment is shown.

The chromatin immunoprecipitation (ChIP) assay was performed using the ChIP-IT Express kit (Active Motif, Carlsbad, CA, United States) as described previously[22]. The following antibodies were used: a rabbit anti-YY1 (C-20; Santa Cruz Biotechnology), anti-C-Jun (N; Santa Cruz Biotechnology), ant-C/EBPβ (Santa Cruz Biotechnology) and a nonspecific rabbit IgG (Santa Cruz Biotechnology). PCR was performed with the following primer, which were designed to amplify the -2048 to -1912 region of the TBX21 promoter: 5’-CCCACTTTGAACATCAGGCAGA-3’ and 5’-CAGCACATCTTTTGAGGTTGGAG-3’.

A reporter plasmid carrying luciferase gene under the control of a 2.2-kb TBX21 promoter fragment carrying the -1993T or -1993C allele was used as described previously[22]. The pCMV-YY1 vector expressing full-size YY1 and its control pCMV6-XL5 were obtained from OriGene.

Harvested cells were suspended in each culture medium supplemented with additional 10% fetal calf serum (FCS). Jurkat cells and Raji cells (2 × 10⁵ cells in 0.5 mL) were cotransfected with 5 μg of the test construct and 25 ng of the pRL-TK Renilla reporter plasmid (Promega) using Nucleofector Kit V(LONZA) according to the manufacturer’s protocol. On the coexpression analysis, 3 μg of pCMV-YY1 or control pCMV6-XL5 was added into the cell suspension. The luciferase activity was measured, as described previously[22].

The isolated PBMCs were plated at a density of 10⁶ cells/ml as described above. siRNA targeted against YY1 (sc-36864) and siRNA consisting of a scrambled sequence that would not lead to specific degradation of any cellular message (control) were purchased from Santa Cruz Biotechnology. The cells in 1 mL of medium were incubated in 5 mmol/L YY1-siRNA in the Nucleofector transfection reagent at room temperature with gentle agitation. Two hours after transfection, fresh medium was added to provide the cells with sufficient nutrients, and the transfected cells were harvested after 48 h for RNA extraction.

The Trizol-purified (Sangon, China) total RNA (2 μg) from PBMCs was used for cDNA synthesis with the RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Canada) following the manufacturer’s instructions. The mRNA expression levels of IFN-γ and of nuclear factors such as YY1 and T-bet were quantified by real-time PCR, and the level of β-actin mRNA was also detected as an internal control for each sample. The relative mRNA expression levels of genes were determined using the comparative (2-ΔΔCt) method. The following primer sequences were used for this analysis: IFN-γ 5’-TCAGATGTAGCGGATAATGGAAC-3’ and 5’-TTCCTTGATGGTCTCCACACTC-3’; T-bet 5’-CCCACTTTGAACATCAGGCAGA-3’ and 5’-CAGCACATCTTTTGAGGTTGGAG-3’; 5’-YY1 GGATAACTCGGCCATGAGAA-3’ and 5’-ATAGGGCCTGTCTCCGGTAT-3’.

The experiments were performed on whole blood using the BD FastImmune CD4 Intracellular Cytokine Detection Kit (cat# 340970, BDIS, San Jose, CA, United States) according to the manufacturer’s instructions. In order to identify Th1 and B effector cells, 0.5 mL heparinized whole blood was stimulated with 2.5 μg/mL PMA and either the anti-CD28/ant-CD49d co-stimulatory monoclonal antibodies (final concentration of 1 μg/mL) or IFN-α2b (2000 U/mL, Schering-Plough, United States)[26]. For the five-color analysis of the CD4⁺ and CD20⁺ cytokine responses, the staining antibody cocktail consisted of anti-IFNγ-FITC/anti-CD4-PerCP(PE)-Cy5.5/anti-CD69-PE/anti-T-bet-Alexa-Fluor 647 (eBioscience, San Diego, United States) and anti-IFNγ-Cy5.5/anti-CD27-PE/anti-CD20-PE- Cy5.5/anti-T-bet-APC (eBioscience). Isotype-matched control antibodies were included to detect non-specific binding to the cells. Each analysis was performed using at least 50000 cells that were gated in the region of the lymphocyte population, as determined by light-scatter properties (forwardscatter versus side-scatter). To analyze the expression of IFN-γ and T-bet in lymphocytes (CD4⁺ T cells and CD20⁺ B cells), cells were gated in both the lymphocyte and CD4⁺/CD20⁺ regions. The dot plots display the events as cytokine+ versus CD69⁺ T or CD27⁺ B cells. The specific responses of the lymphocytes to stimuli were obtained by subtracting the percentage of positive events in the unstimulated sample from that in the activated samples. The isotype-corrected responses of the unstimulated samples were subtracted from those of the activated samples.

Data are expressed as mean ± SD. All statistical analyses were performed in SPSS 19.0 software. The difference between the groups was analyzed by parametric Student’s t-test or nonparametric Mann-Whitney U test. Correlations were examined by Pearson correlation. P values < 0.05 were considered significant.

To determine the potential role of CD4+ T and B cells in the pathogenesis of AIH, 20 patients with active AIH and 35 gender- and age-matched healthy controls were recruited. There was no significant difference in the distribution of age and gender between the patients and healthy controls (Table 1). As expected, the concentrations of serum ALT and AST and total bilirubin in the patients were significantly higher than that in the healthy controls. Furthermore, 15 of 20 patients with active AIH were positive for anti-ANA antibodies, three were positive for anti-SMA antibodies, and two were positive for both. In addition, abnormally higher levels of serum IgG were detected in the patients, demonstrating the liver damage and hypergammaglobulinaemia in the AIH-1 patients.

The results of EMSA showed the formation of two specific DNA-protein complexes (1 and 2) for both -1993T and -1993C allelic probes (Figure 1A and B). The addition of antibody against YY1 led to abrogation of only complex 2 in both the -1993T and -1993C probes (Figure 1A, lanes 3 and 4; Figure 1B, lanes 9 and 10), suggesting that YY1 participates in complex 2 formation. AP1, HSF1 and C/EBPβ did not seem to be involved in the formation of complexes 1 and 2 (Figure 1A, lanes 5-10; Figure 1B, lanes 3-8). A competition assay was performed to confirm whether the -1993C probe interacted more efficiently with the YY1 protein compared with the -1993T. It was found that the complexes formed with the -1993C probe were consistently more intense than these with -1993T (Figure 1C), which was consistent with the results of our study using Jurkat T cells[22]. In comparison with complex 1, we also observed that complex 2 required higher concentrations of competitors to diminish its band intensity. These data indicate that the complexes contain sequence-specific DNA-binding proteins and that the T-1993C SNP resides in a functionally important nucleotide, and is perhaps more relevant for complex 2.

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Figure 1 Identification of transcription factor binding to the T-1993C SNP site of TBX21 promoter in vitro and in vivo. A, B: EMSA analysis with biotin-labeled probes carrying the -1993T and -1993C allele and with nuclear extract from Jurkat cells and Raji cells was performed in the presence of anti-YY1 antibody; C: EMSA with biotin-labeled probes and with nuclear extract from Raji cells was performed in the presence of 100-200-fold excess of unlabeled self-oligonucleotide or YY1 probe; D: In vivo binding of YY1 to the T-1993C SNP site of the TBX21 promoter. ChIP assays with an anti-YY1, anti-C/EBPβ, ant-C-Jun, or control antibody (rabbit IgG) were performed on Jurkat cells or Raji cells. Input DNA or Immunoprecipitated DNA was used as template for PCR amplification of a 137-bp amplicon encompassing TBX21-1993. EMSA: Electrophoretic mobility shift assay; ChIP: Chromatin immunoprecipitation.

ChIP assay was performed to further determine if YY1 binds to the T-1993C element of the TBX21 promoter in vivo. PCR amplification with the primers specific to the TBX21 promoter region, wherein the YY1-binding sites exist at the T-1993C locus, demonstrated a 137-bp product in both Jurkat and Raji chromatin after immunoprecipitation with the anti-YY1 antibody, but not with the ant-C/EBPβ and ant-C-Jun antibodies, or rabbit IgG (Figure 1D). These results demonstrate the direct interaction of YY1 with the TBX21 promoter in a non-cell-specific manner.

Luciferase reporter assay was performed to study transcriptional regulation of the TBX21 promoter constructs carrying either the -1993T or -1993C allele in the Jurkat T cell and Raji B cell lines. The luciferase activity derived from the -1993T promoter was significantly higher than that from the -1993C promoter in both cell lines (P < 0.01) (Figure 2). These data indicate that TBX21-1993C is significantly less active than the -1993T allele in a non-cell-specific manner. We further examined whether the transfection of a YY1 expression vector induced changes in the promoter activity of the -1993T and -1993C constructs in both Jurkat T cells and Raji B cells. We found that the luciferase activity derived from the -1993T promoter was 4-fold lower in the presence of overproduced YY1, and that from the -1993C promoter decreased only by approximately 2-fold (P < 0.01) (Figure 2). These results demonstrated that the YY1 motif upstream of the T-1993C polymorphism plays a strong negative regulatory role in the activity of the TBX21 promoter, and the -1993C promoter responds to YY1 better than the -1993T promoter.

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Figure 2 Effect of YY1 transcription factor on transcriptional activity of TBX21-1993T and -1993C promoter constructs. Luciferase reporter assay showing relative luciferase activity of the -1993T and -1993C promoter constructs following co-transfection of Raji cells or of Jurkat cells with pCMV-YY1 or pCMV6-XL5 (control). The results are expressed as mean ± SD for three independent experiments; ^bP < 0.01.

Flow cytometric analysis revealed that the T-bet expression of circulating CD4⁺ T cells and CD20⁺ B cells in active AIH-1 patients was significantly greater than that in controls (Z = -3.92, P < 0.01, Z = 2.43, P < 0.05,) (Figure 3A and C). The levels of IFN-γ production in circulating CD4⁺ T cells and CD20+ B cells from active AIH-1 patients were significantly higher than that from controls (Z = -4.89, P < 0.01; Z = 2206, P < 0.05) (Figure 3B and D). These data indicate an involvement of Th1 inflammation in Chinese patients with active AIH-1. The disease activity, represented by the IAIHG score, was positively correlated with T-bet expression in CD4⁺ T cells and CD20+ B cells from active AIH-1 patients (Figure 3E and F). Together, these results suggest an association between AIH-1 development and T-bet expression of circulating T cells or B cells.

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Figure 3 Expression of T-bet and IFN-γ in the peripheral blood of AIH-1 patients. A-D: Flow cytometry showing the percentage of cells positive for T-bet and IFN-γ expression among the stimulated CD4⁺ T cells and CD20⁺ B cells from the peripheral bloods of 20 active AIH-1 patients and 35 healthy subjects; E-F: The correlation between the percentage of T-bet-expressing B cells or T cells and the IAIHG score in active AIH-1 patients. IAIHG, the International Autoimmune Hepatitis Group. ^aP < 0.05, ^bP < 0.01 vs controls.

Flow cytometry was performed to analyze T-bet expression and IFN-γ production in circulating T cells and B cells from AIH-1 patients carrying -1993TC and -1993TT genotypes, and from healthy controls carrying -1993TC and -1993TT genotypes (Figure 4). The T-bet expression in CD4⁺ T cells and CD20+ B cells was significantly higher in the -1993TT patients than in the -1993TC patients and the -1993TT controls (Z = -2.75, P < 0.01; Z = -4.33, P < 0.01; Z = -4.21, P < 0.01; Z = -2.75, P < 0.05 respectively) (Figure 5A-D). No significant difference in T-bet expression was observed between in the -1993TC patients and -1993TC controls in both CD4⁺ T cells and CD20⁺ B cells. The levels of IFN-γ production was significantly higher in CD4⁺ T cells and CD20⁺ B cells from the -1993TT patients than from the -1993TC patients and the -1993TT controls (Z = -4.16, P < 0.01; Z = -5.07, P < 0.01; Z = -3.274, P < 0.01; Z = -2.529, P < 0.05 respectively) (Figure 5A-D). No significant difference in IFN-γ production was observed between in the -1993TC patients and -1993TC controls. The results demonstrated that increased IFN-γ and T-bet expression in circulating T and B cells from AIH-1 patients might result primarily from the -1993TT homozygotous patents with AIH-1.

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Figure 4 Representative two-parameter dot plots showing only the cells gated for CD4⁺ T and CD20⁺ B cells from the peripheral bloods of individuals carrying the TBX21-1993TC and -1993TT genotypes. The y-axis of each histogram represents the specific fluorescence of extracellular CD69-phycoerythrin (PE) (T lymphocytes) and CD27-PE (B lymphocytes). The x-axis represents the specific fluorescence of Alexa-Flour 647-T-bet, FITC-IFNγ, or Cy5.5-IFN-γ on four-decade logarithmic scales. The quadrants were assigned using appropriate isotype controls for each intra- and extracellular antibody. A: AIH-1 patients; B: Healthy controls.

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Figure 5 Expression of T-bet and IFN-γ in the peripheral blood of AIH-1 patients carrying -1993TC and -1993TT genotypes. A-D: Flow cytometry showing the percentage of cells positive for T-bet and IFN-γ expression among the stimulated CD4⁺ T cells and CD20⁺ B cells from the peripheral bloods of active AIH-1 patients including 5-1993TC and 15-1993TT genotype carriers, and healthy subjects including 10-1993TC and 25-1993TT genotype carriers. ^aP < 0.05, ^bP < 0.01, vs controls; ^dP < 0.01, TT vs TC.

Knockdown of YY1 with siRNA was performed to analyze whether YY1 affects T-bet and IFN-γ expression of circulating CD4⁺ T and B cells in AIH-1 patients. Real-time PCR assay showed that YY1 siRNA significantly lowered the YY1 mRNA level in PBMCs from AIH patients (t = 13.83, P < 0.01) (Figure 6). The levels of T-bet and IFN-γ mRNA were up-regulated in the YY1-siRNA PBMCs compared with that in the control-siRNA PBMCs (t = -7.61; t = 12.11, P < 0.01 respectively) (Figure 6). These results suggested that YY1 affects type 1 immunity of AIH-1 patients by T-bet-mediated regulation.

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Figure 6 Effects of YY1 knockdown on T-bet and interferon-γ expression in peripheral blood mononuclear cells from autoimmune hepatitis type patients. PBMCs were isolated from 20 AIH-1 patients. PBMCs were transfected with YY1-siRNA or control-siRNA. Forty-eight hours after transfection, the mRNA expression levels of YY1, T-bet and IFN-γ were measured by real-time PCR. ^bP < 0.01 vs control. PBMCs: Peripheral blood mononuclear cell.

Autoimmune hepatitis (AIH) is an autoimmune disease that involves aberrant B and T lymphocyte responses. T-box transcription factor (T-bet) is a key regulator for the lineage commitment in CD4⁺ T hepler 1 and B effector 1 cells by activating the hallmark production of interferon-γ (IFN-γ). Although the role of T-bet has been studied in a variety of autoimmune diseases in mice, its relative importance in human AIH is not characterized. Detailed knowledge about T-bet-medicated immune responses will therefore enhance our understanding of the pathogenesis of AIH and might support the development of new immunomodulatory treatment approaches.

TBX21, which encodes T-bet, harbors many common polymorphisms at a strong linkage disequilibrium, and distinct TBX21 haplotypes are associated with autoimmune diseases in ethnically distinct populations. Our case-control study (84 cases and 318 control subjects) demonstrated a commone single nucleotide polymorphism (SNP) at the -1993 site of the TBX21 gene promoter that was associated with susceptibility to type 1 AIH (AIH-1) in a chinese population. Individuals carrying the -1993C allele had a decreased risk to AIH-1 compared with those without the -1993C allele (OR = 0.22; 95%CI: 0.09-0.56; P = 0.0016). Functional studies are necessary to dissect the mechanisms underlying the contribution of natural genetic variations to TBX21 dysregulation and the susceptibility to AIH-1 development.

In this study, the authors determined the characterization of transcription factor binding to the T-1993C SNP site of the TBX21 promoter in CD4+ T and B cell lines, and measured the expression of T-bet and IFN-γ in peripheral blood CD4⁺ T cells and B cells from active AIH-1 patients carrying -1993TC and -1993TT genotypes, compared with healthy controls carrying -1993TC and -1993TT genotypes, in an attempt to provide functional evidence for the association of the TBX21 T-1993C promoter polymorphism and AIH-1 development.

In vivo, in vitro, and reporter analyses were performed to determine the function of transcription factor Yin-Yang 1(YY1) binding to the T-1993C element of the TBX21 promoter in human CD4⁺ T and B cell lines. Flow cytometry and quantitative real-time PCR were used to analyze T-bet and IFN-γ expressions in CD4⁺ T cells, B cells and monocytes from the peripheral blood of AIH-1 patients including 5-1993TC and 15-1993TT genotype carriers, and healthy controls including 10-1993TC and 25-1993TT genotype carriers. Furthermore, knockdown of YY1 with siRNA was performed to investigate T-bet-medicated regulation of immune response in peripheral blood of AIH-1 patients. The difference between the groups was analyzed by parametric Student’s t-test or nonparametric Mann-Whitney U test. Correlations of T-bet expression in CD4⁺ T cells and B cells from active AIH-1 patients with AIH disease activity, represented by the International Autoimmune Hepatitis Group score, were examined by Pearson correlation.

TBX21-1993C allele created a strong Yin-Yang 1 (YY1)-binding site and decreased TBX21 promoter activity in human CD4⁺ T and B cells. Higher levels of T-bet and IFN-γ were detected in the circulating CD4⁺ T cells and B cells of AIH-1 patients carrying the TBX21-1993 TT genotype compared with the patients carrying the -1993 TC genotype and controls with the -1993 TC genotype. T-bet expression levels of circulating T cells and B cells were also positively correlated with AIH-1 disease activity. Knockdown of YY1 with siRNA caused increased expression of T-bet and IFN-γ in peripheral blood mononuclear cells in AIH-1 patients. This study has limitations, such as a relative small sample size and the lack of functional study of T-bet-expressing T and B cells in the pathogenic process of AIH, as well as no information about T-bet-expressing T and B cells infiltrates in the liver.

Reduced T-bet and IFN-γ expression of circulating CD4⁺ T cells and B cells existed in the individuals carrying the -1993C allele compared with those without the -1993C allele, and played a protective role in AIH-1 development. Moreover, the authors propose a schematic model showing high-affinity binding of YY1 to the -1993C allele site leading to down-regulation of the TBX21 promoter activity, a mechanism for a promoter polymorphism affecting T-bet expression in AIH.

Further studies are necessary to identify polymorphisms in other loci of the genome and to analyze their association with the T-1993C polymorphism for further elucidation of the genomic background of AIH. Furthermore, further longitudinal studies are necessary to analyze the numbers and function of T-bet-expressing T and B cells in the pathogenic process of AIH -1 with a bigger population.