Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3⁺ regulatory T cells

Abstract

Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (T_reg) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8⁺ T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) mice to recombination-activating gene (Rag)1^–/– recipients. We then used this robust established adoptive transfer system and co-transferred CD8⁺ T cells from dnTGF-βRII mice with either C57BL/6 or dnTGF-βRII forkhead box protein 3 (FoxP3⁺) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of T_reg from C57BL/6 but not dnTGF-βRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down-regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 T_reg versus dnTGF-βRII T_reg on the ability to down-regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)-10 in T_reg from C57BL/6 compared to dnTGF-βRII mice. Our data reflect the therapeutic potential of wild-type CD4⁺ FoxP3⁺ T_reg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.

Introduction

Regulatory T cells (T_reg) play a pivotal role in control of the immune response in vivo and in vitro both directly by cell–cell contact and indirectly through the production of anti-inflammatory cytokines, including interleukin (IL)-10 and transforming growth factor (TGF)-β; functional impairment and absence leads to uncontrolled inflammation [1–5]. The transcription factor forkhead box protein 3 (FoxP3), a specific marker for T_reg, is expressed in two T_reg subsets: natural T_reg (nT_reg) cells and adaptive or induced T_reg (iT_reg) cells. Both subsets display similar phenotypical characteristics and comparable inhibitory activity against the immune response, although subtle differences in mRNA transcripts, protein expression, epigenetic modification and stability have been documented, although not universally accepted [6]. Reduction of T_reg suppressive function has been reported in the presence of inflammatory cytokines, including IL-1β and IL-6 [7]. T_reg can also assume an activated/memory phenotype; such cells have been described in humans with type 1 diabetes [8].

We have previously reported the spontaneous appearance of a robust autoimmune cholangitis in the dominant negative form of TGF-β receptor type II (dnTGF-βRII) mice [9]. Adoptive transfer of CD8⁺ T cells from dnTGF-βRII mice to recombination-activating gene (Rag)1^–/– recipients leads to the transfer of autoimmune chalangitis [10]. Interestingly, CD8⁺ T cells from OT-I/dnTGF-βRII/Rag1^–/– mice, in which the entire T cell repertoire was replaced with ovalbumin (OVA)-specific CD8, failed to transfer disease [11]. Importantly, mixed (dnTGF-βRII and B6) bone marrow chimeric mice were protected from biliary disease compared to dnTGF-βRII single bone marrow chimerics [12]. Therefore, we focused our attention on the CD8⁺ T cell population and T regulatory compartments in the development of autoimmune cholangitis. We report herein evaluation of the potential therapeutic efficacy of T_reg. In particular, co-transfer of CD4⁺FoxP3⁺ regulatory T cells from B6 mice [wild-type (WT) T_reg] prevents inflammatory cytokine production, portal inflammation and interlobular biliary cell damage induced by adoptive transfer of CD8 cells from dnTGF-βRII mice to Rag1^–/– mice. Importantly, comprehensive analysis of phenotypical markers led to the identification of effector/memory T_reg cells associated with increased activation markers that include CD25, CD39, CD101, OX40, PD-L1 and Helios, and decreased production of IL-10 in FoxP3^GFP/dnTGF-βRII mice. Indeed, correction of this phenotype was successful despite the intrinsic CD8 T cell defects mediated by the dnTGF-βRII transgene. T_reg cells are now within the scope of clinical use to treat autoimmune diseases and control physiological and pathological immune responses [13]. Thus, our current data provide the foundation for the potential use of normal T_reg to regulate aberrant autoimmune T cell responses by adoptive transfer in autoimmune cholangitis.

Materials and methods

Animals

Our colony of dnTGF-βRII mice onto a C57BL/6 (B6) strain background was bred at the animal facilities of the University of California at Davis [9]. Mice were fed a sterile rodent helicobacter medicated dosing system (three-drug combination) diet (Bio-Serv, Frenchtown, NJ, USA) and maintained in individually ventilated cages under specific pathogen-free conditions. Sulfatrim (Hi-tech Pharmacal, Amityville, NY, USA) was delivered through drinking water. B6.Cg-FoxP3^{tm2 (EGFP) Tch}/J mice (FoxP3^GFP/B6 mice) were bred at the animal facilities of the University of California at Davis. FoxP3^GFP/dnTGF-βRII mice were generated as follows. Male dnTGF-βRII mice were mated with female FoxP3^GFP/B6 mice to obtain FoxP3^GFP/dnTGF-βRII male mice, which were subsequently back-crossed with female FoxP3^GFP/B6 mice to obtain FoxP3^GFP/dnTGF-βRII female mice. The parental dnTGF-βRII and the derived FoxP3^GFP/dnTGF-βRII mice at 3–4 weeks of age were genotyped to confirm the dnTGF-βRII gene in their genomic DNA [9] and the peripheral blood from the derived FoxP3^GFP/dnTGF-βRII mice was analysed by flow cytometry to confirm the expression of the FoxP3 green fluorescent protein (GFP) gene. Male hemizygous FoxP3^GFP/dnTGF-βRII mice were back-crossed onto female FoxP3^GFP/B6 mice, respectively. All protocols were approved by the University of California Animal Care and Use Committee.

Isolation and adoptive transfer of CD8⁺ T cells from dnTGF-βRII mice and CD4⁺FoxP3⁺ regulatory T cells from FoxP3^GFP/dnTGF-βRII mice or FoxP3^GFP/B6 mice

The experimental protocol is summarized in Fig. 1. Female recipient Rag1^–/– mice at 8 weeks of age underwent adoptive transfer with purified splenic CD8⁺ T cells from donor dnTGF-βRII mice (dnRII CD8) and splenic CD4⁺FoxP3⁺ regulatory T cells from FoxP3^GFP/dnTGF-βRII mice or FoxP3^GFP/B6 mice. Purified CD8⁺ T cells were prepared using CD8 microbeads (Miltenyi Biotec, Auburn, CA, USA) and the purity of CD8⁺ T cells was greater than 90%. Purified CD4⁺FoxP3⁺ regulatory T cells were prepared using Cytomation MoFlo Cell Sorter (Beckman Coulter, Brea, CA, USA) after negative selection by CD8α, CD19, CD11c, Gr-1 Dynabeads® (Life Technologies, Grand Island, NY, USA). Single-cell suspensions in 100 μl phosphate-buffered saline (PBS) (1 × 10⁶ of CD8 T cells with or without 1 × 10⁶ of FoxP3 T_reg per mouse) were transferred adoptively via orbital vein injection. Eight weeks following adoptive transfer, all recipients were killed and blood, liver, spleen and mesenteric lymph node were collected. The liver specimens were examined for histopathology. Splenic and hepatic MNCs were analysed by flow cytometry [11].

Fig. 1.

Schematic illustration of the experimental protocol.

Flow cytometry analysis

Liver and spleen infiltrating mononuclear cells (MNCs) were isolated from FoxP3^GFP/dnTGF-βRII and FoxP3^GFP/B6 mice [9,10]. The cells were resuspended in staining buffer [0·2% bovine serum albumin (BSA), 0·04% ethylenediamine tetraacetic acid (EDTA) and 0·05% sodium azide in PBS], divided into 25-μl aliquots and incubated with anti-mouse FcR blocking reagent (BioLegend, San Diego, CA, USA) for 15 min at 4°C. Cells were washed and individual aliquots stained for 30 min at 4°C with a panel of fluorochrome-conjugated monoclonal antibody (mAb) that included the cell surface markers CD4, CD44, CD62L, CD103, CD69, OX40, CD73 and PD-1 (Biolegend), T cell receptor (TCR)-β, CD101, CD39, glucocorticoid-induced TNF-receptor (GITR), glycoprotein A repetitions predominant (GARP), programmed death-ligand 1 [PD-L1, or CD274(B7-H1)], TCR-β and CD25 (eBioscience, San Diego, CA, USA). The cells were then washed with PBS containing 0·2% BSA. For intracellular cytokine staining, cells were resuspended in 10% FBS (RPMI-FBI) and stimulated at 37°C for 4 h with leucocyte activation cocktail in the presence of BD GolgiPlug (BD Pharmingen, San Diego, CA, USA). The cells were stained for surface CD8α and TCR-β, fixed and permeabilized with BD Cytofix/Cytoperm Solution (BD Biosciences, San Diego, CA, USA), then stained for intracellular interferon (IFN)-γ (eBioscience), cytotoxic T lymphocyte antigen-4 (CTLA-4), Helios, IL-10 and tumour necrosis factor (TNF)-α (BioLegend) [14]. A fluorescence activated cell sorter (FACS)can flow cytometer (BD Immunocytometry Systems, San Jose, CA, USA) upgraded for detection of five colours by Cytek Development (Fremont, CA, USA) was used to acquire data, which were analysed with Cellquest PRO software (BD Immunocytometry Systems) [14,15].

Histopathology

The liver and spleen of Rag1^–/– recipient mice were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4-μm sections, deparaffinized and stained with haematoxylin and eosin (H&E) [16]. Portal inflammation, lobular inflammation and bile duct damage were evaluated by a ‘blinded’ pathologist. The severity of portal inflammation and lobular inflammation were scored as follows: 0, normal liver histology; 1, minimal inflammation; 2, mild inflammation; 3, moderate inflammation; and 4, severe inflammation. The severity of bile duct damage was graded as: 0, no significant changes of bile duct; 1, epithelial damage (cytoplasmic change); 2, epithelial damage with nuclear change; 3, chronic non-suppurative destructive cholangitis (CNSDC); and 4, bile duct loss. Frequency was scored as follows: 0, none; 1, 1–10%; 2, 11–20%; 3, 21–50%; and 4, more than 50%.

Cell culture and cytokine analysis

Splenic CD8⁺ T cells were isolated and sorted from Rag1^–/– recipient mice with CD8a (Ly-2) MicroBeads (Miltenyi Biotec Inc., Auburn, CA, USA). Aliquots of 2·0 × 10 [5] CD8⁺ T cells were cultured in 96-well round-bottomed plates in 200 μl of RPMI media supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Gibco-Invitrogen Corp., Grand Island, NY, USA), 100 μg/ml streptomycin, 100 U/ml penicillin and 0·5 μg/ml each of anti-CD3 (BioLegend) and anti-CD28 (BioLegend). The cells were incubated for 72 h at 37°C in a humidified 5% CO₂ incubator, then centrifuged. The supernatant was collected and analysed for concentration of cytokines [14,15]. In addition, total protein was extracted from 30 mg of frozen liver tissue by homogenization in T-Per® Tissue Protein Extraction buffer (Thermo, Rockford, IL, USA) containing a protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The homogenized tissue suspension was centrifuged at 12 096 g for 20 min at 4°C and supernatant fluid stored at −80°C. Using serum, tissue lysate and supernatants from cell cultures, levels of IFN-γ, TNF-α, IL-6 and IL-10 were quantified using a cytokine bead array assay termed the T helper type 1 (Th1)/Th2/Th17 cytokine kit (BD Biosciences). The protein concentration of liver extracts was measured using the BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) [15]. Sera were tested for IL-12/23p40 levels at a dilution of 1:4 utilizing the Quantikine enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA).

Statistical analysis

A two-tailed unpaired t-test and Kruskal–Wallis test [non-parametric analysis of variance (anova)] were used to analyse the data; P-values < 0·05 were considered statistically significant.

Results

FoxP3⁺ regulatory T cells from B6 mice (WT T_reg) prevent autoimmune cholangitis

Liver tissues from each of the studies were obtained at 8 weeks post-adoptive transfer of cells. As seen in Fig. 2, the transfer of dnRII CD8 cells into Rag1^–/– mice as a positive control induced cholangitis with portal inflammation, cholangiocyte damage and bile duct loss. Co-transfer of dnRII CD8 and FoxP3⁺ dnRII T_reg did not ameliorate this damage (Fig. 2). In contrast, however, adoptive transfer of dnRII CD8 together with FoxP3⁺ WT T_reg resulted in a significant reduction in both the severity and frequency of portal pathology and interlobular biliary cell damage (Fig. 2). We note that although four of seven mice in the dnRII CD8⁺WT T_reg group exhibited portal inflammation, the inflammatory scores were minimal and there was no interlobular biliary cell damage (Fig. 2b). Hence, WT T_reg, but not dnTGF-βRII T_reg, successfully reduced dnRII CD8⁺ T cell-mediated autoimmune cholangitis (Fig. 2).

Fig. 2.

Splenic CD4⁺ forkhead box protein 3 (FoxP3)⁺ regulatory T cells (T_reg) from FoxP3^GFP/B6 mice suppresses cholangitis. (a) Representative haematoxylin and eosin (H&E)-stained liver sections from recombination-activating gene (Rag)1^–/– mice at 8 weeks of age underwent adoptive transfer with purified splenic CD8⁺ T cells from donor dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) mice (n = 11) and splenic CD4⁺ FoxP3⁺ regulatory T cells from FoxP3^GFP/dnTGF-βRII (n = 7) or FoxP3^GFP/B6 mice (n = 7). (b) Severity and frequency of portal inflammation and bile duct destruction scores. Data are expressed as individual scores. *P < 0·05; **P < 0·01; Kruskal–Wallis test [non-parametric analysis of variance (anova)].

To further determine whether the improved autoimmune cholangitis was due to decreased hepatic accumulation of dnRII CD8⁺ T cells in the liver of recipients, CD8⁺ T cells from blood, spleen, liver and mesenteric lymph node of adoptive transfer recipients were analysed and compared among the three groups. Our data revealed a significantly decreased frequency of effector memory population (defined as CD44⁺CD62L⁻) in recipients of the dnRII CD8 ⁺ WT T_reg group when compared to recipients of either the dnRII CD8⁺ T cells only or dnRII CD8 ⁺ dnRII T_reg groups in spleen and liver. A reduced frequency of effector memory cells was also found in the peripheral blood and mlNs of dnRII CD8 ⁺ WT T_reg recipients, but the changes did not reach statistical significance when compared with dnRII CD8 ⁺ dnRII T_reg recipients (Fig. 3a). Furthermore, the total count of intrahepatic MNC, CD8⁺ T cells and CD8⁺ effector memory T cells decreased significantly in the dnRII CD8 ⁺ WT T_reg group in comparison to either the dnRII CD8 or dnRII CD8 ⁺ dnRII T_reg groups. It has been reported previously that dnTGF-βRII mice still retain a low level of functional TGF-βRII that can induce some levels of TGF-β signalling in T_reg [17,18]. In agreement with this, reduced intrahepatic CD8⁺ T cells and CD8⁺ effector memory T cells were also detected in the dnRII CD8 ⁺ dnRII T_reg group when compared to the dnRII CD8 group, indicating that dnRII T_reg retained some ability to limit the accumulation of dnRII CD8 effectors. dnRII T_reg, however, could not prevent disease progression (Fig. 3b). Intracellular staining analysis demonstrated that the percentages of IFN-γ and TNF-α-producing CD8⁺ T cells were decreased in liver tissues from dnRII CD8 ⁺ WT T_reg transfer recipients (Fig. 4a). The hepatic production of the proinflammatory cytokines IFN-γ, TNF-α and IL-6 was reduced significantly in dnRII CD8 ⁺ WT T_reg transfer recipients compared to the dnRII CD8 or dnRII CD8 ⁺ WT T_reg groups (Fig. 4b). A similar set of results was obtained in serum samples from the adoptive transfer of WT T_reg but not dnRII T_reg, exemplified by the suppressed production of inflammatory cytokines IFN-γ, TNF-α, IL-6 and IL-12/23p40 by dnRII CD8 (Fig. 4c). Our data clearly support the view that a therapeutic potential of WT T_reg in suppressing dnRII CD8-induced autoimmune cholangitis exists and indicates that in the presence of defects in both T_reg and T effector cells (as seen in the dnTGF-βRII mice), correction of the T_reg defect alone may be sufficient to modify disease successfully.

Fig. 3.

Splenic CD4⁺ forkhead box protein 3 (FoxP3)⁺ regulatory T cells (T_reg) from FoxP3^GFP/B6 mice [wild-type (WT) T_reg] successfully suppresses CD8 T cell activation. (a) Frequency of effector memory (CD44⁺ CD62L^–) subpopulations in CD8 T cell subsets in liver and the periphery. (b) Absolute number of intra-hepatic mononuclear cells (MNCs), CD8 T cells, and effector memory (CD44⁺CD62L^–) subpopulations in CD8 T cell subsets. dnRII CD8 group (n = 11), dnRII CD8 ⁺ dnRII T_reg group (n = 7) and dnRII CD8 ⁺ WT T_reg group (n = 7). Data are expressed as mean ± standard error of the mean. *P < 0·05; **P < 0·01; ***P < 0·001; two-tailed unpaired t-test.

Fig. 4.

Splenic CD4⁺ forkhead box protein 3 (FoxP3)⁺ regulatory T cells (T_reg) from FoxP3^GFP/B6 mice [wild-type (WT) T_reg] successfully suppresses inflammatory cytokine production by CD8 T cells. (a) Intracellular staining analysis for interferon (IFN)-γ and tumour necrosis factor (TNF)-α-expressing CD8 T cells in liver. (b) Level of IFN-γ, TNF-α and interleukin (IL)-6 in liver protein extracts from recipients. (c) Serum levels of IFN-γ, TNF-α, IL12/IL23p40 and IL-6 in recipient mice. dnRII CD8 group (n = 11), dnRII CD8 ⁺ dnRII T_reg group (n = 7) and dnRII CD8 ⁺ WT T_reg group (n = 7). Data are expressed as mean ± standard error of the mean. *P < 0·05; **P < 0·01; ***P < 0·001; two-tailed unpaired t-test.

The phenotype of splenic dnRII T_reg compared to WT T_reg: increased IL-10 production by WT T_reg

To address why dnTGF-βRII T_reg were not sufficient to suppress the activation of dnRII CD8 and protect recipients from dnRII CD8-induced portal inflammation compared to WT T_reg, we examined T_reg frequency, FoxP3 expression and production of IL-10 in splenic FoxP3 ⁺ T_reg. We found that the splenic CD4⁺FoxP3⁺ T_reg population was increased significantly in FoxP3^GFP/dnTGF-βRII mice, while the mean fluorescence intensity (MFI) of FoxP3⁺ was not significantly different between dnRII T_reg and WT T_reg (Fig. 5a). Furthermore, the production of the inhibitory cytokine IL-10 was significantly lower in splenic T_reg from FoxP3^GFP/dnTGF-βRII mice in comparison with FoxP3^GFP/B6 mice (Fig. 5c). We next evaluated the phenotype of T_reg at the site of inflammation (liver), as well as thymus and secondary lymphoid organs. The expression of CD101, CD69, CD39, GARP and Helios were significantly higher, but the expression of CD73 was significantly lower on dnRII T_reg from thymus, spleen, LN and liver compared with WT T_reg (Fig. 5b). The T_reg within the spleen and LN from dnRII mice showed increased expression of GITR and PD-1 but significantly decreased expression of PD-1 in the thymus compared with WT T_reg (Fig. 5b).

Fig. 5.

Phenotypical analysis of splenic dnRII and wild-type (WT) T_reg. (a) Frequency and mean fluorescence intensity (MFI) of CD4⁺ forkhead box protein 3 (FoxP3)⁺ regulatory T cells (T_reg) in spleen from FoxP3^GFP/donor dominant negative form of transforming growth factor beta receptor type II (dnTGF-βRII) (n = 7) and FoxP3^GFP/B6 mice (n = 7). (b) Expression of T_reg activation markers were compared between donor FoxP3^GFP/dnTGF-βRII and FoxP3^GFP/B6 in spleen, lymph node (LN), liver and thymus. CD101, CD103, CD69, OX40, CD73, CD39, glycoprotein A repetitions predominant (GARP), programmed death (PD)-1, glucocorticoid-induced TNF-receptor-related protein (GITR), CD274 (B7-H1) (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), Helios and CD25 as an activation marker. (c) Frequency of IL-10-positive cells in splenic CD4⁺ FoxP3⁺ T_reg from FoxP3^GFP/dnTGF-βRII (n = 3) and FoxP3^GFP/B6 mice. Data are expressed as mean ± standard error of the mean. *P < 0·05; **P < 0·01; ***P < 0·001; two-tailed unpaired t-test.

Discussion

The TGF-β family of cytokines encompass TGF-β1, TGF-β2 and TGF-β3, that are highly pleiotropic with diverse effects on many developmental and physiological processes. TGF-β1 is most abundant in lymphoid organs and has multiple of effects on immunity, including inhibition of T cell proliferation and differentiation and negative effects on macrophage activation and dendritic cell (DC) maturation [19]. Active TGF-β mediates its biological function by binding to TGF-β type I (TGF-βRI) and type II (TGF-βRII) receptors, both of which are serine/threonine kinases. TGF-β engagement with a tetrameric receptor complex consisting of two TGF-βRI molecules and two TGF-βRII molecules activates these receptor kinases, allowing them to phosphorylate downstream targets and to activate different signalling pathways [20]. TGF-β1 has been shown to act as a co-stimulatory factor for the expression of FoxP3 [21], leading to the differentiation of T_reg cells from naive T cells [20,22,23]. Therefore, the disruption of TGFR signalling would lead to T_reg defects such as we have demonstrated herein [12,24–26].

We have reported previously that peripheral blood CD4⁺ CD25^high T_reg were reduced in primary biliary cirrhosis (PBC) patients compared with healthy controls, and the level of FoxP3-expressing T_reg was markedly lower in affected portal tracts of PBC patients compared with patients with chronic hepatitis C (CHC) and autoimmune hepatitis (AIH) [27]. We have postulated that murine autoimmune cholangitis requires defects in both the cytotoxic CD8⁺ T cell effector cells and T_reg cells [12]. Moreover, mixed dnTGF-βRII and B6 bone marrow chimeric mice were protected from biliary disease compared to dnTGF-βRII single bone marrow chimerics [12]. To assess the therapeutic manipulation of CD4⁺FoxP3⁺ T_reg in autoimmune cholangitis, we generated FoxP3^GFP/dnTGF-βRII mice and performed adoptive CD8⁺ T cell transfer from dnTGF-βRII mice together with either dnRII T_reg or WT T_reg. In the present study, we evaluated the suppressive function of WT T_reg in vivo and demonstrated that WT T_reg were sufficient to control abnormal dnRII CD8⁺ T cell activation (Fig. 3a,b) and suppress the proinflammatory cytokine production in peripheral blood and at the inflammatory hepatic site (Fig. 4). Mechanisms underlying T_reg immune suppressive function include direct cell–cell contact and the production of IL-10 and TGF-β. The successful immune suppressive function of WT T_reg in our autoimmune cholangitis model suggests that WT T_reg controlled the disease driven by intrinsic defects within the CD8⁺ T cells via an immunoregulatory pathway, probably including IL-10.

We performed a comprehensive examination of the phenotype of CD4⁺FoxP3⁺ regulatory T cells in FoxP3^GFP/dnTGFβ-RII and FoxP3^GFP/B6 donors. In secondary lymphoid organs such as spleen and LN, there were differences in the expression of several activation markers between dnRII T_reg and WT T_reg. For example, CD101, CD69, CD39, GARP, PD-1, GITR, PD-L1 and CTLA-4 were significantly lower in WT T_reg than in dnRII T_reg mice. CTLA-4 is an important surface molecule linked to the suppressive function of T_reg [28,29]. FoxP3 drives the constitutive expression of CTLA-4 by T_reg. Increased expression level of FoxP3 may promote the expression of CTLA-4 in T_reg from dnTGF-βRII mice. However, the reasons why defective T_reg are associated with increased expression levels of FoxP3 and CTLA-4 need to be further addressed. GARP is essential for the surface expression of latent TGF-β on activated T_reg that represent subsets with highly potent regulatory function [30,31]. The surface expression of GARP on T_reg is greatly enhanced by either TGF-β signal blockage [32] or TCR stimulation [33]. Interestingly, it should be noted that the expression of GARP on the T_reg was not required for suppressive function of T_reg in vitro [33]. Decreased CD73 and CD103 were detected in thymus, liver, spleen and LN from dnRII T_reg compared to WT T_reg (Fig. 2b). It has been documented that adenosine generation catalyzed by CD73 expressed by regulatory T cells mediates immune suppression [34], and CD103⁺ T_reg are potent suppressors of tissue inflammation in several autoimmune diseases [35]. The expression of CD73 [36,37] and CD103 [38–40] on T_reg is known to be inducible by TGF-β. Our findings that CD73 and CD103 expression are reduced on CD4⁺FoxP3⁺ T_reg in dnTGF-βRII mice further emphasizes the regulatory role of TGF-β signalling on the expression of CD73 and CD103. The reduction of CD73 and CD103 in T_reg at the inflammatory site and other organs may be partially responsible for the defective suppressive function of dnRII T_reg. It is of interest to note that while this is the first preclinical evidence of the effectiveness of a cellular-based therapeutic approach in PBC, current data on the role of T_reg abnormalities in PBC remain speculative. Indeed, the effector mechanisms in PBC, as with other examples of mucosal mediated diseases, may have significant individual variations [11,41–48]. We note in the current study the unique and different profile of the phenotypical expression of CD39 and CD73 on dnTGF-βRII T_reg. This differential expression of the two ectonucleotidases may be important for the immunosuppressive T_reg activities. CD39 and CD73 are ectonucleotidases that hydrolyze adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and adenosine 5′-phosphate (AMP) to adenosine, creating a ‘purinergic halo’ surrounding immune cells. While this has not been studied in PBC, we note a variety of previous data which have highlighted differential expressions of a variety of effector pathways in PBC, and we suggest that this is an important area for future research [11,14,15,49–53]. We suggest that further studies are necessary to determine the mechanisms of regulation by CD73 and CD103 on T_reg function.

Conclusion

Taken together, our data demonstrate a distinct T_reg phenotype in dnTGF-βRII mice and show that correction of these T_reg defects with physiologically normal T_reg, was associated with enhanced IL-10 effects, decreased inflammatory cytokines and prevention of disease. Thus, wild-type CD4⁺FoxP3⁺ T_reg have a therapeutic potential in limiting the excessive effector T cell response in autoimmune cholangitis.

Disclosure

There are no competing interests.

Author contributions

H.T., W. Z., G.-X. Y., Y. A., T. T., K. T., P. L., Z. X. L. and T. J. performed the experiments. M. E. G., W. M. R., R. L. C. and A. A. A. designed the experiments. M. E. G., A. A. A., W. M. R. and H. T. wrote the paper.