Regulatory T cells in inflamed liver are dysfunctional in murine primary biliary cholangitis

Abstract

Primary biliary cholangitis (PBC) is a chronic autoimmune disease characterized by immune-mediated destruction of intrahepatic small bile ducts. CD8 T cells play a critical role in biliary destruction. However, regulatory T cells (Tregs) have also been identified in the portal tracts of PBC patients. This study tested the hypothesis that hepatic Tregs in PBC were dysfunctional in suppressing immune responses in disease by using our human PBC-like autoimmune cholangitis (AIC) mouse model induced by 2-octynoic acid-conjugated ovalbumin (2-OA-OVA). Our results showed that female and male mice immunized with 2-OA-OVA developed AIC; however, female AIC mice had more severe liver inflammation and fibrosis than male AIC mice. Levels of functional effector CD8 T cells and their chemoattractants, CXCL9 and CXCL10, in the liver were markedly elevated in female AIC mice than in male AIC mice. These results reinforce that CD8 T cells are the primary effector cells in PBC. The number of hepatic Tregs in AIC mice was also higher than in saline-treated mice, but there was no difference between male and female AIC mice. The suppressive function of AIC Tregs was evident despite a discrepancy in the changes in their co-inhibitory receptors and inhibitory cytokines. However, the expansion of hepatic Tregs by low-dose IL-2 treatment did not reduce immune responses to AIC, which may be due to the dysfunction of Tregs in inhibiting T cells. In conclusion, the function of Tregs in the inflamed liver of PBC was insufficient, and low-dose IL-2 treatment could not restore their function to suppress pathological immune responses. Transferring normal Tregs or directly targeting effector CD8 T cells may be beneficial for treating PBC.

Keywords: CD8 T cell; autoimmune cholangitis; liver inflammation; low-dose IL-2.

Graphical Abstract

Figure 1.

Both male and female mice immunized with 2-OA-OVA developed AIC, whereas female mice had higher disease features. Male and female mice were immunized with 2-OA-OVA or normal saline and examined at 11 weeks post-immunization. (A) Serum levels of anti-PDC-E2 IgG were determined using ELISA. AU, arbitrary unit. (B) IFN-γ and TNF-α mRNA expression in the liver were detected using RT-qPCR. (C) Representative H&E staining (scale bar, 50 µm) and Masson’s trichrome staining (scale bar, 10 µm) of liver sections were shown. The collagen fibers are stained blue. (D) Liver leukocytes were quantified. (E) Collagen I and collagen III mRNA expression in the liver were detected using RT-qPCR. M, male mice; F, female mice. Each dot represents an individual mouse. n = 5‒15 mice per group. All error bars denote ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 2.

Increased cytokines and chemokines in female AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline and examined at 5 weeks post-immunization. (A) IFN-γ and TNF-α mRNA expression in the liver were detected using RT-qPCR. (B) CXCL9 and CXCL10 mRNA expression in the liver were detected using RT-qPCR. M, male mice; F, female mice. Each dot represents an individual mouse. n = 6‒14 mice per group. All error bars denote ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Increased CD8 T cells in female AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline and examined at 5 weeks post-immunization. (A) Lymphocytes in the liver were quantified. (B) Representative flow plots show the gating strategies of different subsets of lymphocytes. (C) Percentages of T, CD4 T, CD8 T, and Tregs on lymphocytes in the liver were detected using flow cytometry. (D) The numbers of hepatic T, CD4 T, CD8 T, and Tregs were quantified. M, male mice; F, female mice. Each dot represents an individual mouse. n = 6‒15 mice per group. All error bars denote ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 4.

Increased effector function of CD8 T cells in female AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline and examined at 5 weeks post-immunization. (A) Representative flow plots show the gating strategies of effector molecules in CD8 T cells. (B) The percentages of IFN-γ, granzyme B, and perforin expression in CD8 T cells were detected. (C) The percentages of IFN-γ expression in CD4 T cells were quantified. M, male mice; F, female mice. For the 2-OA group, each dot represents an individual mouse. For the saline group, each dot represents a pooled sample from 2 to 3 mice. n = 6‒15 mice per group. All error bars denote ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5.

T-cell subsets in AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline and examined at 5 weeks post-immunization. (A) Representative flow plots show the gating strategies of T-cell subsets. (B) The frequency and cell number of hepatic effector and memory CD4 T cells were quantified. (C) The frequency and cell number of hepatic effector and memory CD8 T cells were quantified. (D) The frequency and cell number of hepatic CD4 Trm, Tem, and Tcm cells were quantified. (E) The frequency and cell number of hepatic CD8 Trm, Tem, and Tcm cells were quantified. M, male mice; F, female mice. Each dot represents an individual mouse. n = 7‒8 mice per group. All error bars denote ± SEM. A two-sided unpaired Student’s t-test was used. *P < 0.05; **P < 0.01; ***P < 0.001. #P < 0.05; ##P < 0.01.

Figure 6.

Tregs in AIC mice. Male and female mice were immunized with 2-OA-OVA or normal saline and examined at 5 weeks post-immunization. (A) Representative flow cytometry analysis of CD25, CTLA-4, PD-1, LAG-3, IL-10, and TGF-β (LAP) in hepatic Tregs (red color). The blue color denotes isotype control. (B) Graphical summary of MFI of Foxp3 in Tregs. (C) Graphical summary of frequency and MFI of CD25 expression on Tregs. (D) Graphical summary of frequency and MFI of CTLA-4, PD-1, and LAG-3 on Tregs. (E) Graphical summary of frequency IL-10 and TGF-β (LAP) production in Tregs. (F) Representative histogram of the suppressive assay in T cell proliferation and graphical summary of inhibition percentage by the indicated Tregs of different groups. M, male mice; F, female mice. MFI, mean fluorescence intensity. For the 2-OA group, each dot represents an individual mouse. For the saline group, each dot represents a pooled sample from 2 to 3 mice. n = 3‒14 per group. All error bars denote ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 7.

Hepatic Tregs in AIC did not suppress the progression of AIC, although they were expanded and activated by low-dose IL-2. Mice were injected with AAV-IL-2 (1 × 10⁷ TU/mouse or 2 × 10⁷ TU/mouse) or AAV mock (2 × 10⁷ TU/mouse) 5 days before 2-OA-OVA immunization and examined at 5 weeks post-immunization. (A) Numbers of hepatic Tregs were quantified. (B) Foxp3 expression (MFI) and frequency of CD25⁺ in Tregs were detected. (C) Serum levels of anti-PDC-E2 IgG were determined using ELISA. AU, arbitrary unit. (D) IFN-γ and TNF-α mRNA expression in the liver were detected using RT-qPCR. (E) Lymphocytes in the liver were quantified. (F) The numbers of hepatic NK, NKT, T, CD4 T, and CD8 T cells were quantified. (G) The frequencies of memory (Tmem), effector (Teff), and naïve (Tnaive) phenotypes in CD4 T and CD8 T cells. (H) Graphical summary of frequencies of CD25 and IFN-γ expression in NK, NKT, CD8 T, and CD4 T cells were quantified. (I) Graphical summary of frequency of perforin expression in NK, NKT, and CD8 T cells were quantified. (J) A graphical summary of the frequency of granzyme B expression in NK, NKT, and CD8 T cells was quantified. Group mock means mice were treated with 2 × 10⁷ TU AAV-mock. Group IL-2-1 means mice were treated with 1 × 10⁷ TU AAV-IL-2. Group IL-2-2 means mice were treated with 2 × 10⁷ TU AAV-IL-2. MFI, mean fluorescence intensity. Each dot represents an individual mouse. n = 4‒14 mice per group. All error bars denote ± SEM. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Figure 8.

Dysfunctional Tregs in low-dose IL-2 treated AIC mice. Mice were injected with AAV-IL-2 (1 × 10⁷ TU/mouse or 2 × 10⁷ TU/mouse) or AAV mock (2 × 10⁷ TU/mouse) 5 days before 2-OA-OVA immunization and examined at 5 weeks post-immunization. (A) The frequencies of memory (Tmem), effector (Teff), and naïve (Tnaive) phenotypes in Treg cells. (B-D) Graphical summary of frequency and MFI of CTLA-4, PD-1, and LAG-3 on Tregs. (E) Graphical summary of frequency IL-10 and TGF-β (LAP) production in Tregs. (F) Representative histogram of the suppressive assay in T-cell proliferation and graphical summary of inhibition percentage by the indicated Tregs of different groups. Group mock means mice were treated with 2 × 10⁷ TU AAV-mock. Group IL-2-1 means mice were treated with 1 × 10⁷ TU AAV-IL-2. Group IL-2-2 means mice were treated with 2 × 10⁷ TU AAV-IL-2. MFI, mean fluorescence intensity. Each dot represents an individual mouse. n = 6‒13 mice per group. All error bars denote ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.