The Role of Regulatory CD4 T Cells in Maintaining Tolerance in a Mouse Model of Autoimmune Hepatitis

Abstract

Background: The role of regulatory CD4 T cells (Treg) in immune-mediated liver disease is still under debate. It remains disputed whether Treg suppress T cell-mediated hepatitis in vivo and whether hepatic regulatory T cells are functional in patients with autoimmune hepatitis.

Methods: We used TF-OVA mice, which express ovalbumin in hepatocytes, to investigate the impact of Treg in a model of autoimmune hepatitis. Treg isolated from inflamed livers of TF-OVA mice were tested for their functionality in vitro. By employing double transgenic TF-OVAxDEREG (DEpletion of REGulatory T cells) mice we analyzed whether Treg-depletion aggravates autoimmune inflammation in the liver in vivo.

Results: CD25+Foxp3+ CD4 T cells accumulated in the liver in the course of CD8 T cell-mediated hepatitis. Treg isolated from inflamed livers were functional to suppress CD8 T-cell proliferation in vitro. Depletion of Treg in TF-OVAxDEREG mice dramatically amplified T cell-mediated hepatitis. Repeated administration of antigen-specific CD8 T cells led to a second wave of inflammation only after depletion of Treg.

Conclusion: Our data add to the evidence for an important role of Treg in autoimmune hepatitis and show that Treg reduce the severity of T-cell mediated hepatitis in vivo. They constitute a key immune cell population that actively maintains a tolerogenic milieu in the liver and protects the liver against repeated inflammatory challenges.

Fig 1. Regulatory T cells accumulate in the liver of mice in the course of hepatitis.

4x10⁶ CD8 OT-I T cells were transferred intravenously into TF-OVA mice. Non-parenchymal cells were isolated from liver and spleen at the indicated days and analyzed for the presence of CD4⁺, CD25⁺, and Foxp3⁺ T cells by flow cytometry. Absolute numbers of (A) CD4⁺ T cells and (B) Foxp3⁺CD25⁺CD4⁺ T cells are depicted (mean±SEM from n = 4–25 mice per time point; ***p<0.001, **p<0.01, *p<0.05 by Mann-Whitney test). (C) Immunohistochemistry for CD3 (red membrane staining) and Foxp3 (brown nuclear staining) of livers from mice at the indicated days after transfer of CD8 OT-I T cells (magnification 200x), arrows indicate exemplary CD3⁺Foxp3⁺ cells). Representative images from 3–4 mice per time point are depicted.

Fig 2. Hepatic regulatory T cells display effector-Treg phenotype and suppress the proliferation of CD8 OT-I T cells in vitro.

(A) CFSE-labeled CD8 OT-I T cells were stimulated by APCs isolated from spleens of TF-OVA mice for three days alone or in co-culture with CD4⁺CD25^- T cells (naïve CD4 T cells), CD4⁺CD25⁺ Treg isolated from lymph nodes and spleens of wild type-mice (control-Treg) or CD4⁺CD25⁺ Treg isolated from livers of TF-OVA mice suffering from hepatitis (liver-Treg). Depicted are representative histograms showing proliferating CFSE-labeled CD8 OT-I T cells at day three, cultured with different CD4 T-cell types (gated on CD8⁺CFSE⁺). (B) The percentage of proliferating CD8 T cell cultured with control-Treg, liver-Treg, or naïve CD4 T cells was calculated by CFSE-intensity relative to the proliferation of CD8 T cells (100% = dotted line) cultured alone. Box plots depict the percentage of CD8 T-cell proliferation. N = 5–9. p-values were calculated using one-sample t-test against a hypothetical value of 100%. **p<0.01. (C) Liver-Treg and control-Treg were generated and isolated as above. CD4⁺CD25⁺Foxp3⁺ cells were analyzed for expression of various markers by flow cytometry. Depicted are mean ± SD from n = 4 mice per group, ***p<0.001, **p<0.01, *p<0.05 by unpaired t-test.

Fig 3. Regulatory T cells suppress CD8 T cell-mediated hepatitis in vivo.

4x10⁶ OT-I CD8 T cells were transferred intravenously into TF-OVAxDEREG mice and treated with DT (diphtheria toxin) or PBS as described in methods. (A) Spleens and livers were analyzed for the presence of Foxp3⁺ T cells on day 5. Plots depict data gated on CD4⁺T cells. ALT (B) and bilirubin (C) were measured in serum on days 4 or 5, respectively. Values from individual mice and mean±SEM are depicted. (D) The number of OT-I T cells was estimated by staining for the Vα2 chain, which forms the OT-I TCR, since no clonotypic antibody is available. The number of CD8⁺Vα2⁺ T cells in liver and spleen was determined by flow cytometry. Depicted are mean±SD from 6–7 mice per group. (E) At day 5 after induction of hepatitis, IFN-γ production of CD8 OT-I T cells isolated from liver and spleen was measured after in vitro cultivation in medium or SIINFEKL. Plots depict mean±SD, events are gated on CD8⁺Vα2⁺T cells, data are from 5–7 mice per group. (F) 5 days after the induction of hepatitis, OT-I CD8 Thy1.1⁺ T cells were purified from liver and spleen and specific lysis was analyzed and calculated as described in methods. Plots depict mean±SD from 5 mice per group. (G) Histology was assessed on day 4 using antibodies against CD3, Foxp3 (magnification 100x), and CD8 (magnification 200x). Representative results from 5–6 mice in each group are shown. Statistics in (B-F) were performed using the Mann-Whitney-test; ***p<0.001, **p<0.01, *p<0.05; ns = not significant.

Fig 4. Regulatory T cells maintain tolerance during T-cell mediated hepatitis.

4*10⁶ CD8 OT-I T cells were transferred into TF-OVAxDEREG mice at day 0 and day 21 (A–C). In addition, Treg were depleted by application of DT, either at the time point of the first transfer of CD8 OT-I T cells (B) or at the time point of the second CD8 OT-I T-cell transfer (C). Control mice received DT alone at both time points (D). Depicted are serum ALT levels of mice at the indicated time points. Statistics were performed using the Wilcoxon-signed-rank-test; p-values<0.05 were considered significant. (A-D) Histology of livers was assessed at day 26 using H&E staining (magnification 100x), as well as antibodies against CD3 (magnification 200x; red membrane staining). Depicted are representative pictures from TF-OVAxDEREG mice treated as indicated in A-D. (E) Box plot depicts the number of CD3⁺T cells in liver sections quantified as described in methods of n = 6–7 mice. (F) Box Plot shows the corresponding ALT-levels measured in sera of n = 5–7 mice at day 26. *p<0.05, **p<0.01; Kruskal–Wallis with Dunn’s posttest.