HCV+ hepatocytes induce human regulatory CD4+ T cells through the production of TGF-beta

Abstract

Background: Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease. Impaired T cell responses facilitate and maintain persistent HCV infection. Importantly, CD4(+) regulatory T cells (Tregs) act by dampening antiviral T cell responses in HCV infection. The mechanism for induction and/or expansion of Tregs in HCV is unknown.

Methodology/principal findings: HCV-expressing hepatocytes were used to determine if hepatocytes are able to induce Tregs. The infected liver environment was modeled by establishing the co-culture of the human hepatoma cell line, Huh7.5, containing the full-length genome of HCV genotype 1a (Huh7.5-FL) with activated CD4(+) T cells. The production of IFN-gamma was diminished following co-culture with Huh7.5-FL as compared to controls. Notably, CD4(+) T cells in contact with Huh7.5-FL expressed an increased level of the Treg markers, CD25, Foxp3, CTLA-4 and LAP, and were able to suppress the proliferation of effector T cells. Importantly, HCV(+) hepatocytes upregulated the production of TGF-beta and blockade of TGF-beta abrogated Treg phenotype and function.

Conclusions/significance: These results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses.

Figure 1. HCV⁺ hepatocytes decrease IFN-γ production by CD4⁺ T cells.

A, B) Plate-bound anti-CD3/CD28 activated CD4⁺ T cells were co-cultured with Huh7.5 cells, as well as Huh7.5 cells stably transfected with the non-structural portion of the HCV genome (Huh7.5-SG) or the full HCV genome (Huh7.5-FL). The cells were cultured for 48 hrs in media containing 10 U/ml rhIL-2 at a ratio of 1∶1. The supernatant was analyzed by ELISA. IFN-γ production is presented relative to a no hepatocyte control. Data is representative of 10 healthy CD4⁺ T cell donors examined. C) Primary hepatocytes were cultured on matrigel and exposed to control serum or serum from genotype 1 HCV⁺ patients for 24 hrs. Following infection, cells were washed with media and allowed to proceed with infection for 5 days. RT-PCR analysis of HCV genome was performed to ensure infection had occurred. 5 days after infection, activated CD4⁺ T cells were added to the hepatocyte culture. Supernatant was removed and analyzed by ELISA after 48 hrs of co-culture. ELISA data is representative of 3 separate blood and liver donors.

Figure 2. Huh7.5-FL contact results in an increased abundance of regulatory T cells by phenotype.

A) Representative flow cytometry analysis of CD25 and Foxp3 staining following CD4⁺ T cell/hepatocyte co-culture. Rectangles show double positive gating and numbers reflect percentage of cells in that gate. B) CD25⁺Foxp3^hi data was compiled from 8 healthy CD4⁺ T cell donors. C) Expression of CTLA-4 and LAP in the total CD4⁺ T cell and CD25⁺Foxp3^hi populations was assessed from CD4⁺ T cells co-cultured with Huh7.5-FL. Data is presented in histogram with total CD4⁺ T cells represent in solid grey and CD25⁺Foxp3^hi cells as the black line. Compiled mean fluorescence intensity (MFI) is shown from experiments with 8 CD4⁺ T cell donors. D) CD4⁺ T cell/hepatocyte co-culture was conducted using CD25-depleted CD4⁺ T cells. IFN-γ production is presented relative to no hepatocyte control. Data are compiled from 7 CD4⁺ T cell donors.

Figure 3. Huh7.5-FL induction of a T regulatory phenotype is associated with an increase in CD4⁺ T cell TGF-β production.

A) Cytokines produced by CD4⁺ T cells in hepatocyte co-culture were measured by ELISA (TGF-β and IL-10) or by bead multiplex (IL-17 and IL-5). B) Intracellular TGF-β levels were examined by flow cytometry after hepatocyte co-culture. Gating shows the percentage of TGF-β containing, CD25⁺ T cells. Data are reproducible in 3 independent experiments.

Figure 4. Regulatory T cells developed in Huh7.5-FL co-cultures have suppressive function.

A) CFSE-labeled cell CD4⁺ T cells were used in the CD4⁺ T cell/Huh7.5 co-culture in order to examine proliferative response. Cells considered to have divided demonstrate CFSE dilution. Data was compiled from 5 CD4⁺ T cell donors. B, C) Equal numbers of CD4⁺ T cells were removed after co-culture with Huh7.5 or Huh7.5-FL and placed in co-culture with CFSE-labeled CD4⁺ T cells at a ratio of 1∶1 in the presence of plate-bound anti-CD3/CD28. Data were compiled from 7 CD4⁺ T cell donors. D) Isolated CD25⁺ cells from CD4⁺ T cell/Huh7.5-FL co-culture were incubated with CFSE-labeled T effector responder cells. Data are reproducible in 3 independent experiments.

Figure 5. TGF-β contributes to increased regulatory T cell development.

TGF-β blocking antibody (0.1 µg/mL) was added to the co-culture in order to monitor impairment of Treg development by (A, B) percentage developing a Treg phenotype or (C) IFN-γ production within the co-cultures. Percent suppression is calculated as ((Huh7.5-Huh7.5-FL)/Huh7.5)*100. Data are compiled from 7 CD4⁺ T cell donors.

Figure 6. HCV⁺ hepatocytes cause immunosuppression of CD4⁺ T cells due to enhanced TGF-β production.

A, B) Hepatocyte TGF-β production was assessed by intracellular flow cytometry (A) and by Western blot (B). Data are reproducible in 3 independent experiments. C, D) TGF-β siRNA knockdown was conducted in hepatocytes using 2 different siRNAs targeting TGF-β and a random RNA sequence not found in the human genome as a control. (C) siRNA knockdown was confirmed by intracellular flow staining of TGF-β. (D) IFN-γ production was assessed by ELISA. Data are compiled from 4 CD4⁺ T cell donors.