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

Hall CH, Kassel R, Tacke RS, Hahn YS - PLoS ONE (2010)

Bottom Line: Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease.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.These results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia, USA.

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.

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Related in: MedlinePlus

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.
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pone-0012154-g001: 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.

Mentions: As hepatocytes are the primary site of HCV infection and the establishment of persistent HCV infection is associated with a weak CD4+ antiviral response, we investigated the possibility that HCV-infected hepatocytes directly modulate CD4+ T cell responsiveness. To this end, we established a CD4+ T cell/hepatocyte co-culture using a human hepatoma cell line (Huh7.5), stably transfected with the full HCV genome (Huh7.5-FL) or a subgenomic region of HCV (Huh7.5-SG). To examine CD4+ T cell function, we assessed the production of the key antiviral Th1 cytokine, IFN-γ. Huh7.5 co-culture resulted in an increase in IFN-γ by CD4+ T cells as compared to no hepatocyte treatment. IFN-γ production can be attributed to the CD4+ T cells, as no IFN-γ was produced by the hepatocytes. Interestingly, activated CD4+ T cells produced less IFN-γ when co-cultured with Huh7.5-FL as compared with Huh7.5 or Huh7.5-SG (Fig. 1A). Despite blood donor variability, IFN-γ suppression was statistically significant (Fig. 1B). CD4+ T cells co-cultured with HCV-infected primary hepatocytes also demonstrated decreased IFN-γ production as compared to co-culture with hepatocytes exposed to control serum (Fig. 1C). These results suggest that complete HCV genomic expression within hepatocytes has an immunoregulatory effect on CD4+ T cell function.


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

Hall CH, Kassel R, Tacke RS, Hahn YS - PLoS ONE (2010)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2921368&req=5

pone-0012154-g001: 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.
Mentions: As hepatocytes are the primary site of HCV infection and the establishment of persistent HCV infection is associated with a weak CD4+ antiviral response, we investigated the possibility that HCV-infected hepatocytes directly modulate CD4+ T cell responsiveness. To this end, we established a CD4+ T cell/hepatocyte co-culture using a human hepatoma cell line (Huh7.5), stably transfected with the full HCV genome (Huh7.5-FL) or a subgenomic region of HCV (Huh7.5-SG). To examine CD4+ T cell function, we assessed the production of the key antiviral Th1 cytokine, IFN-γ. Huh7.5 co-culture resulted in an increase in IFN-γ by CD4+ T cells as compared to no hepatocyte treatment. IFN-γ production can be attributed to the CD4+ T cells, as no IFN-γ was produced by the hepatocytes. Interestingly, activated CD4+ T cells produced less IFN-γ when co-cultured with Huh7.5-FL as compared with Huh7.5 or Huh7.5-SG (Fig. 1A). Despite blood donor variability, IFN-γ suppression was statistically significant (Fig. 1B). CD4+ T cells co-cultured with HCV-infected primary hepatocytes also demonstrated decreased IFN-γ production as compared to co-culture with hepatocytes exposed to control serum (Fig. 1C). These results suggest that complete HCV genomic expression within hepatocytes has an immunoregulatory effect on CD4+ T cell function.

Bottom Line: Hepatitis C Virus (HCV) is remarkably efficient at establishing persistent infection and is associated with the development of chronic liver disease.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.These results demonstrate that HCV infected hepatocytes are capable of directly inducing Tregs development and may contribute to impaired host T cell responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Beirne B. Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia, USA.

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.

Show MeSH
Related in: MedlinePlus