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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

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+Foxp3hi 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+Foxp3hi 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+Foxp3hi 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.
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pone-0012154-g002: 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+Foxp3hi 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+Foxp3hi 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+Foxp3hi 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.

Mentions: Human CD4+ T cells have been found to maintain plasticity after activation. Recent studies support the possibility that after the initial activation of naïve CD4+ T cells in the lymph nodes, CD4+ T cells are skewed away from a productive antiviral Th1 phenotype [21] and toward a regulatory phenotype in the liver. Regulatory CD4+ T cell development can occur as late as 72 hours after TCR engagement [22]. Therefore, the decreased IFN-γ production by CD4+ T cells co-cultured with Huh7.5-FL may result from the development of Tregs. Treg development was assessed by the expression of CD25 and Foxp3 in CD4+ T cells co-cultured with Huh7.5, Huh7.5-SG or Huh7.5-FL. As shown in Fig. 2A and Fig. 2B, the intracellular CD25 and Foxp3 staining demonstrated a significant increase in CD25+Foxp3hi populations among CD4+ T cells co-cultured with Huh7.5-FL. Since Foxp3 expression in humans appears to be associated with activation in addition to a regulatory phenotype, we also examined the expression of T cell activation marker, CD69, to determine if Huh7.5-FL merely modulated T cell activation status. CD69 was not upregulated on T cells co-cultured with Huh7.5-FL in comparison to Huh7.5. Rather, CD69 expression decreased with Huh7.5-FL (data not shown). CTLA-4 and LAP (a TGF-β binding protein) have been reported to serve as additional markers for functional CD4+ Tregs [23], [24]. Further characterization of the CD25+Foxp3hi population supported the notion that these were regulatory cells as they also up-regulated both CTLA-4 and LAP (Fig. 2C).


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)

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+Foxp3hi 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+Foxp3hi 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+Foxp3hi 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.
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Related In: Results  -  Collection

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pone-0012154-g002: 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+Foxp3hi 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+Foxp3hi 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+Foxp3hi 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.
Mentions: Human CD4+ T cells have been found to maintain plasticity after activation. Recent studies support the possibility that after the initial activation of naïve CD4+ T cells in the lymph nodes, CD4+ T cells are skewed away from a productive antiviral Th1 phenotype [21] and toward a regulatory phenotype in the liver. Regulatory CD4+ T cell development can occur as late as 72 hours after TCR engagement [22]. Therefore, the decreased IFN-γ production by CD4+ T cells co-cultured with Huh7.5-FL may result from the development of Tregs. Treg development was assessed by the expression of CD25 and Foxp3 in CD4+ T cells co-cultured with Huh7.5, Huh7.5-SG or Huh7.5-FL. As shown in Fig. 2A and Fig. 2B, the intracellular CD25 and Foxp3 staining demonstrated a significant increase in CD25+Foxp3hi populations among CD4+ T cells co-cultured with Huh7.5-FL. Since Foxp3 expression in humans appears to be associated with activation in addition to a regulatory phenotype, we also examined the expression of T cell activation marker, CD69, to determine if Huh7.5-FL merely modulated T cell activation status. CD69 was not upregulated on T cells co-cultured with Huh7.5-FL in comparison to Huh7.5. Rather, CD69 expression decreased with Huh7.5-FL (data not shown). CTLA-4 and LAP (a TGF-β binding protein) have been reported to serve as additional markers for functional CD4+ Tregs [23], [24]. Further characterization of the CD25+Foxp3hi population supported the notion that these were regulatory cells as they also up-regulated both CTLA-4 and LAP (Fig. 2C).

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