In this report, we demonstrated the contribution of HCV+
hepatocytes to regulatory CD4+
T cells development. Numerous studies have reported an increased frequency of circulating Tregs during chronic HCV infection 
. These Tregs are shown to suppress the antiviral activity of CD8+
T cells and facilitate the establishment of chronic HCV infection. However, the mechanism of Treg induction during HCV infection is unknown. Given the identification of a local TGF-β production as a key determinant of Treg development in mucosal immunity 
, local production of TGF-β in the liver may contribute to the development of Tregs during HCV infection. Indeed, our work supports this possibility by demonstrating a novel mechanism of CD4+
Treg development mediated through TGF-β production by HCV+
hepatocytes. Other studies support the finding that HCV proteins can induce the production of TGF-β 
. These findings have clinical relevance as enhanced TGF-β has been identified in HCV infection 
, and appears to influence disease progression as polymorphisms that reduced TGF-β production correlate with HCV clearance
. Additionally, a dramatic decrease in TGF-β production is seen in patients that respond to HCV antivirals
This is the first report to show hepatocyte-mediated induction of regulatory T cells to suppress effector cell proliferation and antiviral activities. Hepatocyte-mediated immune regulation appears to require full HCV genome or the structural portion to exert maximal effect. Although partial genomic expression by Huh7.5-SG does exert a partial increase in TGF-β expression, the strongest effect requires the portion of the genome containing the structural proteins. Previous work may suggest that this difference is due to HCV core expression in Huh7.5-FL 
. Further analysis of genomic expression is necessary in order to define which HCV gene product(s) contribute to the production of TGF-β.
Reduction of TGF-β by antibody or siRNA treatment reveals the important contribution of TGF-β to Treg induction and immunosuppression. Although no virions are produced from Huh7.5-FL cells 
, we cannot discount the possibility that a small amount HCV viral protein may be released and directly interacting with the CD4+
T cells. Previous work from our lab demonstrates the ability of HCV core protein to interact with gC1q receptor on the surface of T cells to disrupt TCR signaling and reduce cell proliferation by interfering with early signaling events in naïve T cell activation 
. Thus, it is unlikely that this mechanism is at work in the hepatocyte/CD4+
T cell co-culture as the fully activatedCD4+
T cells are exposed to the hepatocyte. Additionally, IFN-γ reduction without a pronounced reduction in proliferation is observed by the CD4+
T cells in this co-culture.
Studies of HCV-infected patients have found an increase in HCV-specific Tregs in addition to an increase in Tregs specific for other pathogens such as influenza and tetanus 
. Our study, identifying an enhancement of TGF-β production by HCV+
hepatocytes, suggests that induction of Tregs of many specificities could occur as they travel through the liver. There may be a relative enhancement of Tregs that react to antigen within the liver due to local sequestration. Although HCV is not considered to be an immunosuppressive virus such as HIV, there is growing evidence that HCV infection predisposes one for a poor response to vaccinations 
and increased susceptibility to infection with opportunistic pathogens 
. This could be due in part to the general enhancement of the Treg population.
Hepatic TGF-β production can also account for immunosuppression of other cell types. It is documented that TGF-β can inhibit proliferation and cytokine secretion or can induce a suppressive phenotype in CD8+
T cells depending on the strength of the costimulation 
. This method of CD8+
T cell functional suppression is supported by our recent studies that Huh7.5-FL reduced IFN-γ production by activated CD8+
T cells (R. Kassel, unpublished). Furthermore, Huh7.5-FL TGF-β production may dampen the immune response by inducing IL-10 production by macrophages 
or by preventing the maturation of DCs 
. Further studies are necessary in order to determine the full impact of HCV-infected hepatocytes on regulation of host immunity.
Multiple mechanisms for Treg-mediated suppression of effector T cell functions have been recently reported. Tregs can modulate the activity of T effectors by the production of the immunosuppressive cytokines IL-10 and TGF-β. TGF-β reduces IFN-γ production by activated CD4+
T cells and can also induce the expression of IL-10 
. This mechanism likely contributed to the reduction in IFN-γ, as the CD4+
T cell TGF-β production was substantially enhanced in the Huh7.5-FL co-culture. Tregs can also directly kill effector cells in a cell contact dependent manner. Recent studies report that natural Tregs mediate apoptosis by perforin or granzyme release 
whereas inducible Tregs require Fas/FasL interaction to induce cell death 
. Indeed this mechanism could contribute to local immunosuppression as FasL was upregulated and apoptosis enhanced with Huh7.5-FL co-culture (data not shown). Therefore, multiple suppressive pathways may contribute to the reduction in IFN-γ in our system.
The appearance of Tregs generated during this model of HCV infection is contradictory to the development of liver inflammation and cirrhosis during later stages of disease progression. This conflict could be explained by the contribution of other inflammatory cytokines counteracting the TGF-β produced by the infected hepatocytes. One potential cytokine is IL-6 which accumulates in the liver during chronic liver disease 
. Importantly, high concentrations of IL-6 are able to block TGF-β mediated Treg development and enhance the development of a Th17 population. Enhancement of Th17 cells has been noted in the livers of chronic HCV patients 
. Additionally TGF-β is known to contribute directly to the induction of fibrosis by stimulating collagen production 
. A balance of pro- and anti-inflammatory cytokines is likely to contribute for both CD4+
T cell phenotype and antiviral activity during the course of HCV infection.
Previous clinical studies of HCV infections have clearly demonstrated the importance of Tregs to the suppression of an effective immune response. Our work establishes the capability of HCV+ hepatocytes to induce Tregs from activated CD4+ T cells. siRNA data suggests that the hepatocytes are able to affect this change in part by producing the immunosuppressive cytokine, TGF-β. This immunosuppressive influence of HCV+ hepatocytes can contribute to a weak immune response and the development of chronic infection.