The CD4 ؉ CD25 ؉ regulatory T lymphocytes have been implicated in suppressing T cell immune responses. Our aim was to characterize the frequency, phenotype, function, and specificity of CD4 ؉ CD25 ؉ T cells in hepatitis C virus (HCV) infection. Peripheral CD4 ؉ CD25 ؉ cells from recovered (n ‫؍‬ 15), chronic infected (n ‫؍‬ 30), and normal control (n ‫؍‬ 15) subjects were analyzed ex vivo for quantitation, phenotype, and effect on HCVspecific interferon gamma production and proliferation. CD4 ؉ CD25 ؉ specificity was determined by intracellular cytokine staining for interleukin 10 (IL-10). A higher proportion of CD4 ؉ CD25 ؉ were found in chronic infection (mean, 3.02%) when compared with recovered (1.64%, P ‫؍‬ .001) and normal controls (2.27%, P ‫؍‬ .02). CD4 ؉ CD25 ؉ cells display CD45RO high , CD45RA low , CD28 high , CD62L high , and CD95 high phenotype. HCVspecific interferon gamma activity was enhanced in peripheral blood mononuclear cells depleted of CD4 ؉ CD25 ؉ and suppressed in peripheral blood mononuclear cells enriched with CD4 ؉ CD25 ؉ . Depletion of CD4 ؉ CD25 ؉ cells also enhanced HCV-specific CD4 ؉ and CD8 ؉ T cell proliferation. Cytokine analysis suggested CD4 ؉ CD25 ؉ cells secrete transforming growth factor beta (TGF-␤ 1 ) and IL-10. The inhibitory role for TGF-␤ 1 was confirmed by anti-TGF-␤ 1 . Transwell studies showed CD4 ؉ CD25 ؉ mediated suppression to be dose dependent and requiring cell contact. CD4 ؉ CD25 ؉ cells showed HCV-specificity through IL-10 production, with a frequency ranging from 1.9% to 5.3%. A positive correlation was detected between CD4 ؉ CD25 ؉ T cell frequency and HCV RNA titer, whereas an inverse relation was found with liver inflammatory activity. In conclusion, CD4 ؉ CD25 ؉ T lymphocytes constitute a highly differentiated population and appear to play a role in viral persistence by suppressing HCV-specific T cell responses in a cell-cell contact manner.

The mechanisms of chronic HBV infection and immunopathogenesis are poorly understood due to a lack of a robust small animal model. Here we report the development of a humanized mouse model with both human immune system and human liver cells by reconstituting the immunodeficient A2/NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with human HLA-A2 transgene) with human hematopoietic stem cells and liver progenitor cells (A2/NSG-hu HSC/Hep mice). The A2/NSG-hu HSC/Hep mouse supported HBV infection and approximately 75% of HBV infected mice established persistent infection for at least 4 months. We detected human immune responses, albeit impaired in the liver, chronic liver inflammation and liver fibrosis in infected animals. An HBV neutralizing antibody efficiently inhibited HBV infection and associated liver diseases in humanized mice. In addition, we found that the HBV mediated liver disease was associated with high level of infiltrated human macrophages with M2-like activation phenotype. Importantly, similar M2-like macrophage accumulation was confirmed in chronic hepatitis B patients with liver diseases. Furthermore, gene expression analysis showed that induction of M2-like macrophage in the liver is associated with accelerated liver fibrosis and necrosis in patients with acute HBV-induced liver failure. Lastly, we demonstrate that HBV promotes M2-like activation in both M1 and M2 macrophages in cell culture studies. Our study demonstrates that the A2/NSG-hu HSC/Hep mouse model is valuable in studying HBV infection, human immune responses and associated liver diseases. Furthermore, results from this study suggest a critical role for macrophage polarization in hepatitis B virus-induced immune impairment and liver pathology.

The liver protects the host from gut-derived pathogens yet is tolerant of antigenic challenge from food and commensal sources. Innate responses involving liver macrophages (Kupffer cells) and effector liver natural killer (NK) cells form the first line in this defense. We address the impact of Toll-like receptor (TLR) signaling on the cross talk between these two cells, and reveal how the liver displays a down-regulated inflammatory response to constitutive bacterial elements through the secretion of interleukin (IL) 10 yet retains a vigorous response to viral challenge. The data support the model that (a) human liver Kupffer cells respond to TLR ligands and indirectly activate NK cells; (b) the activation depends on cell–cell contact; (c) the Kupffer cells synthesize NK cell activating signals, among which IL-18 is critical, and NK cell inhibitory factors, including IL-10; (d) ligands that signal via myeloid differentiation factor 88 induce IL-10, giving a blunted response in the NK cells; and (e) ligands that signal via the Toll–IL-1 receptor domain–containing adaptor inducing interferon (IFN) β–IFN regulatory factor 3 pathway induce less IL-10, and also directly potentiate the stimulatory effect of IL-18 on NK cells, resulting in enhanced activation. Subversion of cellular mechanisms of innate immune response against viruses may be important for hepatotropic viruses (e.g., hepatitis B and C) to develop persistence.

Background and aimsHBV infection represents a major health problem worldwide, but the immunological mechanisms by which HBV causes chronic persistent infection remain only partly understood. Recently, cell subsets with suppressive features have been recognised among monocytes and natural killer (NK) cells. Here we examine the effects of HBV on monocytes and NK cells.MethodsMonocytes and NK cells derived from chronic HBV-infected patients and healthy controls were purified and characterised for phenotype, gene expression and cytokines secretion by flow cytometry, quantitative real-time (qRT)-PCR, ELISA and western blotting. Culture and coculture of monocytes and NK cells were used to determine NK cell activation, using intracellular cytokines staining.ResultsIn chronic HBV infection, monocytes express higher levels of PD-L1, HLA-E, interleukin (IL)-10 and TGF-β, and NK cells express higher levels of PD-1, CD94 and IL-10, compared with healthy individuals. HBV employs hepatitis B surface antigen (HBsAg) to induce suppressive monocytes with HLA-E, PD-L1, IL-10 and TGF-β expression via the MyD88/NFκB signalling pathway. HBV-treated monocytes induce NK cells to produce IL-10, via PD-L1 and HLA-E signals. Such NK cells inhibit autologous T cell activation.ConclusionsOur findings reveal an immunosuppressive cascade, in which HBV generates suppressive monocytes, which initiate regulatory NK cells differentiation resulting in T cell inhibition.