HCV+ Hepatocytes Induce Human Regulatory CD4+ T Cells through the Production of TGF-β

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-c 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-b and blockade of TGF-b 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

Liver Is Able to Activate Naïve CD8+ T Cells with Dysfunctional Anti-Viral Activity in the Murine System

The liver possesses distinct tolerogenic properties because of continuous exposure to bacterial constituents and nonpathogenic food antigen. The central immune mediators required for the generation of effective immune responses in the liver environment have not been fully elucidated. In this report, we demonstrate that the liver can indeed support effector CD8+ T cells during adenovirus infection when the T cells are primed in secondary lymphoid tissues. In contrast, when viral antigen is delivered predominantly to the liver via intravenous (IV) adenovirus infection, intrahepatic CD8+ T cells are significantly impaired in their ability to produce inflammatory cytokines and lyse target cells. Additionally, intrahepatic CD8+ T cells generated during IV adenovirus infection express elevated levels of PD-1. Notably, lower doses of adenovirus infection do not rescue the impaired effector function of intrahepatic CD8+ T cell responses. Instead, intrahepatic antigen recognition limits the generation of potent anti-viral responses at both priming and effector stages of the CD8+ T cell response and accounts for the dysfunctional CD8+ T cell response observed during IV adenovirus infection. These results also implicate that manipulation of antigen delivery will facilitate the design of improved vaccination strategies to persistent viral infection

Hepatitis D double reflex testing of all hepatitis B carriers in low-HBV- and high-HBV/HDV-prevalence countries

Hepatitis D virus (HDV) infection occurs as a coinfection with hepatitis B and increases the risk of hepatocellular carcinoma, decompensated cirrhosis, and mortality compared to hepatitis B virus (HBV) monoinfection. Reliable estimates of the prevalence of HDV infection and disease burden are essential to formulate strategies to find coinfected individuals more effectively and efficiently. The global prevalence of HBV infections was estimated to be 262,240,000 in 2021. Only 1,994,000 of the HBV infections were newly diagnosed in 2021, with more than half of the new diagnoses made in China. Our initial estimates indicated a much lower prevalence of HDV antibody (anti-HDV) and HDV RNA positivity than previously reported in published studies. Accurate estimates of HDV prevalence are needed. The most effective method to generate estimates of the prevalence of anti-HDV and HDV RNA positivity and to find undiagnosed individuals at the national level is to implement double reflex testing. This requires anti-HDV testing of all hepatitis B surface antigen-positive individuals and HDV RNA testing of all anti-HDV-positive individuals. This strategy is manageable for healthcare systems since the number of newly diagnosed HBV cases is low. At the global level, a comprehensive HDV screening strategy would require only 1,994,000 HDV antibody tests and less than 89,000 HDV PCR tests. Double reflex testing is the preferred strategy in countries with a low prevalence of HBV and those with a high prevalence of both HBV and HDV. For example, in the European Union and North America only 35,000 and 22,000 cases, respectively, will require anti-HDV testing annually

Clinical and virological characteristics of hospitalised COVID-19 patients in a German tertiary care centre during the first wave of the SARS-CoV-2 pandemic: a prospective observational study

Purpose: Adequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. The purpose of this work was to identify risk factors associated with need for invasive mechanical ventilation (IMV), to analyse viral kinetics in patients with and without IMV and to provide a comprehensive description of clinical course. Methods: A cohort of 168 hospitalised adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care centre was analysed. Results: Forty-four per cent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95% CI 1.10-1.37, p < 0.01) and history of hypertension (aOR 5.55, 95% CI 2.00-16.82, p < 0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26-46.75, vs 18 days, IQR 16-46.75, respectively, p < 0.01). Median duration of hospitalisation was 9 days (IQR 6-15.5) for non-IMV and 49.5 days (IQR 36.8-82.5) for IMV patients. Conclusions: Our results indicate a short duration of symptoms before admission as a risk factor for severe disease that merits further investigation and different viral load kinetics in severely affected patients. Median duration of hospitalisation of IMV patients was longer than described for acute respiratory distress syndrome unrelated to COVID-19

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

<p>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.</p

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

<p>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.</p

TGF-β contributes to increased regulatory T cell development.

<p>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.</p

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

<p>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.</p

Antigen presentation by liver parenchyma cells induces suboptimal differentiation of CD8⁺ T cells.

<p>2×10⁶ CFSE labeled Thy1.1⁺OT-1⁺CD8⁺ T cells were adoptively transferred into naïve Thy1.2⁺ C57BL/6 mice reconstituted with BalbC or C57BL/6 bone marrow and then the recipient mice were infected with 5×10⁸ PFU Ad-OVA via IV injection one day later. (A) At 48 hours p.i., liver cells were isolated and the proliferative response of OVA-specific CD8⁺ T cells was evaluated. The plots are gated on Thy1.1⁺CD8⁺ T cells. (B) The expression of CD25 and OVA tetramer by proliferating OVA-specific CD8⁺ was evaluated directly <i>ex vivo</i>. The ability of dividing Thy1.1⁺CD8⁺ T cells to produce IFN-γ, and granzyme-B following peptide restimulation was assessed using flow cytometry. The plots are gated on Thy1.1⁺CD8⁺ T cells. Data are representative of two independent experiments.</p

IV adenovirus administration results in the diminished CD8⁺ T cell responses in the liver.

<p>(A, B) C57BL/6 mice were infected with 5×10⁸ PFU Ad-OVA via either SubQ or IV inoculation. At days 7 and 14 p.i., liver leukocytes were isolated and the percentage of OVA tet⁺CD8⁺ T cells (A) was determined by direct <i>ex vivo</i> staining. The percentage of CD8⁺ T cells producing IFN-γ (B) was assessed following OVA peptide restimulation for 5 hours in the presence of monensin. Data are representative of at least two independent experiments for each time point (<i>n</i> = 3/group). (C) C57BL/6 mice were infected with 5×10⁸ PFU Ad-LacZ via either SubQ or IV administration. At days 7 and 14 p.i., the percentage of βgal tet⁺CD8⁺ T cells and IFN-γ-producing CD8⁺ T cells was determined. Data are presented as averages ± SEM (<i>n</i> = 3/group). (D) C57BL/6 mice were infected with Ad-LacZ via IV inoculation at 10⁸, 10⁷, 10⁶, or 10⁵ PFU per mouse. The percentage of βgal tet⁺CD8⁺ T cells and IFN-γ⁺CD8⁺ T cells in the liver was determined at day 7 p.i. Data are representative of three independent experiments. (E) C57BL/6 mice were infected with 5×10⁸ PFU Ad-OVA via SubQ, IV, or IN immunization. At 7 days p.i., the percentage of OVA tet⁺CD8⁺ T cells and IFN-γ⁺CD8⁺ T cells resident in the liver was calculated. Data are representative of two independent experiments.</p