Metabolic Activation of Intrahepatic CD8+ T Cells and NKT Cells Causes Nonalcoholic Steatohepatitis and Liver Cancer via Cross-Talk with Hepatocytes

SummaryHepatocellular carcinoma (HCC), the fastest rising cancer in the United States and increasing in Europe, often occurs with nonalcoholic steatohepatitis (NASH). Mechanisms underlying NASH and NASH-induced HCC are largely unknown. We developed a mouse model recapitulating key features of human metabolic syndrome, NASH, and HCC by long-term feeding of a choline-deficient high-fat diet. This induced activated intrahepatic CD8+ T cells, NKT cells, and inflammatory cytokines, similar to NASH patients. CD8+ T cells and NKT cells but not myeloid cells promote NASH and HCC through interactions with hepatocytes. NKT cells primarily cause steatosis via secreted LIGHT, while CD8+ and NKT cells cooperatively induce liver damage. Hepatocellular LTβR and canonical NF-κB signaling facilitate NASH-to-HCC transition, demonstrating that distinct molecular mechanisms determine NASH and HCC development

Enolase represents a metabolic checkpoint controlling the differential exhaustion programmes of hepatitis virus-specific CD8 + T cells

Objective: Exhausted T cells with limited effector function are enriched in chronic hepatitis B and C virus (HBV and HCV) infection. Metabolic regulation contributes to exhaustion, but it remains unclear how metabolism relates to different exhaustion states, is impacted by antiviral therapy, and if metabolic checkpoints regulate dysfunction. Design: Metabolic state, exhaustion and transcriptome of virus-specific CD8+ T cells from chronic HBV-infected (n=31) and HCV-infected patients (n=52) were determined ex vivo and during direct-acting antiviral (DAA) therapy. Metabolic flux and metabolic checkpoints were tested in vitro. Intrahepatic virus-specific CD8+ T cells were analysed by scRNA-Seq in a HBV-replicating murine in vivo model of acute and chronic infection. Results: HBV-specific (core18-27, polymerase455-463) and HCV-specific (NS31073-1081, NS31406-1415, NS5B2594-2602) CD8+ T cell responses exhibit heterogeneous metabolic profiles connected to their exhaustion states. The metabolic state was connected to the exhaustion profile rather than the aetiology of infection. Mitochondrial impairment despite intact glucose uptake was prominent in severely exhausted T cells linked to elevated liver inflammation in chronic HCV infection and in HBV polymerase455-463 -specific CD8+ T cell responses. In contrast, relative metabolic fitness was observed in HBeAg-negative HBV infection in HBV core18-27-specific responses. DAA therapy partially improved mitochondrial programmes in severely exhausted HCV-specific T cells and enriched metabolically fit precursors. We identified enolase as a metabolic checkpoint in exhausted T cells. Metabolic bypassing improved glycolysis and T cell effector function. Similarly, enolase deficiency was observed in intrahepatic HBV-specific CD8+ T cells in a murine model of chronic infection. Conclusion: Metabolism of HBV-specific and HCV-specific T cells is strongly connected to their exhaustion severity. Our results highlight enolase as metabolic regulator of severely exhausted T cells. They connect differential bioenergetic fitness with distinct exhaustion subtypes and varying liver disease, with implications for therapeutic strategies

Improving Therapeutic Vaccination against Hepatitis B—Insights from Preclinical Models of Immune Therapy against Persistent Hepatitis B Virus Infection

Chronic hepatitis B affects more than 250 million individuals worldwide, putting them at risk of developing liver cirrhosis and liver cancer. While antiviral immune responses are key to eliminating hepatitis B virus (HBV) infections, insufficient antiviral immunity characterized by failure to eliminate HBV-infected hepatocytes is associated with chronic hepatitis B. Prophylactic vaccination against hepatitis B successfully established protective immunity against infection with the hepatitis B virus and has been instrumental in controlling hepatitis B. However, prophylactic vaccination schemes have not been successful in mounting protective immunity to eliminate HBV infections in patients with chronic hepatitis B. Here, we discuss the current knowledge on the development and efficacy of therapeutic vaccination strategies against chronic hepatitis B with particular emphasis on the pathogenetic understanding of dysfunctional anti-viral immunity. We explore the development of additional immune stimulation measures within tissues, in particular activation of immunogenic myeloid cell populations, and their use for combination with therapeutic vaccination strategies to improve the efficacy of therapeutic vaccination against chronic hepatitis B

Improving Therapeutic Vaccination against Hepatitis B—Insights from Preclinical Models of Immune Therapy against Persistent Hepatitis B Virus Infection

Chronic hepatitis B affects more than 250 million individuals worldwide, putting them at risk of developing liver cirrhosis and liver cancer. While antiviral immune responses are key to eliminating hepatitis B virus (HBV) infections, insufficient antiviral immunity characterized by failure to eliminate HBV-infected hepatocytes is associated with chronic hepatitis B. Prophylactic vaccination against hepatitis B successfully established protective immunity against infection with the hepatitis B virus and has been instrumental in controlling hepatitis B. However, prophylactic vaccination schemes have not been successful in mounting protective immunity to eliminate HBV infections in patients with chronic hepatitis B. Here, we discuss the current knowledge on the development and efficacy of therapeutic vaccination strategies against chronic hepatitis B with particular emphasis on the pathogenetic understanding of dysfunctional anti-viral immunity. We explore the development of additional immune stimulation measures within tissues, in particular activation of immunogenic myeloid cell populations, and their use for combination with therapeutic vaccination strategies to improve the efficacy of therapeutic vaccination against chronic hepatitis B

In Vivo Bioluminescence Imaging of HBV Replicating Hepatocytes Allows for the Monitoring of Anti-Viral Immunity

Immunity against hepatitis B virus (HBV) infection is complex and not entirely understood so far, including the decisive factors leading to the development of chronic hepatitis B. This lack of a mechanistic understanding of HBV-specific immunity is also caused by a limited number of suitable animal models. Here, we describe the generation of a recombinant adenovirus expressing an HBV 1.3-overlength genome linked to luciferase (Ad-HBV-Luc) allowing for precise analysis of the quantity of infected hepatocytes. This enables sensitive and close-meshed monitoring of HBV-specific CD8 T cells and the onset of anti-viral immunity in mice. A high dose of Ad-HBV-Luc developed into chronic hepatitis B accompanied by dysfunctional CD8 T cells characterized by high expression of PD1 and TOX and low expression of KLRG1 and GzmB. In contrast, a low dose of Ad-HBV-Luc infection resulted in acute hepatitis with CD8 T cell-mediated elimination of HBV-replicating hepatocytes associated with elevated sALT levels and increased numbers of cytotoxic HBV-specific CD8 T cells. Thus, the infectious dose was a critical factor to induce either acute self-limited or chronic HBV infection in mice. Taken together, the new Ad-HBV-Luc vector will allow for highly sensitive and time-resolved analysis of HBV-specific immune responses during acute and chronic infection

In Vivo Bioluminescence Imaging of HBV Replicating Hepatocytes Allows for the Monitoring of Anti-Viral Immunity

Immunity against hepatitis B virus (HBV) infection is complex and not entirely understood so far, including the decisive factors leading to the development of chronic hepatitis B. This lack of a mechanistic understanding of HBV-specific immunity is also caused by a limited number of suitable animal models. Here, we describe the generation of a recombinant adenovirus expressing an HBV 1.3-overlength genome linked to luciferase (Ad-HBV-Luc) allowing for precise analysis of the quantity of infected hepatocytes. This enables sensitive and close-meshed monitoring of HBV-specific CD8 T cells and the onset of anti-viral immunity in mice. A high dose of Ad-HBV-Luc developed into chronic hepatitis B accompanied by dysfunctional CD8 T cells characterized by high expression of PD1 and TOX and low expression of KLRG1 and GzmB. In contrast, a low dose of Ad-HBV-Luc infection resulted in acute hepatitis with CD8 T cell-mediated elimination of HBV-replicating hepatocytes associated with elevated sALT levels and increased numbers of cytotoxic HBV-specific CD8 T cells. Thus, the infectious dose was a critical factor to induce either acute self-limited or chronic HBV infection in mice. Taken together, the new Ad-HBV-Luc vector will allow for highly sensitive and time-resolved analysis of HBV-specific immune responses during acute and chronic infection

MHC Class I-Restricted TCR-Transgenic CD4+ T Cells Against STEAP1 Mediate Local Tumor Control of Ewing Sarcoma In Vivo

In this study we report the functional comparison of T cell receptor (TCR)-engineered major histocompatibility complex (MHC) class I-restricted CD4+ versus CD8+ T cells targeting a peptide from six transmembrane epithelial antigen of the prostate 1 (STEAP1) in the context of HLA-A*02:01. STEAP1 is a tumor-associated antigen, which is overexpressed in many cancers, including Ewing sarcoma (EwS). Based on previous observations, we postulated strong antitumor potential of tumor-redirected CD4+ T cells transduced with an HLA class I-restricted TCR against a STEAP1-derived peptide. We compared CD4+ T cell populations to their CD8+ counterparts in vitro using impedance-based xCELLigence and cytokine/granzyme release assays. We further compared antitumor activity of STEAP130-TCR transgenic (tg) CD4+ versus CD8+ T cells in tumor-bearing xenografted Rag2−/−γc−/− mice. TCR tgCD4+ T cells showed increased cytotoxic features over time with similar functional avidity compared to tgCD8+ cells after 5–6 weeks of culture. In vivo, local tumor control was equal. Assessing metastatic organotropism of intraveniously (i.v.) injected tumors, only tgCD8+ cells were associated with reduced metastases. In this analysis, EwS-redirected tgCD4+ T cells contribute to local tumor control, but fail to control metastatic outgrowth in a model of xenografted EwS

Pioglitazone-mediated peroxisome proliferator-activated receptor γ activation aggravates murine immune-mediated hepatitis

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) regulates target gene expression upon ligand binding. Apart from its effects on metabolism, PPARγ activity can inhibit the production of pro-inflammatory cytokines by several immune cells, including dendritic cells and macrophages. In chronic inflammatory disease models, PPARγ activation delays the onset and ameliorates disease severity. Here, we investigated the effect of PPARγ activation by the agonist Pioglitazone on the function of hepatic immune cells and its effect in a murine model of immune-mediated hepatitis. Cytokine production by both liver sinusoidal endothelial cells (IL-6) and in T cells ex vivo (IFNγ) was decreased in cells from Pioglitazone-treated mice. However, PPARγ activation did not decrease pro-inflammatory tumor necrosis factor alpha TNFα production by Kupffer cells after Toll-like receptor (TLR) stimulation ex vivo. Most interestingly, although PPARγ activation was shown to ameliorate chronic inflammatory diseases, it did not improve hepatic injury in a model of immune-mediated hepatitis. In contrast, Pioglitazone-induced PPARγ activation exacerbated D-galactosamine (GalN)/lipopolysaccharide (LPS) hepatitis associated with an increased production of TNFα by Kupffer cells and increased sensitivity of hepatocytes towards TNFα after in vivo Pioglitazone administration. These results unravel liver-specific effects of Pioglitazone that fail to attenuate liver inflammation but rather exacerbate liver injury in an experimental hepatitis model

Targeted antigen delivery to dendritic cells elicits robust antiviral T cell-mediated immunity in the liver.

Hepatotropic viruses such as hepatitis C virus cause life-threatening chronic liver infections in millions of people worldwide. Targeted in vivo antigen-delivery to cross-presenting dendritic cells (DCs) has proven to be extraordinarily efficient in stimulating antigen-specific T cell responses. To determine whether this approach would as well be suitable to induce local antiviral effector T cells in the liver we compared different vaccine formulations based on either the targeting of DEC-205 or TLR2/6 on cross-presenting DCs or formulations not involving in vivo DC targeting. As read-outs we used in vivo hepatotropic adenovirus challenge, histology and automated multidimensional fluorescence microscopy (MELC). We show that targeted in vivo antigen delivery to cross-presenting DCs is highly effective in inducing antiviral CTLs capable of eliminating virus-infected hepatocytes, while control vaccine formulation not involving DC targeting failed to induce immunity against hepatotropic virus. Moreover, we observed distinct patterns of CD8+ T cell interaction with virus-infected and apoptotic hepatocytes in the two DC-targeting groups suggesting that the different vaccine formulations may stimulate distinct types of effector functions. Our findings represent an important step toward the future development of vaccines against hepatotropic viruses and the treatment of patients with hepatic virus infection after liver transplantation to avoid reinfection