Attenuated effector T cells are linked to control of chronic HBV infection.

Hepatitis B virus (HBV)-specific CD8+ T cells play a dominant role during acute-resolving HBV infection but are functionally impaired during chronic HBV infection in humans. These functional deficits have been linked with metabolic and phenotypic heterogeneity, but it has remained unclear to what extent different subsets of HBV-specific CD8+ T cells still suppress viral replication. We addressed this issue by deep profiling, functional testing and perturbation of HBV-specific CD8+ T cells during different phases of chronic HBV infection. Our data revealed a mechanism of effector CD8+ T cell attenuation that emerges alongside classical CD8+ T cell exhaustion. Attenuated HBV-specific CD8+ T cells were characterized by cytotoxic properties and a dampened effector differentiation program, determined by antigen recognition and TGFß signaling, and were associated with viral control during chronic HBV infection. These observations identify a distinct subset of CD8+ T cells linked with immune efficacy in the context of a chronic human viral infection with immunotherapeutic potential.

Tumor-derived prostaglandin E2 programs cDC1 dysfunction to impair intratumoral orchestration of anti-cancer T cell responses.

Type 1 conventional dendritic cells (cDC1s) are critical for anti-cancer immunity. Protective anti-cancer immunity is thought to require cDC1s to sustain T cell responses within tumors, but it is poorly understood how this function is regulated and whether its subversion contributes to immune evasion. Here, we show that tumor-derived prostaglandin E2 (PGE2) programmed a dysfunctional state in intratumoral cDC1s, disabling their ability to locally orchestrate anti-cancer CD8+ T cell responses. Mechanistically, cAMP signaling downstream of the PGE2-receptors EP2 and EP4 was responsible for the programming of cDC1 dysfunction, which depended on the loss of the transcription factor IRF8. Blockade of the PGE2-EP2/EP4-cDC1 axis prevented cDC1 dysfunction in tumors, locally reinvigorated anti-cancer CD8+ T cell responses, and achieved cancer immune control. In human cDC1s, PGE2-induced dysfunction is conserved and associated with poor cancer patient prognosis. Our findings reveal a cDC1-dependent intratumoral checkpoint for anti-cancer immunity that is targeted by PGE2 for immune evasion.

Intrahepatic myeloid-cell aggregates enable local proliferation of CD8(+) T cells and successful immunotherapy against chronic viral liver infection.

Chronic infection is difficult to overcome because of exhaustion or depletion of cytotoxic effector CD8(+) T cells (cytotoxic T lymphoytes (CTLs)). Here we report that signaling via Toll-like receptors (TLRs) induced intrahepatic aggregates of myeloid cells that enabled the population expansion of CTLs (iMATEs: 'intrahepatic myeloid-cell aggregates for T cell population expansion') without causing immunopathology. In the liver, CTL proliferation was restricted to iMATEs that were composed of inflammatory monocyte-derived CD11b(+) cells. Signaling via tumor-necrosis factor (TNF) caused iMATE formation that facilitated costimulation dependent on the receptor OX40 for expansion of the CTL population. The iMATEs arose during acute viral infection but were absent during chronic viral infection, yet they were still induced by TLR signaling. Such hepatic expansion of the CTL population controlled chronic viral infection of the liver after vaccination with DNA. Thus, iMATEs are dynamic structures that overcome regulatory cues that limit the population expansion of CTLs during chronic infection and can be used in new therapeutic vaccination strategies.

TOX reinforces the phenotype and longevity of exhausted T cells in chronic viral infection.

Cytotoxic T cells are essential mediators of protective immunity to viral infection and malignant tumours and are a key target of immunotherapy approaches. However, prolonged exposure to cognate antigens often attenuates the effector capacity of T cells and limits their therapeutic potential1-4. This process, known as T cell exhaustion or dysfunction1, is manifested by epigenetically enforced changes in gene regulation that reduce the expression of cytokines and effector molecules and upregulate the expression of inhibitory receptors such as programmed cell-death 1 (PD-1)5-8. The underlying molecular mechanisms that induce and stabilize the phenotypic and functional features of exhausted T cells remain poorly understood9-12. Here we report that the development and maintenance of populations of exhausted T cells in mice requires the thymocyte selection-associated high mobility group box (TOX) protein13-15. TOX is induced by high antigen stimulation of the T cell receptor and correlates with the presence of an exhausted phenotype during chronic infections with lymphocytic choriomeningitis virus in mice and hepatitis C virus in humans. Removal of its DNA-binding domain reduces the expression of PD-1 at the mRNA and protein level, augments the production of cytokines and results in a more polyfunctional T cell phenotype. T cells with this deletion initially mediate increased effector function and cause more severe immunopathology, but ultimately undergo a massive decline in their quantity, notably among the subset of TCF-1+ self-renewing T cells. Altogether, we show that TOX is a critical factor for the normal progression of T cell dysfunction and the maintenance of exhausted T cells during chronic infection, and provide a link between the suppression of effector function intrinsic to CD8 T cells and protection against immunopathology.

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.

Gut microbiota promote liver regeneration through hepatic membrane phospholipid biosynthesis.

BACKGROUND & AIMS: Hepatocyte growth and proliferation depends on membrane phospholipid biosynthesis. Short-chain fatty acids (SCFAs) generated by bacterial fermentation, delivered through the gut-liver axis, significantly contribute to lipid biosynthesis. We therefore hypothesized that dysbiotic insults like antibiotic treatment not only affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. METHODS: Stable isotope labeling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wild-type mice, in mice treated with broad-spectrum antibiotics, in germ-free mice and mice colonized with minimal microbiota. The microbiome was analyzed by 16S rRNA gene sequencing and microbial culture. Gut content, liver, blood and primary hepatocyte organoids were tested by mass spectrometry-based lipidomics, quantitative reverse-transcription PCR (qRT-PCR), immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of patients subjected to surgical intervention to induce hyperplasia were analyzed by qRT-PCR for lipogenic enzymes. RESULTS: Three days of antibiotic treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but a massive increase of Proteobacteria, accompanied by decreased colonic SCFAs. After PHx, antibiotic-treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the lipogenic enzyme SCD1 was upregulated after PHx but delayed by antibiotic treatment. Germ-free mice essentially recapitulated the phenotype of antibiotic treatment. Phospholipid biosynthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCFAs induced the growth of murine hepatocyte organoids and hepatic SCD1 expression in mice. Further, SCD1 was required for proliferation of human hepatoma cells and was associated with liver regeneration in human patients. CONCLUSION: Gut microbiota are pivotal for hepatic membrane phospholipid biosynthesis and liver regeneration. IMPACT AND IMPLICATIONS: Gut microbiota affect hepatic lipid metabolism through the gut-liver axis, but the underlying mechanisms are poorly understood. Perturbations of the gut microbiome, e.g. by antibiotics, impair the production of bacterial metabolites, which normally serve as building blocks for membrane lipids in liver cells. As a consequence, liver regeneration and survival after liver surgery is severely impaired. Even though this study is preclinical, its results might allow physicians in the future to improve patient outcomes after liver surgery, by modulation of gut microbiota or their metabolites.

The CGRP receptor component RAMP1 links sensory innervation with YAP activity in the regenerating liver.

The effectiveness of liver regeneration limits surgical therapies of hepatic disorders and determines patient outcome. Here, we investigated the role of the neuropeptide calcitonin gene-related peptide (CGRP) for liver regeneration after acute or chronic injury. Mice deficient for the CGRP receptor component receptor activity-modifying protein 1 (RAMP1) were subjected to a 70% partial hepatectomy or repeated intraperitoneal injections of carbon tetrachloride. RAMP1 deficiency severely impaired recovery of organ mass and hepatocyte proliferation after both acute and chronic liver injury. Mechanistically, protein expression of the transcriptional coactivators Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) was decreased in regenerating livers of RAMP1-deficient mice. Lack of RAMP1 was associated with hyperphosphorylation of YAP on Ser127 and Ser397, which regulates YAP functional activity and protein levels. Consequently, expression of various YAP-controlled cell cycle regulators and hepatocyte proliferation were severely reduced in the absence of RAMP1. In vitro, CGRP treatment caused increased YAP protein expression and a concomitant decline of YAP phosphorylation in liver tissue slice cultures of mouse and human origin and in primary human hepatocytes. Thus, our results indicate that sensory nerves represent a crucial control element of liver regeneration after acute and chronic injury acting through the CGRP-RAMP1 pathway, which stimulates YAP/TAZ expression and activity.

A dual role for hepatocyte-intrinsic canonical NF-κB signaling in virus control.

BACKGROUND & AIMS: Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver-resident macrophages. However, hepatocytes, the parenchymal cells of the liver, also possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct antiviral mechanisms employed by hepatocytes. METHODS: Using lymphocytic choriomeningitis virus (LCMV) as a model of liver infection, we first assessed the role of myeloid cells by depletion prior to infection. We investigated the role of hepatocyte-intrinsic innate immune signaling by infecting mice lacking canonical NF-κB signaling (IkkßΔHep) specifically in hepatocytes. In addition, mice lacking hepatocyte-specific interferon-α/ß signaling-(IfnarΔHep), or interferon-α/ß signaling in myeloid cells-(IfnarΔMyel) were infected. RESULTS: Here, we demonstrate that LCMV activates NF-κB signaling in hepatocytes. LCMV-triggered NF-κB activation in hepatocytes did not depend on Kupffer cells or TNFR1 signaling but rather on Toll-like receptor signaling. LCMV-infected IkkßΔHep livers displayed strongly elevated viral titers due to LCMV accumulation within hepatocytes, reduced interferon-stimulated gene (ISG) expression, delayed intrahepatic immune cell influx and delayed intrahepatic LCMV-specific CD8+ T cell responses. Notably, viral clearance and ISG expression were also reduced in LCMV-infected primary hepatocytes lacking IKKß, demonstrating a hepatocyte-intrinsic effect. Similar to livers of IkkßΔHep mice, enhanced hepatocytic LCMV accumulation was observed in livers of IfnarΔHep mice, whereas IfnarΔMyel mice were able to control LCMV infection. Hepatocytic NF-κB signaling was also required for efficient ISG induction in HDV-infected dHepaRG cells and interferon-α/ß-mediated inhibition of HBV replication in vitro. CONCLUSIONS: Together, these data show that hepatocyte-intrinsic NF-κB is a vital amplifier of interferon-α/ß signaling, which is pivotal for strong early ISG responses, immune cell infiltration and hepatic viral clearance. LAY SUMMARY: Innate immune cells have been ascribed a primary role in controlling viral clearance upon hepatic infections. We identified a novel dual role for NF-κB signaling in infected hepatocytes which was crucial for maximizing interferon responses and initiating adaptive immunity, thereby efficiently controlling hepatic virus replication.

Reduced mitochondrial resilience enables non-canonical induction of apoptosis after TNF receptor signaling in virus-infected hepatocytes.

BACKGROUND & AIMS: Selective elimination of virus-infected hepatocytes occurs through virus-specific CD8 T cells recognizing peptide-loaded MHC molecules. Herein, we report that virus-infected hepatocytes are also selectively eliminated through a cell-autonomous mechanism. METHODS: We generated recombinant adenoviruses and genetically modified mouse models to identify the molecular mechanisms determining TNF-induced hepatocyte apoptosis in vivo and used in vivo bioluminescence imaging, immunohistochemistry, immunoblot analysis, RNAseq/proteome/phosphoproteome analyses, bioinformatic analyses, mitochondrial function tests. RESULTS: We found that TNF precisely eliminated only virus-infected hepatocytes independently of local inflammation and activation of immune sensory receptors. TNF receptor I was equally relevant for NF-kB activation in healthy and infected hepatocytes, but selectively mediated apoptosis in infected hepatocytes. Caspase 8 activation downstream of TNF receptor signaling was dispensable for apoptosis in virus-infected hepatocytes, indicating an unknown non-canonical cell-intrinsic pathway promoting apoptosis in hepatocytes. We identified a unique state of mitochondrial vulnerability in virus-infected hepatocytes as the cause for this non-canonical induction of apoptosis through TNF. Mitochondria from virus-infected hepatocytes showed normal biophysical and bioenergetic functions but were characterized by reduced resilience to calcium challenge. In the presence of unchanged TNF-induced signaling, reactive oxygen species-mediated calcium release from the endoplasmic reticulum caused mitochondrial permeability transition and apoptosis, which identified a link between extrinsic death receptor signaling and cell-intrinsic mitochondrial-mediated caspase activation. CONCLUSION: Our findings reveal a novel concept in immune surveillance by identifying a cell-autonomous defense mechanism that selectively eliminates virus-infected hepatocytes through mitochondrial permeability transition. LAY SUMMARY: The liver is known for its unique immune functions. Herein, we identify a novel mechanism by which virus-infected hepatocytes can selectively eliminate themselves through reduced mitochondrial resilience to calcium challenge.

Cytokine-inducible promoters to drive dynamic transgene expression: The "Smart Graft" strategy.

BACKGROUND: Ubiquitous expression of T-cell regulatory transgenes such as the cytotoxic T lymphocyte-associated antigen 4 (CTLA4) or the high-affinity variant LEA29Y improves xeno graft survival. Such donor pigs are however immunocompromised and susceptible to infection. Continous high expression of CTLA4 or LEA29Y in the graft could also compromise the health status of recipients. The novel "Smart Graft" strategy is likely to avoid these problems by controlling the expression of T-cell regulatory transgenes as and when required. METHODS: Candidate promoters inducible by inflammatory cytokines were identified by in silico screening for potential NF-κB binding sites. Basal promoter levels and responsiveness to TNFα and IL1ß were quantified by expression of secreted embryonic alkaline phosphatase in cultured cells. Promoters were modified to increase responsiveness by removing regulatory elements or adding SP-1 or NF-κB binding sites and again tested in vitro. The most promising promoters were then assessed in vivo. Porcine cells expressing inducible Renilla luciferase constructs were transplanted into immunodeficient NOD-Scid-IL2 receptor gammanull (NSG) mice. Following engraftment, the recipient's immune system was reconstituted by splenocyte transfer raising an immune response to the porcine xenograft. The resulting induction of promoter activity was detected by in vivo bioimaging. RESULTS: Three human (hTNFAIP1, hVCAM1 and hCCL2), and one porcine promoter (pA20) were chosen for in vitro tests. In all experiments, the semi-synthetic and inducible ELAM promoter as well as the CAG promoter were used as references. In contrast to hTNFAIP1 and hVCAM1 the ELAM, hCCL2 and pA20 promoters showed significant induction after cytokine challenge. The hCCL2 and pA20 promoters were further optimized, resulting in increased responsiveness to TNFα and IL1ß. Cytokine-dependent upregulation of promoter activity was tested in vivo, where the ELAM and the optimized hCCL2 promoters showed a 2-fold upregulation, while one of the improved A20 promoters showed almost 10-fold upregulation. Our results also revealed more than 4-fold cytokine inducibility of the CAG promoter. CONCLUSION: This is the first in vivo comparison of existing and newly designed cytokine-inducible promoters. Optimization of promoter structure resulted in almost 10-fold inducibility of promoter activity. Such a rapid and dynamically regulated response to inflammation and cell damage could reduce initial graft rejection, making the "Smart Graft" approach a useful means of modulating the expression of immune regulatory transgenes to avoid deleterious effects on porcine and human health. Expressing transgenes in this fashion could provide a safer organ for transplantation.

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.

Outcome of Antiviral Immunity in the Liver Is Shaped by the Level of Antigen Expressed in Infected Hepatocytes.

The liver bears unique immune properties that support both immune tolerance and immunity, but the mechanisms responsible for clearance versus persistence of virus-infected hepatocytes remain unclear. Here, we dissect the factors determining the outcome of antiviral immunity using recombinant adenoviruses that reflect the hepatropism and hepatrophism of hepatitis viruses. We generated replication-deficient adenoviruses with equimolar expression of ovalbumin, luciferase, and green fluorescent protein driven by a strong ubiquitous cytomegalovirus (CMV) promoter (Ad-CMV-GOL) or by 100-fold weaker, yet hepatocyte-specific, transthyretin (TTR) promoter (Ad-TTR-GOL). Using in vivo bioluminescence to quantitatively and dynamically image luciferase activity, we demonstrated that Ad-TTR-GOL infection always persists, whereas Ad-CMV-GOL infection is always cleared, independent of the number of infected hepatocytes. Failure to clear Ad-TTR-GOL infection involved mechanisms acting during initiation as well as execution of antigen-specific immunity. First, hepatocyte-restricted antigen expression led to delayed and curtailed T-cell expansion-10,000-fold after Ad-CMV-GOL versus 150-fold after Ad-TTR-GOL-infection. Second, CD8 T-cells primed toward antigens selectively expressed by hepatocytes showed high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression levels similar to that seen in chronic hepatitis B. Third, Ad-TTR-GOL but not Ad-CMV-GOL-infected hepatocytes escaped being killed by effector T-cells while still inducing high PD-1/Tim-3/LAG-3/CTLA-4/CD160 expression, indicating different thresholds of T-cell receptor signaling relevant for triggering effector functions compared with exhaustion. Conclusion: Our study identifies deficits in the generation of CD8 T-cell immunity toward hepatocyte-expressed antigens and escape of infected hepatocytes expressing low viral antigen levels from effector T-cell killing as independent factors promoting viral persistence. This highlights the importance of addressing both the restauration of CD8 T-cell dysfunction and overcoming local hurdles of effector T-cell function to eliminate virus-infected hepatocytes.

Mannose receptor induces T-cell tolerance via inhibition of CD45 and up-regulation of CTLA-4.

The mannose receptor (MR) is an endocytic receptor involved in serum homeostasis and antigen presentation. Here, we identify the MR as a direct regulator of CD8(+) T-cell activity. We demonstrate that MR expression on dendritic cells (DCs) impaired T-cell cytotoxicity in vitro and in vivo. This regulatory effect of the MR was mediated by a direct interaction with CD45 on the T cell, inhibiting its phosphatase activity, which resulted in up-regulation of cytotoxic T-lymphocyte-associated Protein 4 (CTLA-4) and the induction of T-cell tolerance. Inhibition of CD45 prevented expression of B-cell lymphoma 6 (Bcl-6), a transcriptional inhibitor that directly bound the CTLA-4 promoter and regulated its activity. These data demonstrate that endocytic receptors expressed on DCs contribute to the regulation of T-cell functionality.

RIG-I Activation Protects and Rescues from Lethal Influenza Virus Infection and Bacterial Superinfection.

Influenza A virus infection causes substantial morbidity and mortality in seasonal epidemic outbreaks, and more efficient treatments are urgently needed. Innate immune sensing of viral nucleic acids stimulates antiviral immunity, including cell-autonomous antiviral defense mechanisms that restrict viral replication. RNA oligonucleotide ligands that potently activate the cytoplasmic helicase retinoic-acid-inducible gene I (RIG-I) are promising candidates for the development of new antiviral therapies. Here, we demonstrate in an Mx1-expressing mouse model of influenza A virus infection that a single intravenous injection of low-dose RIG-I ligand 5'-triphosphate RNA (3pRNA) completely protected mice from a lethal challenge with influenza A virus for at least 7 days. Furthermore, systemic administration of 3pRNA rescued mice with pre-established fulminant influenza infection and prevented the fatal effects of a streptococcal superinfection. Type I interferon, but not interferon-λ, was required for the therapeutic effect. Our results suggest that the use of RIG-I activating oligonucleotide ligands has the clinical potential to confine influenza epidemics when a strain-specific vaccine is not yet available and to reduce lethality of influenza in severely infected patients.

Liver sinusoidal endothelial cell cross-priming is supported by CD4 T cell-derived IL-2.

BACKGROUND & AIMS: Liver sinusoidal endothelial cells (LSECs) are prominent liver-resident antigen (cross-)presenting cells. LSEC cross-priming of naïve CD8 T cells does not require CD4 T cell help in contrast to priming by dendritic cells (DC) but leads to the formation of memory T cells that is preceded by transient Granzyme B (GzmB) expression. Here we provide evidence for a so far unrecognized CD4 T helper cell function in LSEC-induced CD8 T cell activation. METHODS: Naïve CD8 T cells and differentiated T helper 1 (Th1) cells were stimulated by antigen-presenting LSEC, and GzmB expression in CD8 T cells was determined by flow cytometry. To identify molecular pathways mediating this GzmB expression, mechanistic proof-of-concept experiments were conducted using stimulatory anti-CD3 antibody together with Hyper-IL-6. RESULTS: We demonstrate that LSECs simultaneously function in antigen co-presentation to CD8 and CD4 T cells. Such co-presentation revealed a function of Th1 cells to increase GzmB expression in CD8 T cells after LSEC but not DC cross-priming. IL-2 released from Th1 cells was required but not sufficient for rapid GzmB induction in CD8 T cells. T cell receptor together with IL-6 trans-signaling was necessary for IL-2 to mediate rapid GzmB induction. CONCLUSIONS: Our findings indicate that LSECs can serve as a platform to facilitate CD4-CD8 T cell crosstalk enhancing the immune function of LSECs to cross-prime CD8 T cells. IL-6 trans-signaling-mediated responsiveness for IL-2 inducing sustained GzmB expression in CD8 T cells reveals unique mechanisms of CD4 T cell help and CD8 T cell differentiation through liver-resident antigen-presenting cells. LAY SUMMARY: Our findings demonstrate that LSEC co-present antigen to CD8 and CD4 T cells and thereby enable CD4 T cell help for LSEC-priming of CD8 T cells. This CD4 T cell help selectively enhances the rapid upregulation of GzmB and effector function of LSEC-primed CD8 T cells thereby enhancing functional differentiation towards CD8 effector T cells.

Pancreatic Premalignant Lesions Secrete Tissue Inhibitor of Metalloproteinases-1, Which Activates Hepatic Stellate Cells Via CD63 Signaling to Create a Premetastatic Niche in the Liver.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) metastasizes to liver at early stages, making this disease highly lethal. Tissue inhibitor of metalloproteinases-1 (TIMP1) creates a metastasis-susceptible environment in the liver. We investigated the role of TIMP1 and its receptor CD63 in metastasis of early-stage pancreatic tumors using mice and human cell lines and tissue samples. METHODS: We obtained liver and plasma samples from patients in Germany with chronic pancreatitis, pancreatic intra-epithelial neoplasia, or PDAC, as well as hepatic stellate cells (HSCs). We performed studies with Ptf1a+/Cre;Kras+/LSL-G12D;Trp53loxP/loxP (CPK) mice, Pdx-1+/Cre;Kras+/LSL-G12D;Trp53+/LSL-R172H (KPC) mice, and their respective healthy littermates as control, and Cd63-/- mice with their wild-type littermates. KPC mice were bred with Timp1-/- mice to produce KPCxTimp1-/- mice. TIMP1 was overexpressed and CD63 was knocked down in mice using adenoviral vectors AdTIMP1 or AdshCD63, respectively. Hepatic susceptibility to metastases was determined after intravenous inoculation of syngeneic 9801L pancreas carcinoma cells. Pancreata and liver tissues were collected and analyzed by histology, immunohistochemical, immunoblot, enzyme-linked immunosorbent assay, and quantitative polymerase chain reaction analyses. We analyzed the effects of TIMP1 overexpression or knockdown and CD63 knockdown in transduced human primary HSCs and HSC cell lines. RESULTS: Chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients expressed higher levels of TIMP1 protein than normal pancreas. The premalignant pancreatic lesions that developed in KPC and CPK mice expressed TIMP1 and secreted it into the circulation. In vitro and in vivo, TIMP1 activated human or mouse HSCs, which required interaction between TIMP1 and CD63 and signaling via phosphatidylinositol 3-kinase, but not TIMP1 protease inhibitor activity. This signaling pathway induced expression of endogenous TIMP1. TIMP1 knockdown in HSCs reduced their activation. Cultured TIMP1-activated human and mouse HSCs began to express stromal-derived factor-1, which induced neutrophil migration, a marker of the premetastatic niche. Mice with pancreatic intra-epithelial neoplasia-derived systemic increases in TIMP1 developed more liver metastases after injections of pancreatic cancer cells than mice without increased levels of TIMP1. This increase in formation of liver metastases from injected pancreatic cancer cells was not observed in TIMP1 or CD63 knockout mice. CONCLUSIONS: Expression of TIMP1 is increased in chronic pancreatitis, pancreatic intra-epithelial neoplasia, and PDAC tissues from patients. TIMP1 signaling via CD63 leads to activation of HSCs, which create an environment in the liver that increases its susceptibility to pancreatic tumor cells. Strategies to block TIMP1 signaling via CD63 might be developed to prevent PDAC metastasis to the liver.

IAP antagonization promotes inflammatory destruction of vascular endothelium.

In this study, we show for the first time that the therapeutic antagonization of inhibitor of apoptosis proteins (IAPs) inhibits B16 melanoma growth by disrupting tumor vasculature. Specifically, the treatment of mice bearing B16 melanoma with an IAP antagonist compound A (Comp A) inhibits tumor growth not by inducing direct cytotoxicity against B16 cells but rather by a hitherto unrecognized antiangiogenic activity against tumor vessels. Our detailed analysis showed that Comp A treatment induces NF-κB activity in B16 tumor cells and facilitates the production of TNF. In the presence of Comp A, endothelial cells (ECs) become highly susceptible to TNF and undergo apoptotic cell death. Accordingly, the antiangiogenic and growth-attenuating effects of Comp A treatment were completely abolished in TNF-R knockout mice. This novel targeting approach could be of clinical value in controlling pathological neoangiogenesis under inflammatory condition while sparing blood vessels under normal condition.

T Cells Engineered to Express a T-Cell Receptor Specific for Glypican-3 to Recognize and Kill Hepatoma Cells In Vitro and in Mice.

BACKGROUND & AIMS: Cancer therapies are being developed based on our ability to direct T cells against tumor antigens. Glypican-3 (GPC3) is expressed by 75% of all hepatocellular carcinomas (HCC), but not in healthy liver tissue or other organs. We aimed to generate T cells with GPC3-specific receptors that recognize HCC and used them to eliminate GPC3-expressing xenograft tumors grown from human HCC cells in mice. METHODS: We used mass spectrometry to obtain a comprehensive peptidome from GPC3-expressing hepatoma cells after immune-affinity purification of human leukocyte antigen (HLA)-A2 and bioinformatics to identify immunodominant peptides. To circumvent GPC3 tolerance resulting from fetal expression, dendritic cells from HLA-A2-negative donors were cotransfected with GPC3 and HLA-A2 RNA to stimulate and expand antigen-specific T cells. RESULTS: Peptide GPC3367 was identified as a predominant peptide on HLA-A2. We used A2-GPC3367 multimers to detect, select for, and clone GPC3-specific T cells. These clones bound the A2-GPC3367 multimer and secreted interferon-γ when cultured with GPC3367, but not with control peptide-loaded cells. By genomic sequencing of these T-cell clones, we identified a gene encoding a dominant T-cell receptor. The gene was cloned and the sequence was codon optimized and expressed from a retroviral vector. Primary CD8(+) T cells that expressed the transgenic T-cell receptor specifically bound GPC3367 on HLA-A2. These T cells killed GPC3-expressing hepatoma cells in culture and slowed growth of HCC xenograft tumors in mice. CONCLUSIONS: We identified a GPC3367-specific T-cell receptor. Expression of this receptor by T cells allows them to recognize and kill GPC3-positive hepatoma cells. This finding could be used to advance development of adoptive T-cell therapy for HCC.

Escherichia coli-induced immune paralysis is not exacerbated during chronic filarial infection.

Sepsis initially starts with a systemic inflammatory response (SIRS phase) and is followed by a compensatory anti-inflammatory response syndrome (CARS) that causes impaired adaptive T-cell immunity, immune paralysis and an increased susceptibility to secondary infections. In contrast, parasitic filariae release thousands of microfilariae into the peripheral blood without triggering inflammation, as they induce regulatory, anti-inflammatory host responses. Hence, we investigated the impact of chronic filarial infection on adaptive T-cell responses during the SIRS and CARS phases of a systemic bacterial infection and analysed the development of T-cell paralysis following a subsequent adenovirus challenge in BALB/c mice. Chronic filarial infection impaired adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ responses in the absence of a bacterial challenge and led to higher numbers of splenic CTLA-4(+)  CD4(+) T cells, whereas splenic T-cell expression of CD69 and CD62 ligand, serum cytokine levels and regulatory T-cell frequencies were comparable to naive controls. Irrespective of filarial infection, the SIRS phase dominated 6-24 hr after intravenous Escherichia coli challenge with increased T-cell activation and pro-inflammatory cytokine production, whereas the CARS phase occurred 6 days post E. coli challenge and correlated with high levels of transforming growth factor-ß and increased CD62 ligand T-cell expression. Escherichia coli-induced impairment of adenovirus-specific CD8(+) T-cell cytotoxicity and interferon-γ production was not additionally impaired by chronic filarial infection. This suggests that filarial immunoregulation does not exacerbate E. coli-induced T-cell paralysis.

Transfer of MHC-class-I molecules among liver sinusoidal cells facilitates hepatic immune surveillance.

BACKGROUND & AIMS: In the liver, antigen-presenting cell populations such as Kupffer cells, liver dendritic cells, and liver sinusoidal endothelial cells (LSECs) participate through cross-presentation to CD8 T cells (CTLs) in hepatic immune-regulation and immune-surveillance. The participation of hepatic stellate cells (HSCs) in immune regulation is controversial. Here we studied HSC's contribution to antiviral CTL immunity. METHODS: Flow cytometric analysis of MHC-I molecules at the cell surface of liver cells from mice with cell-type restricted MHC-I expression. Mice with HSC-restricted MHC-I expression were infected with a hepatotropic virus and analyzed for development of viral hepatitis after CTL transfer. RESULTS: HSCs transferred MHC-I molecules to LSECs and these molecules were employed for LSEC cross-presentation to CTLs. Such transfer of MHC-I molecules was sufficient to support in vivo LSEC cross-presentation of soluble antigens to CTLs. Importantly, this transfer of MHC-I molecules contributed to anti-viral CTL immunity leading to development of immune-mediated hepatitis. CONCLUSIONS: Our findings demonstrate transfer of MHC-I molecules among sinusoidal liver cell populations as a potent mechanism to increase anti-viral CTL effector function. The transfer of MHC-I molecules from HSCs supplies LSECs with additional MHC-I molecules for their own cell-intrinsic cross-presentation. Such cross-allocation of MHC-I molecules in liver cell populations is distinct from cross-dressing that occurs among immune cell populations in lymphoid tissues where peptide-loaded MHC-I molecules are transferred. Our findings thus reveal a novel mechanism that increases local cross-presentation and CTL effector function in the liver, which may be instrumental for immune-surveillance during viral infection of antigen-presenting liver cells.