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A better understanding of mechanisms that regulate CD8

A better understanding of mechanisms that regulate CD8 + T cell responses to therapeutic vaccines is needed to develop approaches to enhance vaccine efficacy for chronic viral infections and cancers. We show here that NK cell depletion enhanced antigen-specific T cell responses to chimp adenoviral vector (ChAdOx) vaccination in a mouse model of chronic HBV infection (CHB). Probing the mechanism underlying this negative regulation, we observed that CHB drove parallel up-regulation of programmed cell death ligand 1 (PD-L1) on liver-resident NK cells and programmed cell death 1 (PD-1) on intrahepatic T cells. PD-L1–expressing liver-resident NK cells suppressed PD-1 hi CD8 + T cells enriched within the HBV-specific response to therapeutic vaccination. Cytokine activation of NK cells also induced PD-L1, and combining cytokine activation with PD-L1 blockade resulted in conversion of NK cells into efficient helpers that boosted HBV-specific CD8 + T cell responses to therapeutic vaccination in mice and to chronic infection in humans. Our findings delineate an immunotherapeutic combination that can boost the response to therapeutic vaccination in CHB and highlight the broader importance of PD-L1–dependent regulation of T cells by cytokine-activated NK cells.

The field of immunometabolism has attracted growing attention as an area at the heart of immune regulation. Upon activation, T cells undergo significant metabolic changes allowing them to mediate effector responses. The advent of chimeric antigen receptor T cell-adoptive therapy has shown some striking clinical efficacy but fails to induce sufficient antitumor response in many patients. Solid tumors put up significant opposition creating a microenvironment deficient of oxygen and glucose, depriving T cells of energy and pushing them to exhaustion. Here, we focus on immune suppressive mechanisms related to hypoxia in the tumor microenvironment and the resulting metabolic changes in T cells. New therapeutic approaches such as generating chimeric antigen receptor T cells able to withstand the challenging solid tumor microenvironment are needed.

Summary: Tissue-resident memory T cells have critical roles in long-term pathogen and tumor immune surveillance in the liver. We investigate the role of autophagy in equipping human memory T cells to acquire tissue residence and maintain functionality in the immunosuppressive liver environment. By performing ex vivo staining of freshly isolated cells from human liver tissue, we find that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8+ T cells. CD8+ T cells with increased autophagy are those best able to proliferate and mediate cytotoxicity and cytokine production. Conversely, blocking autophagy induction results in the accumulation of depolarized mitochondria, a feature of exhausted T cells. Primary hepatic stellate cells or the prototypic hepatic cytokine interleukin (IL)-15 induce autophagy in parallel with tissue-homing/retention markers. Inhibition of T cell autophagy abrogates tissue-residence programming. Thus, upregulation of autophagy adapts CD8+ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence. : Swadling et al. show that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8+ T cells and that in vitro TRM programming requires autophagy induction. Upregulation of autophagy adapts CD8+ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence. Keywords: autophagy, T cell, liver, tissue-resident, hepatitis B virus, mitophagy, IL-15, immunometabolism

Upon activation naïve T cells undergo metabolic changes to support the differentiation into subsets of effector or regulatory cells, and enable subsequent metabolic adaptations to form memory. Interfering with these metabolic alterations leads to abrogation or reprogramming of T cell differentiation, demonstrating the importance of these pathways in T cell development. It has long been appreciated that the conversion of a healthy cell to a cancerous cell is accompanied by metabolic changes, which support uncontrolled proliferation. Especially in solid tumors these metabolic changes significantly influence the tumor microenvironment (TME) and affect tumor infiltrating immune cells. The TME is often hypoxic and nutrient depleted, additionally tumor cells produce co-inhibitory signals, together suppressing the immune response. Interestingly, viruses can stimulate a metabolism akin to that seen in tumor cells in their host cells and even in neighboring cells (e.g., via transfer of virally modified extracellular vesicles). Thus, viruses create their own niche which favors viral persistence and propagation, while again keeping the immune response at bay. In this review we will focus on the mechanisms employed by tumor cells and viruses influencing T cell metabolic regulation and the impact they have on shaping T cell fate.

Summary Tissue-resident memory T cells have critical roles in long-term pathogen and tumor immune surveillance in the liver. We investigate the role of autophagy in equipping human memory T cells to acquire tissue residence and maintain functionality in the immunosuppressive liver environment. By performing ex vivo staining of freshly isolated cells from human liver tissue, we find that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8+ T cells. CD8+ T cells with increased autophagy are those best able to proliferate and mediate cytotoxicity and cytokine production. Conversely, blocking autophagy induction results in the accumulation of depolarized mitochondria, a feature of exhausted T cells. Primary hepatic stellate cells or the prototypic hepatic cytokine interleukin (IL)-15 induce autophagy in parallel with tissue-homing/retention markers. Inhibition of T cell autophagy abrogates tissue-residence programming. Thus, upregulation of autophagy adapts CD8+ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence., Graphical Abstract, Highlights • An increased rate of basal autophagy is a hallmark of liver-resident CD8+ T cells • Enhanced T cell autophagy can be imprinted by IL-15 or hepatic stellate cells • Autophagy induction is required for tissue-residence programming in vitro • Enhanced autophagy maintains TRM mitochondrial fitness in the liver, Swadling et al. show that an increased rate of basal autophagy is a hallmark of intrahepatic lymphocytes, particularly liver-resident CD8+ T cells and that in vitro TRM programming requires autophagy induction. Upregulation of autophagy adapts CD8+ T cells to combat mitochondrial depolarization, optimize functionality, and acquire tissue residence.

Summary T cells are a fundamental component of the adaptive immune response in the context of both acute and chronic viral infection. Tight control over the metabolic processes within T cells provides an additional level of immune regulation that is interlinked with nutrient sensing and the continued balancing of co-stimulatory and co-inhibitory signals. Underpinning T cell responsiveness for viral control are a number of phenotypic and functional adaptations ensuring adequate nutrient uptake and their utilization. T cells responding to persistent viral infections often exhibit a profile associated with immune cell exhaustion and a dysregulated metabolic profile, driven by a combination of chronic antigenic stimulation and signals from the local microenvironment. Understanding alterations in these metabolic processes provides an important basis for immunotherapeutic strategies to treat persistent infections.

Upon activation T cells engage glucose metabolism to fuel the costly effector functions needed for a robust immune response. Consequently, the availability of glucose can impact on T cell function. The glucose concentrations used in conventional culture media and common metabolic assays are often artificially high, representing hyperglycaemic levels rarely present in vivo. We show here that reducing glucose concentration to physiological levels in culture differentially impacted on virus-specific compared to generically activated human CD8 T cell responses. In virus-specific T cells, limiting glucose availability significantly reduced the frequency of effector-cytokine producing T cells, but promoted the upregulation of CD69 and CD103 associated with an increased capacity for tissue retention. In contrast the functionality of generically activated T cells was largely unaffected and these showed reduced differentiation towards a residency phenotype. Furthermore, T cells being cultured at physiological glucose concentrations were more susceptible to viral infection. This setting resulted in significantly improved lentiviral transduction rates of primary cells. Our data suggest that CD8 T cells are exquisitely adapted to their niche and provide a reminder of the need to better mimic physiological conditions to study the complex nature of the human CD8 T cell immune response.

Infection with hepatitis B virus (HBV) results in disparate degrees of tissue injury: the virus can either replicate without pathological consequences or trigger immune-mediated necroinflammatory liver damage. We investigated the potential for myeloid-derived suppressor cells (MDSCs) to suppress T cell-mediated immunopathology in this setting. Granulocytic MDSCs (gMDSCs) expanded transiently in acute resolving HBV, decreasing in frequency prior to peak hepatic injury. In persistent infection, arginase-expressing gMDSCs (and circulating arginase) increased most in disease phases characterized by HBV replication without immunopathology, whilst L-arginine decreased. gMDSCs expressed liver-homing chemokine receptors and accumulated in the liver, their expansion supported by hepatic stellate cells. We provide in vitro and ex vivo evidence that gMDSCs potently inhibited T cells in a partially arginase-dependent manner. L-arginine-deprived T cells upregulated system L amino acid transporters to increase uptake of essential nutrients and attempt metabolic reprogramming. These data demonstrate the capacity of expanded arginase-expressing gMDSCs to regulate liver immunopathology in HBV infection.

HBV-DNA integration frequently occurs in HBV-related hepatocellular carcinoma (HCC), but whether HBV antigens are expressed in HCC cells and can be targeted by immune therapeutic strategies remains controversial. Here, we first characterized HBV antigen expression in HCC metastases, occurring in a patient who had undergone liver transplantation for HBV-related HCC. We then deployed for the first time in HCC autologous T cells, genetically modified to express an HBsAg specific T cell receptor, as therapy against chemoresistant extrahepatic metastases. We confirmed that HBV antigens were expressed in HCC metastases (but not in the donor liver) and demonstrated that tumour cells were recognized in vivo by lymphocytes, engineered to express an HBV-specific T cell receptor (TCR). Gene-modified T cells survived, expanded and mediated a reduction in HBsAg levels without exacerbation of liver inflammation or other toxicity. Whilst clinical efficacy was not established in this subject with end-stage metastatic disease, we confirm the feasibility of providing autologous TCR-redirected therapy against HCC and advocate this strategy as a novel therapeutic opportunity in hepatitis B-associated malignancies.

In this review we discuss the divergent role of two closely related cytokines, IL-12 and IL-23, in shaping immune responses. In light of current therapeutic developments using biologic agents to block these two pathways, a better understanding of the immunological function of these cytokines is pivotal.

Hepatic NK cells eliminate HBV-specific T cells dependent on TRAIL and TRAIL-R2 interactions to limit antiviral immunity in chronic infection., Antiviral T cell responses in hepatotropic viral infections such as hepatitis B virus (HBV) are profoundly diminished and prone to apoptotic deletion. In this study, we investigate whether the large population of activated NK cells in the human liver contributes to this process. We show that in vitro removal of NK cells augments circulating CD8+ T cell responses directed against HBV, but not against well-controlled viruses, in patients with chronic hepatitis B (CHB). We find that NK cells can rapidly eliminate HBV-specific T cells in a contact-dependent manner. CD8+ T cells in the liver microcirculation are visualized making intimate contact with NK cells, which are the main intrahepatic lymphocytes expressing TNF-related apoptosis-inducing ligand (TRAIL) in CHB. High-level expression of the TRAIL death receptor TRAIL-R2 is found to be a hallmark of T cells exposed to the milieu of the HBV-infected liver in patients with active disease. Up-regulation of TRAIL-R2 renders T cells susceptible to caspase-8–mediated apoptosis, from which they can be partially rescued by blockade of this death receptor pathway. Our findings demonstrate that NK cells can negatively regulate antiviral immunity in chronic HBV infection and illustrate a novel mechanism of T cell tolerance in the human liver.

Impaired function of virus-specific T cells resulting from virus persistence is one of the major mechanisms underlying the development of chronic hepatitis B viral infection. Previously, we found that IL-2 can restore the effector function of T cells rendered tolerant by Ag persistence. However, systemic administration of IL-2 induces organ pathology and expansion of T regulatory cells. In this study, we show that nano-APC with engineered HLA alleles and IL-2 deliver peptide–MHC complexes, costimulatory molecules, and IL-2 to Ag-responding T cells, resulting in enhanced expression of CD25 and activation of TCR signaling pathways, while suppressing PD-1 expression on viral-responding CD8 T cells from chronic hepatitis B virus patients. The enhanced activation of CD4 and CD8 T cells induced by IL-2–nano-APC was Ag dependent and IL-2–nano-APC did not affect T regulatory cells. At a size of 500 nm, the nano-APC effectively induce immune synapse formation on Ag-specific T cells and accumulate as free particles in the lymphoid organs. These attributes of IL-2–nano-APC or other bioadjuvant-engineered nano-APC have profound implications for their use as a therapeutic strategy in the treatment of chronic hepatitis B virus infection or other chronic viral diseases.

P>Adenosine is a well-described anti-inflammatory modulator of immune responses within peripheral tissues. Extracellular adenosine accumulates in inflamed and damaged tissues and inhibits the effector functions of various immune cell populations, including CD8 T cells. However, it remains unclear whether extracellular adenosine also regulates the initial activation of naive CD8 T cells by professional and semi-professional antigen-presenting cells, which determines their differentiation into effector or tolerant CD8 T cells, respectively. We show that adenosine inhibited the initial activation of murine naive CD8 T cells after alpha CD3/CD28-mediated stimulation. Adenosine caused inhibition of activation, cytokine production, metabolic activity, proliferation and ultimately effector differentiation of naive CD8 T cells. Remarkably, adenosine interfered efficiently with CD8 T-cell priming by professional antigen-presenting cells (dendritic cells) and semi-professional antigen-presenting cells (liver sinusoidal endothelial cells). Further analysis of the underlying mechanisms demonstrated that adenosine prevented rapid tyrosine phosphorylation of the key kinase ZAP-70 as well as Akt and ERK1/2 in naive alpha CD3/CD28-stimulated CD8 cells. Consequently, alpha CD3/CD28-induced calcium-influx into CD8 cells was reduced by exposure to adenosine. Our results support the notion that extracellular adenosine controls membrane-proximal T-cell receptor signalling and thereby also differentiation of naive CD8 T cells. These data raise the possibility that extracellular adenosine has a physiological role in the regulation of CD8 T-cell priming and differentiation in peripheral organs.

Cross-presentation is an important function of immune competent cells, such as dendritic cells (DCs), macrophages, and an organ-resident liver cell population, i.e., liver sinusoidal endothelial cells (LSECs). Here, we characterize in direct comparison to DCs the distinct dynamics and kinetics of cross-presentation employed by LSECs, which promote tolerance induction in CD8 T cells. We found that LSECs were as competent in cross-presenting circulating soluble antigen ex vivo as DCs at a per-cell basis. However, antigen uptake in vivo was 100-fold more pronounced in LSECs, indicating distinct mechanisms of cross-presentation. In contrast to mannose-receptor–mediated antigen uptake and routing into stable endosomes dedicated to cross-presentation in DCs, we observed distinct antigen-uptake and endosomal routing with high antigen turnover in LSECs that resulted in short-lived cross-presentation. Receptor-mediated endocytosis did not always lead to cross-presentation, because immune-complexed antigen taken up by the Fc-receptor was not cross-presented by LSECs, indicating that induction of CD8 T cell tolerance by LSECs is impaired in the presence of preexisting immunity. Conclusion: These results provide a mechanistic explanation how organ-resident LSECs accommodate continuous scavenger function with the capacity to cross-present circulating antigens using distinct kinetics and dynamics of antigen-uptake, routing and cross-presentation compared to DCs. (HEPATOLOGY 2009.)

Bacillus subtilis strain 168 produces the extremely stable lantibiotic sublancin 168, which has a broad spectrum of bactericidal activity. Both sublancin 168 production and producer immunity are determined by the SPβ prophage. While the sunA and sunT genes for sublancin 168 production have been known for several years, the genetic basis for sublancin 168 producer immunity has remained elusive. Therefore, the present studies were aimed at identifying an SPβ gene(s) for sublancin 168 immunity. By systematic deletion analysis, we were able to pinpoint one gene, named yolF , as the sublancin 168 producer immunity gene. Growth inhibition assays performed using plates and liquid cultures revealed that YolF is both required and sufficient for sublancin 168 immunity even when heterologously produced in the sublancin-sensitive bacterium Staphylococcus aureus . Accordingly, we propose to rename yolF to sunI (for su blanci n i mmunity). Subcellular localization studies indicate that the SunI protein is anchored to the membrane with a single N-terminal membrane-spanning domain that has an N out -C in topology. Thus, the bulk of the protein faces the cytoplasm of B. subtilis . This topology has not yet been reported for known bacteriocin producer immunity proteins, which implies that SunI belongs to a novel class of bacteriocin antagonists.

Dissemination of soluble molecules or antigens via the blood stream is considered to lead to a uniform distribution in the various organs of the body, but organ-specific microarchitecture and vascularization may influence this. Following intravenous injection of alpha wCD3 epsilon antibody (alpha CD3 epsilon Ab) we observed clear differences in antibody binding to Fc gamma receptor (Fc gamma R)(+) antigen-presenting cells (APCs) or T lymphocytes in different organs. Significant binding of blood-borne alpha CD3 epsilon Ab was only detected in the spleen and liver and not in the thymus or lymph node. In the spleen, only 10% of dendritic cells/macrophages and 40% of T-cell receptor (TCR)-beta(+) cells were positive for alpha CD3 epsilon Ab, and, dependent on Fc gamma R-mediated cross-linking of alpha CD3 epsilon Ab, a similar percentage of splenic TCR-beta(+) cells were stimulated and became CD69(+). Stimulation of TCR-beta(+) cells in the liver was at least as efficient as in the spleen, but almost all T cells and all scavenger liver sinusoidal endothelial cells bound alpha CD3 epsilon Ab. In contrast to CD69 up-regulation, only CD4(+) natural killer T (NKT) cells and CD11a(high) CD8(+) T cells were activated by alpha CD3 epsilon Ab and expressed interferon (IFN)-gamma. Again, IFN-gamma release from NKT/T cells was at least as efficient in the liver as in the spleen. Taken together, our results support the notion that the combination of extensive hepatic vascularization and very high scavenger activity allows the liver to fulfill its metabolic tasks and to promote stimulation of the large but widely distributed hepatic population of NKT/T cells.

The treatment of HCV has been transformed by the capacity of direct acting antivirals (DAAs) to eliminate the infection by attacking the virus effectively. In this issue Martin et al. reveal that these drugs can also harness the immune system by releasing it from the burden of chronic antigen load [1]. Their important findings underscore a detrimental immunoregulatory component to the effects of interferon-alpha (IFN-a), which was not able to boost antiviral T cells in the manner now demonstrated with DAAs, even when it achieved viral clearance (Fig. 1). CD8 T cells are well-recognised to be critical for the control of viruses such as HCV but to be depleted and dysfunctional (exhausted) in chronic infection [2–5]. There are clear demonstrations of the inverse relationship between duration and level of viraemia and T cell responses in murine and human persistent viral infections [2,3,6]. However it has been difficult to determine to what extent the persistent, high antigen load in these settings contributes to the failure of the T cell response [7] or is rather a result of it. A vital issue in the attempt to cure persistent viral infection is whether removal of antigen will be sufficient to allow recovery of T cells with antiviral efficacy. Alternatively, T cells may be permanently scarred by, for example, stable imprinting of PD-1 [8], or subject to other ongoing tolerising mechanisms, which are particularly prevalent in the liver environment [9]. Previous data from the HCV field had suggested that whilst T cells show variable recovery following spontaneous or therapeutic removal of antigen in acute infection, they might no longer be amenable to rescue in chronic infection [4,10,11]. Thus patients with IFN-a induced clearance of chronic HCV had no consistent recovery of virus-specific T cells [10–12]. Now the Thimme group demonstrate that when the antigen load is reduced by IFN-free therapy, there is a striking recovery of the capacity of HCV-specific T cells to expand in culture. Responders to DAAs had a significant increase in the number of detectable HCV-specific responses, and in their frequencies after in vitro expansion

Optimal immune activation of naïve CD8 T cells requires signal 1 mediated by the T cell receptor, signal 2 mediated by co-stimulation and signal 3 provided by pro-inflammatory cytokines. However, the potential for signal 3 cytokines to rescue anti-viral responses in functionally exhausted T cells has not been defined. We investigated the effect of using third signal cytokines IL-12 or IFN-α to rescue the exhausted CD8 T cell response characteristic of patients persistently infected with hepatitis B virus (HBV). We found that IL-12, but not IFN-α, potently augmented the capacity of HBV-specific CD8 T cells to produce effector cytokines upon stimulation by cognate antigen. Functional recovery mediated by IL-12 was accompanied by down-modulation of the hallmark inhibitory receptor PD-1 and an increase in the transcription factor T-bet. PD-1 down-regulation was observed in HBV but not CMV-specific T cells, in line with our finding that the highly functional CMV response was not further enhanced by IL-12. IL-12 enhanced a number of characteristics of HBV-specific T cells important for viral control: cytotoxicity, polyfunctionality and multispecificity. Furthermore, IL-12 significantly decreased the pro-apoptotic molecule Bim, which is capable of mediating premature attrition of HBV-specific CD8 T cells. Combining IL-12 with blockade of the PD-1 pathway further increased CD8 functionality in the majority of patients. These data provide new insights into the distinct signalling requirements of exhausted T cells and the potential to recover responses optimised to control persistent viral infections., Author Summary Persistent viral infections continue to cause major morbidity and mortality; chronic hepatitis B virus infection alone accounts for more than a million deaths annually. Such infections are characterised by a failure of viral control perpetuated by exhaustion of the T cell response. Here we show that the cytokine IL-12 can act as a potent “third signal” to rescue antiviral function in exhausted T cells. IL-12 has previously been shown to enhance naïve T cell responses but this is the first demonstration of its capacity to boost the disabled antiviral response in a persistent viral infection. IL-12 was able to down-regulate PD-1, a key inhibitory receptor driving T cell exhaustion, resulting in the recovery of hepatitis B virus-specific responses able to mediate multiple antiviral functions. Control responses in the same patients directed against the well-controlled cytomegalovirus did not require IL-12 to function efficiently. Our findings therefore elucidate a role for IL-12 in re-programming functionally exhausted T cells in persistent viral infections.

Background & Aims: A better understanding of the immunomodulatory effects of PegIFNa therapy could allow more rational optimisation of future therapeutic approaches in chronic HBV infection. In this study, we evaluated dynamic changes in the innate and adaptive arms of the immune system induced by PegIFNa. Methods: PBMC were obtained from a cohort of patients with eAg-negative CHB before, during and after PegIFNa treatment. The number, phenotype and function of global and virus-specific T cells and NK cells were analyzed by flow cytometry and serum cytokines by ELISA or CBA. Results: The absolute number of CD8 T cells was strikingly reduced on PegIFNa therapy (p

The S-type lectin galectin-9 binds to the negative regulatory molecule Tim-3 on T cells and induces their apoptotic deletion or functional inactivation. We investigated whether galectin-9/Tim-3 interactions contribute to the deletion and exhaustion of the antiviral T cell response in chronic hepatitis B virus infection (CHB). We found Tim-3 to be expressed on a higher percentage of CD4 and CD8 T cells from patients with CHB than healthy controls (p

An excess of coinhibitory signals has been proposed to drive the T-cell exhaustion characteristic of persistent viral infections. In this study we examined the contribution of the coinhibitory receptor cytotoxic T lymphocyte antigen-4 (CTLA-4) to CD8 T cell tolerance in chronic hepatitis B virus (HBV) infection (CHB). CD8 T cells in patients with CHB have an increased propensity to express the coinhibitory receptor CTLA-4 and this correlates with viral load. CTLA-4 is up-regulated on those HBV-specific CD8 T cells with the highest levels of the proapoptotic protein Bim, which we have previously shown mediates their premature attrition; abrogation of CTLA-4-mediated coinhibition can reduce Bim expression. Longitudinal study of CHB patients beginning antiviral therapy reveals that HBV DNA suppression induces transient reconstitution of HBV-specific CD8 T cells but does not reprogram their CTLA-4hiBimhi tolerogenic phenotype. Blocking CTLA-4 is able to increase the expansion of interferon gamma (IFN-γ)-producing HBV-specific CD8 T cells in both the peripheral and intrahepatic compartments. The rescue of anti-HBV responses by either CTLA-4 or PD-L1 blockade is nonredundant. Conclusion: CTLA-4 is expressed by HBV-specific CD8 T cells with high levels of Bim and helps to drive this proapoptotic phenotype. CTLA-4 blockade could form one arm of a therapeutic approach to modulate the diverse patterns of coregulation of T-cell exhaustion in this heterogeneous disease. (HEPATOLOGY 2011;)

Cross-presentation of soluble Ag on MHC class I molecules to naive CD8 T cells by liver sinusoidal endothelial cells (LSECs) leads to induction of T cell tolerance that requires interaction between coinhibitory B7-H1 on LSECs and programmed cell death-1 on CD8 T cells. In this study, we investigate whether cross-presentation of high as well as low Ag concentrations allowed for LSEC-induced tolerance. Ag concentration directly correlated with the cross-presentation capacity of murine LSECs and thus strength of TCR stimulation. Although LSEC cross-presentation at low-Ag concentrations resulted in tolerance, they induced differentiation into effector T cells (CTL) at high-Ag concentrations. CTL differentiation under these conditions was not caused by increased expression of costimulatory CD80/86 on cross-presenting LSECs but was determined by early IL-2 release from naive CD8 T cells. B7-H1 signals from LSECs and TCR avidity reciprocally controlled early T cell release of IL-2 and CTL differentiation. B7-H1 expression directly correlated with cross-presentation at low- but not high-Ag concentrations, indicating an imbalance between TCR and coinhibitory signals regulating T cell release of IL-2. Exogenous IL-2 overrode coinhibitory B7-H1–mediated signals by LSECs and induced full CTL differentiation. Our results imply that LSEC-mediated T cell tolerance can be broken in situations where T cells bearing high-avidity TCR encounter LSECs cross-presenting high numbers of cognate MHC class I peptide molecules, such as during viral infection of the liver. Furthermore, we attribute a novel costimulatory function to IL-2 acting in a T cell autonomous fashion to promote local induction of immunity in the liver even in the absence of CD80/86 costimulation.

There is a pressing need to develop new immunotherapeutic interventions in chronic hepatitis B virus (HBV) infection in order to limit the high costs and risks of toxicity or viral resistance associated with maintenance antiviral treatment. Here we review recent advances in our understanding of the molecular defects underlying the profound T cell depletion characteristic of these patients. We propose that T cells are driven to apoptosis by the combination of a persistent, high antigen load and excessive inhibitory signals encountered in the hepatic microenvironment. The feasibility of boosting sustained antiviral control by targeted reversal of key tolerising mechanisms is discussed.

Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naive CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2Kb mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naive CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance. Conclusion: Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naive CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance. (HEPATOLOGY 2009.)

Liver sinusoidal endothelial cells (LSEC) are unique organ-resident antigen-presenting cells capable of cross-presentation and subsequent tolerization of naive CD8+ T cells. We investigated the molecular mechanisms underlying this tolerance induction in naive CD8+ T cells. MHC class I–restricted antigen presentation by LSEC led to initial stimulation of naive CD8+ T cells, which up-regulated CD69, CD25, CD44, and programmed death (PD)-1 and proliferated similar to dendritic cell (DC)–activated CD8+ T cells. Importantly, cognate interaction with naive CD8+ T cells triggered increased expression of co-inhibitory B7-H1 but not co-stimulatory CD80/86 molecules exclusively on LSEC but not DC. This matured phenotype of B7-H1high CD80/86low was critical for induction of CD8+ T cell tolerance by LSEC: B7-H1–deficient LSEC, that failed to interact with PD-1 on stimulated T cells, were incapable of inducing CD8+ T cell tolerance. Moreover, increased costimulation via CD28 interfered with tolerance induction, indicating that the noninducible low expression levels of CD80/86 on LSEC supported B7-H1–dependent tolerance induction. LSEC-tolerized CD8+ T cells had a distinctive phenotype from naive and activated T cells with CD25low, CD44high, CD62Lhigh. They also expressed the homeostatic cytokine receptors CD127, CD122, and high levels of Bcl-2, indicating survival rather than deletion of tolerant CD8+ T cells. On adoptive transfer into congenic animals, tolerized CD8+ T cells failed to show specific cytotoxicity in vivo. Conclusion: Cognate interaction of LSEC with naive CD8+ T cells elicits a unique tolerogenic maturation of LSEC and permissiveness of T cells for tolerogenic signals, demonstrating that LSEC-induced tolerance is an active and dynamic process. (HEPATOLOGY 2007.)

Hepatocellular carcinoma (HCC) is a common and highly lethal tumor that is currently the third-leading cause of cancer-related deaths (1). Hepatitis B virus (HBV) is responsible for more than 50% of HCC cases worldwide, making it the second most important known carcinogen for all types of cancer. Although prevention of HBV infection by implementation of universal infant vaccination strategies is starting to have an impact on the subsequent incidence of HCC (1), there remains a huge burden of disease in store for the 400 million people estimated to be already infected with this virus. There is therefore a pressing need to develop a better understanding of how HBV infection triggers the development of HCC, so that earlier intervention can prevent this complication of persistent infection. Remarkable findings by Sitia et al. in PNAS (2) shed new light on the pathogenesis of HBV-related liver cancer, revealing a surprising effect of antiplatelet drugs in preventing its development.

and expression intensity (MFI). Cross-linking of 2B4 had diverse effects depending on 2B4 expression levels. Expression of 2B4 and SAP correlated inversely, cells with high 2B4 expression showed a lower SAP content than 2B4lo cells and vice versa. Expression of SAP could be modulated by cytokine stimuli as expression intensity increased after 6 hours of incubation with IL-2 or IFNa. However, on continuous stimulation the SAP content decreased after 12 hours leading to a low expression level during following time points. Interestingly, 2B4hi cells showed a stronger loss of SAP after 12 hours as compared to 2B4lo cells. Conclusion: Cross-linking of 2B4 has diverse effects on CD8+ T cells either leading to increase or decrease of function. This regulation of CD8+ T cell function via 2B4 seems to be achieved through modulation of SAP expression possibly determining the outcome of 2B4 engagement.