Engaging stimulatory immune checkpoint interactions in the tumour immune microenvironment of primary liver cancers - how to push the gas after having released the brake.

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the first and second most common primary liver cancer (PLC). For decades, systemic therapies consisting of tyrosine kinase inhibitors (TKIs) or chemotherapy have formed the cornerstone of treating advanced-stage HCC and CCA, respectively. More recently, immunotherapy using immune checkpoint inhibition (ICI) has shown anti-tumour reactivity in some patients. The combination regimen of anti-PD-L1 and anti-VEGF antibodies has been approved as new first-line treatment of advanced-stage HCC. Furthermore, gemcibatine plus cisplatin (GEMCIS) with an anti-PD-L1 antibody is awaiting global approval for the treatment of advanced-stage CCA. As effective anti-tumour reactivity using ICI is achieved in a minor subset of both HCC and CCA patients only, alternative immune strategies to sensitise the tumour microenvironment of PLC are waited for. Here we discuss immune checkpoint stimulation (ICS) as additional tool to enhance anti-tumour reactivity. Up-to-date information on the clinical application of ICS in onco-immunology is provided. This review provides a rationale of the application of next-generation ICS either alone or in combination regimen to potentially enhance anti-tumour reactivity in PLC patients.

Induction of broad multifunctional CD8+ and CD4+ T cells by hepatitis B virus antigen-based synthetic long peptides ex vivo.

Introduction: Therapeutic vaccination based on synthetic long peptides (SLP®) containing both CD4+ and CD8+ T cell epitopes is a promising treatment strategy for chronic hepatitis B infection (cHBV). Methods: We designed SLPs for three HBV proteins, HBcAg and the non-secreted proteins polymerase and X, and investigated their ability to induce T cell responses ex vivo. A set of 17 SLPs was constructed based on viral protein conservation, functionality, predicted and validated binders for prevalent human leukocyte antigen (HLA) supertypes, validated HLA I epitopes, and chemical producibility. Results: All 17 SLPs were capable of inducing interferon gamma (IFNÉ£) production in samples from four or more donors that had resolved an HBV infection in the past (resolver). Further analysis of the best performing SLPs demonstrated activation of both CD8+ and CD4+ multi-functional T cells in one or more resolver and patient sample(s). When investigating which SLP could activate HBV-specific T cells, the responses could be traced back to different peptides for each patient or resolver. Discussion: This indicates that a large population of subjects with different HLA types can be covered by selecting a suitable mix of SLPs for therapeutic vaccine design. In conclusion, we designed a set of SLPs capable of inducing multifunctional CD8+ and CD4+ T cells ex vivo that create important components for a novel therapeutic vaccine to cure cHBV.

Emerging organoid-immune co-culture models for cancer research: from oncoimmunology to personalized immunotherapies.

In the past decade, treatments targeting the immune system have revolutionized the cancer treatment field. Therapies such as immune checkpoint inhibitors have been approved as first-line treatment in a variety of solid tumors such as melanoma and non-small cell lung cancer while other therapies, for instance, chimeric antigen receptor (CAR) lymphocyte transfer therapies, are still in development. Although promising results are obtained in a small subset of patients, overall clinical efficacy of most immunotherapeutics is limited due to intertumoral heterogeneity and therapy resistance. Therefore, prediction of patient-specific responses would be of great value for efficient use of costly immunotherapeutic drugs as well as better outcomes. Because many immunotherapeutics operate by enhancing the interaction and/or recognition of malignant target cells by T cells, in vitro cultures using the combination of these cells derived from the same patient hold great promise to predict drug efficacy in a personalized fashion. The use of two-dimensional cancer cell lines for such cultures is unreliable due to altered phenotypical behavior of cells when compared with the in vivo situation. Three-dimensional tumor-derived organoids, better mimic in vivo tissue and are deemed a more realistic approach to study the complex tumor-immune interactions. In this review, we present an overview of the development of patient-specific tumor organoid-immune co-culture models to study the tumor-specific immune interactions and their possible therapeutic infringement. We also discuss applications of these models which advance personalized therapy efficacy and understanding the tumor microenvironment such as: (1) Screening for efficacy of immune checkpoint inhibition and CAR therapy screening in a personalized manner. (2) Generation of tumor reactive lymphocytes for adoptive cell transfer therapies. (3) Studying tumor-immune interactions to detect cell-specific roles in tumor progression and remission. Overall, these onco-immune co-cultures might hold a promising future toward developing patient-specific therapeutic approaches as well as increase our understanding of tumor-immune interactions.

Systemic T-cell and humoral responses against cancer testis antigens in hepatocellular carcinoma patients.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths worldwide due to high recurrence rates after curative treatment and being frequently diagnosed at an advanced stage. Immune-checkpoint inhibition (ICPI) has yielded impressive clinical successes in a variety of solid cancers, however results in treatment of HCC have been modest. Vaccination could be a promising treatment to synergize with ICPI and enhance response rates. Cancer testis antigens (CTAs) were recently discovered to be widely expressed in HCC and expression in macroscopically tumor-free tissues correlated with recurrence, implying the presence of micro-satellites. To determine whether CTAs are immunogenic in HCC patients, we analyzed systemic T-cell and humoral responses against seven CTAs in 38 HCC patients using a multitude of techniques; flowcytometry, ELISA and whole antigen and peptide stimulation assays. CTA-specific T-cells were detected in all (25/25) analyzed patients, of which most had a memory phenotype but did not exhibit unequivocal signs of chronic stimulation or recent antigen encounter. Proliferative CD4+ and CD8+ T-cell responses against these CTAs were found in 14/16 analyzed HCC patients. CTA-peptide stimulation-induced granzyme B, IL2, and TNFa in 8/8 analyzed patients, including two MAGEA1 peptides included based on in silico prediction. Finally, IgG responses were observed in 13/32 patients, albeit with low titers. The presence of CD4+ and CD8+ T-cells and IgG responses shows the immunogenicity of these CTAs in HCC-patients. We hypothesize that vaccines based on these tumor-specific antigens may boost preexisting CTA-specific immunity and could enhance therapeutic efficacy of ICPI in advanced HCC.

Immunopeptidome of hepatocytes isolated from patients with HBV infection and hepatocellular carcinoma.

Background & Aims: Antigen-specific immunotherapy is a promising strategy to treat HBV infection and hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by human leucocyte antigen (HLA)-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumour-associated antigens (TAA) to guide development of antigen-specific immunotherapy. Methods: Primary human hepatocytes were isolated with high purity from (HBV-infected) non-tumour and HCC tissues using a newly designed perfusion-free procedure. Hepatocyte-derived HLA-bound peptides were identified by unbiased mass spectrometry (MS), after which source proteins were subjected to Gene Ontology and pathway analysis. HBV antigen and TAA-derived HLA peptides were searched for using targeted MS, and a selection of peptides was tested for immunogenicity. Results: Using unbiased data-dependent acquisition (DDA), we acquired a high-quality HLA-I peptidome of 2 × 105 peptides that contained 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAA that were exclusive to tumours. Of these, 3 HBV- and 12 TAA-derived HLA peptides were detected by targeted MS in the sample they were originally identified in by DDA. Moreover, 2 HBV- and 2 TAA-derived HLA peptides were detected in samples in which no identification was made using unbiased MS. Finally, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides. Conclusions: We present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Identified HBV-derived and TAA-derived peptides directly aid development of antigen-specific immunotherapy for chronic HBV infection and HCC. The described methodology can also be applied to personalise immunotherapeutic treatment of liver diseases in general. Lay summary: Immunotherapy that aims to induce immune responses against a virus or tumour is a promising novel treatment option to treat chronic HBV infection and liver cancer. For the design of successful therapy, it is essential to know which fragments (i.e. peptides) of virus-derived and tumour-specific proteins are presented to the T cells of the immune system by diseased liver cells and are thus good targets for immunotherapy. Here, we have isolated liver cells from patients who have chronic HBV infection and/or liver cancer, analysed what peptides are presented by these cells, and assessed which peptides are able to drive immune responses.

Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.

BACKGROUND AND AIMS: HEV infection is the most common cause of liver inflammation, but the pathogenic mechanisms remain largely unclear. We aim to explore whether HEV infection activates inflammasomes, crosstalk with antiviral interferon response, and the potential of therapeutic targeting. APPROACH AND RESULTS: We measured IL-1ß secretion, the hallmark of inflammasome activation, in serum of HEV-infected patients and rabbits, and in cultured macrophage cell lines and primary monocyte-derived macrophages. We found that genotypes 3 and 4 HEV infection in rabbits elevated IL-1ß production. A profound increase of IL-1ß secretion was further observed in HEV-infected patients (1,733 ± 1,234 pg/mL; n = 70) compared to healthy persons (731 ± 701 pg/mL; n = 70). Given that macrophages are the drivers of inflammatory response, we found that inoculation with infectious HEV particles robustly triggered NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in primary macrophages and macrophage cell lines. We further revealed that the ORF2 capsid protein and the formed integral viral particles are responsible for activating inflammasome response. We also identified NF-κB signaling activation as a key upstream event of HEV-induced NLRP3 inflammasome response. Interestingly, inflammasome activation antagonizes interferon response to facilitate viral replication in macrophages. Pharmacological inhibitors and clinically used steroids can effectively target inflammasome activation. Combining steroids with ribavirin simultaneously inhibits HEV and inflammasome response without cross-interference. CONCLUSIONS: HEV infection strongly activates NLRP3 inflammasome activation in macrophages, which regulates host innate defense and pathogenesis. Therapeutic targeting of NLRP3, in particular when combined with antiviral agents, represents a viable option for treating severe HEV infection.

CD4+ T Cells in Chronic Hepatitis B and T Cell-Directed Immunotherapy.

The impaired T cell responses observed in chronic hepatitis B (HBV) patients are considered to contribute to the chronicity of the infection. Research on this impairment has been focused on CD8+ T cells because of their cytotoxic effector function; however, CD4+ T cells are crucial in the proper development of these long-lasting effector CD8+ T cells. In this review, we summarize what is known about CD4+ T cells in chronic HBV infection and discuss the importance and opportunities of including CD4+ T cells in T cell-directed immunotherapeutic strategies to cure chronic HBV.

Empirical Evaluation of the Use of Computational HLA Binding as an Early Filter to the Mass Spectrometry-Based Epitope Discovery Workflow.

Immunopeptidomics is used to identify novel epitopes for (therapeutic) vaccination strategies in cancer and infectious disease. Various false discovery rates (FDRs) are applied in the field when converting liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectra to peptides. Subsequently, large efforts have recently been made to rescue peptides of lower confidence. However, it remains unclear what the overall relation is between the FDR threshold and the percentage of obtained HLA-binders. We here directly evaluated the effect of varying FDR thresholds on the resulting immunopeptidomes of HLA-eluates from human cancer cell lines and primary hepatocyte isolates using HLA-binding algorithms. Additional peptides obtained using less stringent FDR-thresholds, although generally derived from poorer spectra, still contained a high amount of HLA-binders and confirmed recently developed tools that tap into this pool of otherwise ignored peptides. Most of these peptides were identified with improved confidence when cell input was increased, supporting the validity and potential of these identifications. Altogether, our data suggest that increasing the FDR threshold for peptide identification in conjunction with data filtering by HLA-binding prediction, is a valid and highly potent method to more efficient exhaustion of immunopeptidome datasets for epitope discovery and reveals the extent of peptides to be rescued by recently developed algorithms.

Human type 1 and type 2 conventional dendritic cells express indoleamine 2,3-dioxygenase 1 with functional effects on T cell priming.

Dendritic cells (DCs) are key regulators of the immune system that shape T cell responses. Regulation of T cell induction by DCs may occur via the intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO), which catalyzes conversion of the essential amino acid tryptophan into kynurenine. Here, we examined the role of IDO in human peripheral blood plasmacytoid DCs (pDCs), and type 1 and type 2 conventional DCs (cDC1s and cDC2s). Our data demonstrate that under homeostatic conditions, IDO is selectively expressed by cDC1s. IFN-γ or TLR ligation further increases IDO expression in cDC1s and induces modest expression of the enzyme in cDC2s, but not pDCs. IDO expressed by conventional DCs is functionally active as measured by kynurenine production. Furthermore, IDO activity in TLR-stimulated cDC1s and cDC2s inhibits T cell proliferation in settings were DC-T cell cell-cell contact does not play a role. Selective inhibition of IDO1 with epacadostat, an inhibitor currently tested in clinical trials, rescued T cell proliferation without affecting DC maturation status or their ability to cross-present soluble antigen. Our findings provide new insights into the functional specialization of human blood DC subsets and suggest a possible synergistic enhancement of therapeutic efficacy by combining DC-based cancer vaccines with IDO inhibition.

Designing the next-generation therapeutic vaccines to cure chronic hepatitis B: focus on antigen presentation, vaccine properties and effect measures.

In the mid-90s, hepatitis B virus (HBV)-directed immune responses were for the first time investigated in detail and revealed suboptimal T-cell responses in chronic HBV patients. Based on these studies, therapeutic vaccination exploiting the antigen presentation capacity of dendritic cells to prime and/or boost HBV-specific T-cell responses was considered highly promising. Now, 25 years later, it has not yet delivered this promise. In this review, we summarise what has been clinically tested in terms of antigen targets and vaccine forms, how the immunological and therapeutic effects of these vaccines were assessed and what major clinical and immunological findings were reported. We combine the lessons learned from these trials with the most recent insights on HBV antigen presentation, T-cell responses, vaccine composition, antiviral and immune-modulatory drugs and disease biomarkers to derive novel opportunities for the next generation of therapeutic vaccines designed to cure chronic HBV either alone or in combination therapy.

Neoantigen Quantity and Quality in Relation to Pancreatic Cancer Survival.

INTRODUCTION: Factors underlying antitumor immunity in pancreatic cancer (PC) are poorly understood. We hypothesized that not neoantigen quantity, but quality, is related to immune cell infiltration and survival. METHODOLOGY: We performed genomic and transcriptomic profiling of paired normal, tumor tissue of 13 patients with PC with distinct survival times. Additionally, neoantigens prediction and immunological profiling were performed. RESULTS: The proportion of neoantigens with a low similarity-to-self score was higher in short-term survivors (p < 0.0001), while mutational load and burden, similarity-to-known-pathogens, and immunogenicity of neoantigens were not associated with immune cell infiltration or survival. DISCUSSION: No tumor mutational load or neoantigen quantity, but low similarity-to-self score, was associated with immune cell infiltration and survival.

The Potential of Soluble Human Leukocyte Antigen Molecules for Early Cancer Detection and Therapeutic Vaccine Design.

Human leukocyte antigen (HLA) molecules are essential for anti-tumor immunity, as they display tumor-derived peptides to drive tumor eradication by cytotoxic T lymphocytes. HLA molecules are primarily studied as peptide-loaded complexes on cell membranes (mHLA) and much less attention is given to their secretion as soluble HLA-peptide complexes (sHLA) into bodily fluids. Yet sHLA levels are altered in various pathologies including cancer, and are thus of high interest as biomarkers. Disconcordance in results across studies, however, hampers interpretation and generalization of the relationship between sHLA levels and cancer presence, thereby impairing its use as a biomarker. Furthermore, the question remains to what extent sHLA complexes exert immunomodulatory effects and whether shifts in sHLA levels contribute to disease or are only a consequence of disease. sHLA complexes can also bear tumor-derived peptides and recent advancements in mass spectrometry now permit closer sHLA peptide cargo analysis. sHLA peptide cargo may represent a "liquid biopsy" that could facilitate the use of sHLA for cancer diagnosis and target identification for therapeutic vaccination. This review aims to outline the contradictory and unexplored aspects of sHLA and to provide direction on how the full potential of sHLA as a quantitative and qualitative biomarker can be exploited.

Opportunities for Conventional and in Situ Cancer Vaccine Strategies and Combination with Immunotherapy for Gastrointestinal Cancers, A Review.

Survival of gastrointestinal cancer remains dismal, especially for metastasized disease. For various cancers, especially melanoma and lung cancer, immunotherapy has been proven to confer survival benefits, but results for gastrointestinal cancer have been disappointing. Hence, there is substantial interest in exploring the usefulness of adaptive immune system education with respect to anti-cancer responses though vaccination. Encouragingly, even fairly non-specific approaches to vaccination and immune system stimulation, involving for instance influenza vaccines, have shown promising results, eliciting hopes that selection of specific antigens for vaccination may prove useful for at least a subset of gastrointestinal cancers. It is widely recognized that immune recognition and initiation of responses are hampered by a lack of T cell help, or by suppressive cancer-associated factors. In this review we will discuss the hurdles that limit efficacy of conventional cancer therapeutic vaccination methods (e.g., peptide vaccines, dendritic cell vaccination). In addition, we will outline other forms of treatment (e.g., radiotherapy, chemotherapy, oncolytic viruses) that also cause the release of antigens through immunogenic tumor cell death and can thus be considered unconventional vaccination methods (i.e., in situ vaccination). Finally, we focus on the potential additive value that vaccination strategies may have for improving the effect immunotherapy. Overall, a picture will emerge that although the field has made substantial progress, successful immunotherapy through the combination with cancer antigen vaccination, including that for gastrointestinal cancers, is still in its infancy, prompting further intensification of the research effort in this respect.

Design of TLR2-ligand-synthetic long peptide conjugates for therapeutic vaccination of chronic HBV patients.

Synthetic long peptide (SLP) vaccination is a promising new treatment strategy for patients with a chronic hepatitis B virus (HBV) infection. We have previously shown that a prototype HBV-core protein derived SLP was capable of boosting CD4+ and CD8+ T cell responses in the presence of a TLR2-ligand in chronic HBV patients ex vivo. For optimal efficacy of a therapeutic vaccine in vivo, adjuvants can be conjugated to the SLP to ensure delivery of both the antigen and the co-stimulatory signal to the same antigen-presenting cell (APC). Dendritic cells (DCs) express the receptor for the adjuvant and are optimally equipped to efficiently process and present the SLP-contained epitopes to T cells. Here, we investigated TLR2-ligand conjugation of the prototype HBV-core SLP. Results indicated that TLR2-ligand conjugation reduced cross-presentation efficiency of the SLP-contained epitope by both monocyte-derived and naturally occurring DC subsets. Importantly, cross-presentation was improved after optimization of the conjugate by either shortening the SLP or by placing a valine-citrulline linker between the TLR2-ligand and the long SLP, to facilitate endosomal dissociation of SLP and TLR2-ligand after uptake. HBV-core SLP conjugates also triggered functional patient T cell responses ex vivo. These results provide an import step forward in the design of a therapeutic SLP-based vaccine to cure chronic HBV.

Discovery and Selection of Hepatitis B Virus-Derived T Cell Epitopes for Global Immunotherapy Based on Viral Indispensability, Conservation, and HLA-Binding Strength.

Immunotherapy represents an attractive option for the treatment of chronic hepatitis B virus (HBV) infection. The HBV proteins polymerase (Pol) and HBx are of special interest for antigen-specific immunotherapy because they are essential for viral replication and have been associated with viral control (Pol) or are still expressed upon viral DNA integration (HBx). Here, we scored all currently described HBx- and Pol-derived epitope sequences for viral indispensability and conservation across all HBV genotypes. This yielded 7 HBx-derived and 26 Pol-derived reported epitopes with functional association and high conservation. We subsequently predicted novel HLA-binding peptides for 6 HLA supertypes prevalent in HBV-infected patients. Potential epitopes expected to be the least prone to immune escape were subjected to a state-of-the-art in vitro assay to validate their HLA-binding capacity. Using this method, a total of 13 HLA binders derived from HBx and 33 binders from Pol were identified across HLA types. Subsequently, we demonstrated interferon gamma (IFN-γ) production in response to 5 of the novel HBx-derived binders and 17 of the novel Pol-derived binders. In addition, we validated several infrequently described epitopes. Collectively, these results specify a set of highly potent T cell epitopes that represent a valuable resource for future HBV immunotherapy design.IMPORTANCE Multiple HBV-derived T cell epitopes have been reported, which can be useful in a therapeutic vaccination strategy. However, these epitopes are largely restricted to HLA-A*02, which is not dominantly expressed in populations with high HBV prevalence. Thus, current epitopes are falling short in the development of a global immunotherapeutic approach. Therefore, we aimed to identify novel epitopes for 6 HLA supertypes most prevalent in the infected population. Moreover, established epitopes might not all be equally effective as they can be subject to different levels of immune escape. It is therefore important to identify targets that are crucial in viral replication and conserved in the majority of the infected population. Here, we applied a stringent selection procedure to compose a combined overview of existing and novel HBV-derived T cell epitopes most promising for viral eradication. This set of T cell epitopes now lays the basis for the development of globally effective HBV antigen-specific immunotherapies.

Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane.

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular matrix (ECM) and the intracellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nanoclusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular domain. This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular domain and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nanoclusters localized to a meso-scale meshwork pattern.

Intracellular Galectin-9 Controls Dendritic Cell Function by Maintaining Plasma Membrane Rigidity.

Endogenous extracellular Galectins constitute a novel mechanism of membrane protein organization at the cell surface. Although Galectins are also highly expressed intracellularly, their cytosolic functions are poorly understood. Here, we investigated the role of Galectin-9 in dendritic cell (DC) surface organization and function. By combining functional, super-resolution and atomic force microscopy experiments to analyze membrane stiffness, we identified intracellular Galectin-9 to be indispensable for plasma membrane integrity and structure in DCs. Galectin-9 knockdown studies revealed intracellular Galectin-9 to directly control cortical membrane structure by modulating Rac1 activity, providing the underlying mechanism of Galectin-9-dependent actin cytoskeleton organization. Consequent to its role in maintaining plasma membrane structure, phagocytosis studies revealed that Galectin-9 was essential for C-type-lectin receptor-mediated pathogen uptake by DCs. This was confirmed by the impaired phagocytic capacity of Galectin-9-null murine DCs. Together, this study demonstrates a novel role for intracellular Galectin-9 in modulating DC function, which may be evolutionarily conserved.

Elevated serum levels of soluble CD14 in HBeAg-positive chronic HBV patients upon Peginterferon treatment are associated with treatment response.

Pegylated IFNα (PEG-IFN) is one of the treatment options for chronic HBV (CHB) patients. However, the high patient treatment burden and limited response rate together clearly ask for biomarkers to predict PEG-IFN response. Soluble CD14 (sCD14) is considered a marker for immune activation and has been shown to predict clinical outcome of HIV infection. However, studies on sCD14 in CHB infection are inconclusive, and its relationship with clinical outcome is largely unknown. Here, we measured sCD14 levels in CHB patients and investigated whether changes in sCD14 level related to PEG-IFN response. Serum sCD14 levels were determined in 15 healthy controls, 15 acute self-limited HBV, 60 CHB patients in different disease phases and 94 HBeAg+ CHB patients at week 0 and week 12 of a 52-week PEG-IFN treatment. Response to PEG-IFN treatment was defined as HBeAg seroconversion or HBeAg loss at 26 weeks post-treatment. The mean sCD14 level in acute HBV patients (3.0 µg/mL) was significantly higher than in CHB patients (2.4 µg/mL) and healthy controls (2.4 µg/mL). In CHB patients receiving PEG-IFN, a significant increase in sCD14 was found after 12-week treatment (median week 0:2.1 µg/mL; week 12:3.7 µg/mL). After 12-week treatment, the fold change (FC = w12/w0) in sCD14 was significantly higher in responders compared to nonresponders (HBeAg seroconversion: median FCresponder  = 2.1 vs FCnonresponder  = 1.6; HBeAg loss: median FCresponder  = 2.2 vs FCnonresponder  = 1.5). Receiver operating characteristic curves demonstrated that FC-sCD14wk12/wk0 levels can be of significant value as a stopping rule to select patients at week 12 who are not likely to benefit from further PEG-IFN treatment.

TLR7 polymorphism, sex and chronic HBV infection influence plasmacytoid DC maturation by TLR7 ligands.

TLR7 agonists are of high interest for the treatment of cancer, auto-immunity and chronic viral infections. They are known to activate plasmacytoid dendritic cells (pDCs) to produce high amounts of Type I Interferon (IFN) and to facilitate T and B cell responses, the latter with the help of maturation markers such as CD40, CD80 and CD86. The TLR7 single nucleotide polymorphism (SNP) rs179008 (GLn11Leu), sex and chronic viral infection have all been reported to influence pDC IFN production. It is unknown, however, whether these factors also influence pDC phenotypic maturation and thereby IFN-independent pDC functions. Furthermore, it is unclear whether SNP rs179008 influences HBV susceptibility and/or clearance. Here we investigated whether the SNP rs179008, sex and HBV infection affected phenotypic maturation of pDCs from 38 healthy individuals and 28 chronic HBV patients. In addition, we assessed SNP prevalence in a large cohort of healthy individuals (n = 231) and chronic HBV patients (n = 1054). Consistent with previous reports, the rs179008 variant allele was largely absent in Asians and more prevalent in Caucasians. Among Caucasians, the SNP was equally prevalent in healthy and chronically infected males. The SNP was, however, significantly more prevalent in healthy females than in those with chronic HBV infection (42 versus 28%), suggesting that in females it may offer protection from chronic infection. Ex vivo experiments demonstrated that induction of the co-stimulatory molecules CD40 and CD86 by TLR7 ligands, but not TLR9 ligands, was augmented in pDCs from healthy SNP-carrying females. Furthermore, CD80 and CD86 upregulation was more pronounced in females independent of the SNP. Lastly, our data suggested that chronic HBV infection impairs pDC maturation. These findings provide insight into factors determining TLR7 responses, which is important for further clinical development of TLR7-based therapies.